EDITORIAL ADVISORY BOARD
Special Edition n Cleaning Validation
III
Gamal Amer, Ph.D.
Validation and Process
Associates, Inc.
Louis A. Angelucci, III
Foster Wheeler Corporation
George N. Brower
Analex Corporation
Kenneth G. Chapman
Drumbeat Dimensions, Inc.
Dennis Christensen
Consultant
Robert C. Coleman
US Food & Drug Administration
Shahid Dara
Independent Consultant
PCI, Pharmachem International
William E. Hall, Ph.D.
Hall & Associates
Eldon Henson
Boehringer Ingelheim
Animal Health
JAY H. KING
LifeScan, a Johnson & Johnson Company
JOHN G. LANESE, Ph.D.
The Lanese Group, Inc.
Barbara Mullendore
AstraZeneca
ROBERT A. NASH, Ph.D.
St. John’s University
Charlie Neal, Jr.
BE&K
David R. Dills
Medtronic Xomed
TOD E. RANSDELL
Bio-Rad Laboratories
Michael Ferrante
Catalytica Pharmaceuticals
Patricia Stewart
Flaherty
Bayer Corporation
Roberta D. Goode
Consultant
CYNTHIA GREEN
Northwest Regulatory Support
Daniel Harpaz, Ph.D.
MELVIN R. SMITH
Independent Consultant
ROBERT W. STOTZ, Ph.D.
Validation Technologies, Corporation
ERIC D. VEIT
Johnson & Johnson
David W. Vincent
Validation Technologies, Inc.
Editor and Publisher
Glenn Melvin
Vice President
Terri Kulesa
Production Director
Edward Eick
Associate Publisher
Brandon Melvin
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Institute of Validation Technology
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ISSN 1079-6630
CONTENTS
TABLE
OF
Special Edition n Cleaning Validation III
Equipment Cleaning Validation: Microbial Control Issues . . . . . . . . . . . . . . . . . . . . . . . 6
Cleaning Validation: Maximum Allowable Residue: Question and Answer . . . . . . . 13
by
by
Destin A. LeBlanc, M.A.
William E. Hall, Ph.D.
Development of Total Organic Carbon (TOC) Analysis
for Detergent Residue Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
by
James G. Jin and Cheryl Woodward
Total Organic Carbon Analysis for Cleaning Validation
in Pharmaceutical Manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Karen A. Clark
by
Detergent Selection – A First Critical Step in Developing
a Validated Cleaning Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
by
Mark Altier
Analysis Cleaning Validation Samples: What Method? . . . . . . . . . . . . . . . . . . . . . . . . . . 35
by
Herbert J. Kaiser, Ph.D., Maria Minowitz, M.L.S.
Control and Monitoring of Bioburden in
Biotech/Pharmaceutical Cleanrooms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
by Raj Jaisinghani, Greg Smith and Gerald Macedo
A Cleaning Validation Program for the ELIFA System . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
by
LeeAnne Macaulay, Jeff Morier, Patti Hosler and Danuta Kierek-Jaszczuk, Ph.D.
BONUS
A Cleaning Validation Master Plan for Oral Solid Dose
Pharmaceutical Manufacturing Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
by Julie A. Thomas
Proposed Validation standard — VS-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Special Edition: Cleaning Validation III
5
Equipment Cleaning Validation:
Microbial Control Issues
By Destin A. LeBlanc, M.A.
Cleaning Validation Technologies
v
T
he PDA spring conference
taminants.” How­­ever, Section 6.7 of
was held in Las Vegas,
this document that covers “Micro­­bio­
}…it is
Nevada in March 20, 2001.
As­pects” focuses exclusively
becoming more logical
The conference showcased clean­
on the same issue discussed in the
common for
ing validation, residue limits, bio­
FDA guidance document, namely
burden, micro­bial limits, and sani­
the issue of preventing microbial pro­
regulatory
tization. This paper is based on a
liferation during storage.
authorities
pre­sentation at that conference.
As a practical matter, microbial
The initial focus of regulatory
residues on equipment surfaces are
to cite
documents relating to cleaning
part of the contaminants that should
manufacturers
validation for process equipment
be reduced to an acceptable level;
in pharmaceutical manufacturing
that acceptable level being what is
for deficiencies
in­volved measuring residues of the
safe for the manufacture of the sub­
related to
drug active and the cleaning agent.
sequently manufactured pro­duct.
For example, the introduction to
Unfortunately, very little has been
microbial
the Food and Drug Ad­mini­stra­tion
written on what is a safe level for
control
in
(FDA) guidance document on clean­­
microorganisms following cleaning
ing validation1 states: “This guide is
and/or sanitation.3,4 Part of the reason
cleaning
intended to cover equipment clean­
for this is that microbial resi­dues are
validation
ing for chemical residues only.”
significantly different from chemi­
While admitting that microbial re­s­
cal re­sidues. Chemical resi­dues are
programs.~
i­dues are beyond the scope of the
“in­ert” in the sense that it is easy to
guideline, that guidance document
cal­culate (especially using scenarios
further states, “microbiological aspects of equip­
of uniform contamination in the subsequently manu­
ment cleaning should be considered,” particularly factured product) the potential levels and effects of
with reference to preventive measures so that micro­
those chemical residues in the subsequently manu­
bial proliferation does not occur during storage. The factured pro­duct should they be transferred to that
European PIC/S document,2 that was issued several
subsequently manufactured pro­duct. With microbial
years later, does explicitly mention microbial re­si­
residues left after the cleaning process, the situation
dues. In Section 6.2.1, contaminants to be re­moved
is somewhat different. Because microorganisms are
in­clude “the previous products, residues of cleaning living organisms, those left as residues on equipment
agents as well as the control of potential microbial con­ may change in number after the cleaning process, but
6
Institute of Validation Technology
Destin A. LeBlanc, M.A.
before the manufacture of the subsequently manu­
the cleaned equipment. However, many times this
factured pro­duct. Those microbes transferred to the does not include any assessment as to the effect of
subsequently manufactured product may also change that unchanged bioburden level on the subsequently
in number after they are incorporated into the subse­
manufactured product.
quently manufactured product in the manufacturing
This paper will address issues covering ap­proaches
step. This change may be a significant reduction in
to control of microorganisms in process equipment,
bioburden, either due to drying of the equipment or
setting of acceptance limits, sampling techniques, and
due to a preservative in the finished drug product, approaches to providing acceptable documentation.
for example. This change may also involve rapid
Microbial Control Measures
proliferation, either due to suitable growth conditions
in wet equipment during storage, or due to suitable
Control measures to reduce the bioburden on
growth conditions in the finished drug product. Or,
they may result in no significant change in microbial cleaned process equipment include control of bio­
level, because the bioburden was due to bacterial burden of raw materials, the cleaning process itself,
spores (that will survive readily in
dried equipment), or because the
}Some companies will measure the
subsequently manufactured product
was a dry product (with low water
change in microbial levels on
activity). There­fore, knowing the
equipment surfaces during storage
levels of microorganisms left on the
equipment following cleaning does
of the cleaned equipment. However,
not necessarily give one the full
many times this does not include any
story of the po­ten­tial hazards of those
microbial residues. Addi­tional in­for­
assessment as to the effect
mation is required to assess those
of that unchanged bioburden
potential hazards.
Why has microbial evaluation
level on the subsequently
during cleaning of process equip­
manufactured product.~
ment been a little discussed topic?
Part of the reason is that it is not a
significant problem in process man­
ufacturing. Yes, it could conceivably be a problem if a separate sanitizing step, and drying of the equip­
cleaning and storage were inadequate. How­ever, for ment following cleaning. Bioburden of raw materials
the most part, cleaning and storage of pro­cess equip­ in­cludes the active, excipients, water, and any process­
ing aids. In many cases, the manufacturer may have
ment, in so far as it applies to microbial residues,
probably is done relatively well in most pharmaceu­ little control over the bioburden of raw materials other
tical manufacturing facilities. On the other hand, it is than to accept a specification by the raw material sup­
becoming more common for regulatory authorities to plier. The most critical raw materials probably will be
cite manufacturers for deficiencies related to micro­ natural products, in which there may be considerable
bial control in cleaning validation programs. One variation in the levels and types of microorganisms.
A solid monitoring program to control in­coming bio­
reason for this seeming anom­aly is that while firms
are adequately controlling microbial contamination of burden of raw material is necessary. If there could be
process equipment, there may be little documentation significant variation in bioburden, then that should
to support this. This lack of documentation includes be addressed in the cleaning validation Performance
any measurement of microbial residues during the Qualification (PQ) trials. At least one PQ trial should
cleaning validation and/or during routine monitoring. utilize the worst-case incoming bioburden of raw
Some companies will measure the change in micro­ materials to demonstrate adequate cleaning and micro­
bial levels on equipment surfaces during storage of bial control under those conditions.
Special Edition: Cleaning Validation III
7
Destin A. LeBlanc, M.A.
A second means of microbial control is the cleaning
process itself. The conditions of aqueous cleaning
are often hostile to microbial survival. These con­ditions
include high temperature (commonly 60-80ºC), pH
extremes (>11 and <4), and the presence of oxidizers
(such as sodium hypochlorite in biotechnology manu­
facture). In addition, the presence of surfactants in the
cleaning solution can assist in providing good physical
removal of microbes (without necessarily killing them).
Good cleaning is also beneficial to microbial control in
that chemical residues left behind can provide a physi­
cal “microbial trap” to allow microorganisms to survive
even in the presence of chemical sanitizers. Those
chemical residues left behind might also serve as a
nutrient source that allows microbes to proliferate dur­
ing improper storage. Based on the author’s experience,
in most cases, effective control of microorganisms in
pharmaceutical process equipment can be achieved
with the use of an effective cleaning process, without
the need for a separate chemical sanitizing step.
In some cases, a separate sanitizing step may be
necessary. This may include sanitation by steam or by
chemical sanitizers. Suitable chemical sanitizers for
process equipment include sodium hypochlorite (chlo­
rine bleach), quaternary ammonium compounds, alco­
hol (ethyl or isopropyl), hydrogen peroxide, and per­
acetic acid. It should be noted that, with the exception
of alcohol and hydrogen peroxide, additional rinses
would be necessary to remove any chemical residues
of the sanitizer from the equipment. Those chemical
residues may also have to be evaluated as residues to
be measured in the cleaning validation protocol. For
such chemical treatments, it is not an expectation that
the equipment be sterile. Unless the final rinse is with
sterile water, microorganisms will be reintroduced
into the equipment from the use of Water-for-Injection
(WFI) or purified water as the final rinse.
Some companies will use an alternative to sanitizing
immediately after cleaning. This usually involves sani­
tizing after storage and immediately before use. This
may be used in situations where it is difficult to control
microbial recontamination or proliferation during stor­
age. It should be noted that control of storage condi­
tions, if possible, is preferable. The practice of relying
solely on a separate sanitizing step immediately before
manufacture should be discouraged. If this is practiced,
then the sanitization step should be shown to be effec­
tive in reducing bioburden under the worst-case storage
8
Institute of Validation Technology
conditions (“initial” bioburden, time, temperature, and
humidity). Needless to say, if the chemical sanitizing
step is performed im­mediately prior to manufacture of
the subsequently manufactured product, then removal
of the sanitizer chemical residues to an acceptable level
should also be demonstrated.
A fourth consideration for control of microor­
ganisms is drying the process equipment surfaces
following the final rinse. Drying the surfaces will
further reduce the levels of vegetative organisms on
the surface. In addition, drying will assist in prevent­
ing microbial proliferation during storage. Drying
can be achieved by heated air, heated nitrogen, or
by rinsing with alcohol. In all cases, the process can
be assisted by application of a vacuum (to speed the
evaporation of the water or, in the case of an alcohol
rinse, of the alcohol itself).
Limits for Microbes
As mentioned earlier, it is possible to reasonably
predict levels of chemical residues in subsequently
manufactured products based on the levels present on
equipment surfaces.5,6 With microorganisms, it is pos­
sible to measure levels on equipment surfaces; how­
ever, the effect of those residues will depend on what
happens to those microorganisms once they come in
contact with the subsequently manufactured product.
Areas that may have to be evaluated include the species
(including the so-called “objectionable” organisms),
type of organism (vegetative bacteria versus bacterial
spore, for ex­ample), the presence of preservatives in that
subsequently manufactured product, the water activity
of the subsequently manufactured product, as well as
any subsequent sterilization process performed on that
product. As a general rule, if the water activity is less
than 0.6, then it can be expected that microorganisms
will not proliferate (although they may continue to sur­
vive without reproducing).7 Water activity is a physicalchemical measurement that ex­presses the water vapor
pressure above the test sample as a fraction of the water
vapor pressure of pure water at the same temperature
as the test sample. For aqueous products with a neutral
pH, microbial proliferation can generally be expected
unless there is a preservative in the product. If there
is a possibility of microbial proliferation because the
product is unpreserved and neutral, then that should be
addressed in setting limits.
Destin A. LeBlanc, M.A.
Three methods to set microbial limits will be
ad­dressed. The first (Case I) involve limits where the
sub­sequent product does not allow microbial prolif­
eration and is not subject to any further sterilization
process. The second (Case II) involves subsequently
manufactured products that are terminally sterilized.
The third (Case III) involves subsequently manufac­
tured products that are processed aseptically.
Case I Limits
If the subsequently manufactured product does not
allow microbial proliferation, then the determination
of acceptable microbial limits in the cleaned equip­
ment can be calculated using the same principles used
for chemical residues with one important exception.
This process involves first determining the accep­
tance limit in the subsequently manufactured product.
This limit is typically given in Colony Forming Units
(CFU) per gram of product. Once this is determined,
then the limit per surface area of equipment (assum­
ing uniform contamination) can be calculated based
on the batch size of the subsequently manufactured
product and the equipment surface area.
How is the limit in the subsequently manufactured
product determined? For chemical residues, it is based
on dosing information for actives or toxicity in­for­mation
for cleaning agents. Such concepts cannot be directly
applied to microbes. Fortunately, there are two good
sources of information relating to levels of microorgan­
isms in products. One is the manufacturer’s own Quality
Control (QC) specifications for the product, that may
include a limit for bioburden in the product. A second
source is information given in the proposed United
States Pharmacopeia (USP) <1111> relating to
“Microbial Attributes of Non­sterile Pharma­copeial
Articles.”8 Examples of those limits are given below:
Solid oral: ≤1000 CFU/g
Liquid oral;≤100 CFU/g
Topicals: ≤100 CFU/g
Note: Although these limits were discussed and
proposed in the Pharmacopeial Forum, these spe­
cific recommendations were not adopted officially
as part of the 24th edition of the USP.
Unfortunately, this is where the one exception to
the conventional treatment arises. When one looks at
the bioburden in a finished drug product, the equip­
ment surfaces are not the only source of bioburden.
One must also consider the raw materials themselves,
as well as the primary packaging, as potential sources
of microorganisms. The best way to deal with this
issue is to develop information on the bio­burden of the
raw materials and the primary packaging, and factor
these into the limits calculation. For example, if one
were dealing with an oral liquid, one might calculate
the contribution from the raw materials (assuming
the upper limit bioburden for each raw material) as a
maximum of 27 CFU/g. At the same time the contribu­
tion from the primary packaging is determined to be 3
CFU/g. Therefore, the amount allowed from equipment
surfaces would be 70 CFU/g (100 minus 27 minus 3).
An additional safety factor should be used to account
for the significant variability in microbiological enu­
meration. An appropriate factor may be on the order
of 5. There­fore, in this case, the limit (in CFU/g) that
would be allowed solely due to the cleaned equipment
surfaces would be 14 CFU/g (obtained by dividing 70
by 5). Higher safety factors also could be considered.
These numbers are given for illustration purposes only.
It should be realized that the contribution percentage
allowed from cleaned equipment would vary depend­
ing on the contributions from the raw materials and the
primary packaging.
Once the limit in the subsequently manufactured
product allowed from the cleaned equipment sur­
faces is determined, the next step is to determine the
limit per surface area (CFU/cm2). This is calculated
exactly as it would be for chemical residues:
Limit per surface area = LSP x MBS
SA
where
LSP = Limit in the subsequent product
MBS = Minimum batch size
SA = Product contact surface area
In the example above, if the batch size is 200 kg
and the product contact surface area is 260,000 cm2,
then the microbial surface limit of the cleaned equip­
ment is:
Limit per surface area =(70 CFU/g)(200,000g) = 54 CFU/ cm2
(260,000 cm2)
Special Edition: Cleaning Validation III
9
Destin A. LeBlanc, M.A.
If sampling were done with a typical contact plate
of 25 cm2, this would correspond to a limit of over
1300 CFU per contact plate. Since it is reasonable
to count a maximum of only 250 CFU on a typical
contact plate, this would clearly be in the TNTC (too
numerous to count) category. Needless to say, this will
vary with the limit in the subsequently manufactured
product, the portion allowed from cleaned surfaces, the
safety factor used, batch size, and the shared surface
area. However, under most reasonable scenarios, the
calculated limit due to microorganisms on the cleaned
equipment surfaces will be significantly above what
should be (and can be) achieved by proper cleaning.
As a general rule, a good cleaning process should
produce surfaces that contain no more than 25 CFU
per contact plate (<1 CFU/cm2). When failures occur,
generally they will be gross failures, with counts gen­
erally above 100 CFU per-plate.
Case II Limits
This involves setting limits for cleaned equipment
when the product subsequently manufactured in that
equipment is to be sterilized. In this case, the microbial
limit in the subsequently manufactured product can be
established based on the assumed bioburden of that
product at the time of sterilization. In other words, any
validated sterilization process depends on an assumed
bioburden of the item being sterilized. That assumed
bioburden then becomes the limit in the subsequently
manufactured product. Once that limit in the subse­
quently manufactured product is established, then the
calculations are the same as for Case I – a certain por­
tion of that total limit is allowed from cleaned equip­
ment surfaces, a safety factor is applied, and then the
limit per surface area is calculated using the minimum
subsequent product batch size and the product contact
surface area. It is significant that this issue is actually
addressed in the FDA’s cleaning validation guidance
document; that states:
“…it is important to note that control of bio­
burden through adequate cleaning and storage of
equipment is important to ensure that subsequent
sterilization or sanitization procedures achieve
the necessary assurance of sterility.” 9
Case III Limits
This third case involves setting limits on equip­
10
Institute of Validation Technology
ment surfaces where the subsequently manufactured
product is aseptically produced. This case is slightly
different from Case II in that it is the equipment itself,
and not the product, which is subsequently sterilized.
This case is relatively straightforward, because the
microbial limits on the surfaces of cleaned equipment
are established based on the assumed bioburden of the
equipment surfaces for sterilization validation of that
equipment. No information on batch sizes or surface
areas is necessary. The assumed bioburden for the
sterilization validation can be used directly for limit
purposes. The only adjustment may be the incorpora­
tion of a safety factor (to accommodate normal varia­
tion in microbiological enumeration).
Measurement Techniques
Conventional tools used for microbial enumeration
from surfaces can be used. These include rinse water
sampling (usually with membrane filtration), swab­
bing (with desorption of the swab into a sterile solu­
tion and then a pour plate count), and use of a con­tact
plate. The choice of recovery medium and incubation
conditions is usually dictated by the expected organ­
isms. As a general rule, the initial focus is on aerobic
bacteria. However, if anaerobic bac­teria or molds/
yeasts are suspected problems, these should be also
evaluated.
One issue that does not translate directly from
chemical residue measurements is the idea of deter­
mining percent recovery using the sampling method.
In the measurement of chemical residues, the target
residue is spiked onto a model surface and the quan­
titative percent recovery is determined. The amount
re­covered as a percent of the amount spiked is consid­
ered the sampling method percent recovery. Per­cent
recoveries in chemical sampling measurement are
generally above 50 percent. This percent recovery is
then used to convert an analyzed sample value; for
example, if a chemical residue measured by a swab­
bing technique gives 0.6 µg of residue, then with a 50
percent recovery, this actually represents the possibil­
ity of 1.2 µg being on that surface. This concept can­
not be applied directly to microbiological sampling.
The reason for this is partly the inherent variability in
microbiological testing. If one measured 10 CFU in
one test and 5 CFU in a duplicate test (a 50 percent
difference), one would be hard pressed to say that
Destin A. LeBlanc, M.A.
those numbers are significantly different. In addition,
how would one actually measure the percent recovery
in a microbiological test? If a model surface is spiked
with a specific number of a certain bacterium, and
then that surface is allowed to dry and is sampled,
just the process of drying might cause a low recovery
of bacteria (due to the dying of vegetative bacteria by
drying). In addition, what species of bacteria would
be used for the recovery study?
It is recognized that microbiological sampling
methods may understate the number of microbes on
a surface (indeed the concept of a CFU, that may
limits should be included in the validation protocol,
and measured as part of the three PQ trials. One
should also include the absence of “ob­jectionable”
organisms as part of the acceptance criteria.
To deal with processes for which cleaning valida­
tion has already been completed, but for which no
microbial evaluation has been done, there are two
strategies available. The objective of each is to devel­
op documentation that the cleaning process consis­
tently provides equipment surfaces with acceptable
bioburden. One option is to perform a cleaning
validation PQ, measuring only bioburden on sur­
faces for comparison to calculated
acceptance limits. The other option
}One issue that does not translate
is to initiate a routine microbiologi­
cal mon­itoring program as part of
directly from chemical residue
the monitoring of cleaning. This
measurements is the idea of
may involve something as simple
as monitoring the bioburden in the
determining percent recovery
final rinse water to demonstrate con­
using the sampling method.~
sistency. This data, combined with
product QC data on bioburden, may
satisfy the need for adequate docu­
contain any number of bacteria, also clouds the issue).
mentation.
There are two ways to view such an issue. One is to
One should also consider one’s motivation for
make it clear that whatever variation exists in measur­ wanting to obtain assur­ance that the bioburden is
ing micro­organisms on surfaces is probably equally an ac­ceptably low after cleaning. If the im­petus for action
issue when one sets limits based on product limits or
is due to lack of data, one should resist the impulse to
sterilization bioburden limits. Therefore, the variabili­
immediately add a sanitizer into the cleaning program.
ty issue becomes a “wash.” The other perspective is to
The focus should be on developing data to demonstrate
ac­count for such variation by choosing extremely high
the sufficiency of the current cleaning process. Adding
safety factors. In the calculation example for Case I, a separate sanitizing step only complicates matters by
a factor of 5 was used as a safety factor. Even if that
adding additional residue concerns. If the impetus for
safety factor were increased to 10 or 20, the calculated action is due to observed high microbial counts on
acceptance limits would have still been ex­tremely
equipment surfaces or (more likely) in manufactured
high, and still beyond what one should achieve with a
product, then it is important to determine by careful
well-designed cleaning program.
investigation whether that unacceptable contamination
is due to issues with the cleaning process, with stor­
Documentation Strategies
age, or to both. In such a case, a separate sanitizing
step should only be added if the data fully support it.
How these issues will be addressed will depend on
Conclusion
the stage of the cleaning process development. For a
new process being designed, the best strategy is to pre­
Bioburden on cleaned equipment is an impor­
pare a calculation of microbial limits, and then design
the cleaning process to meet those acceptance criteria. tant concern in the cleaning process. Fortunately,
Included in that evaluation should be any change in most aqueous cleaning processes, properly designed,
bioburden (in particular, any increase or proliferation) should provide low and acceptable bioburden levels
on storage of the equipment. The micro­bial acceptance on equipment surfaces following the cleaning pro­cess.
Special Edition: Cleaning Validation III
11
Destin A. LeBlanc, M.A.
Proper drying and storage should provide assurance
that microbial proliferation does not occur be­fore
the manufacture of the subsequently manufactured
product in that equipment. Any scientifically justi­
fied determination of acceptable bioburden levels,
particularly for non-sterile products, is generally far
higher than what should be achieved in conventional
practice. This is becoming more of a regulatory and
compliance issue, not because microbial contami­
nation is a widespread pro­blem, but rather because
pharmaceutical manufacturers may lack appropriate
documentation to support their practices. This can
easily be remedied by a separate validation protocol
to address microbial issues, or by routine monitoring
to demonstrate consistency. o
About the Author
Destin A. LeBlanc, M.A., is with Cleaning Validation
Technologies, providing consulting in the area of
pharmaceutical cleaning validation. He has 25
years experience with cleaning and microbial control technologies. He is a graduate of the University
of Michigan and the University of Iowa. He can be
reached by phone at 210-481-7865, and by e-mail
at destin@cleaningvalidation.com.
References
1. FDA. “Guide to Inspections of Validation of Cleaning Pro­
cesses.” 1993.
2. Pharmaceutical Inspection Cooperation Scheme. Recom­men­
da­tions on Cleaning Validation. Document PR 1/99-2. Geneva,
Switzerland. April 1, 2000.
3. A.M. Cundell. Microbial Monitoring. Presented at the 4th IIR
Cleaning Validation Conference, October 20-22, 1997. (http://
microbiol.org/files/PMFList/clean.ppt, accessed May 29, 2001).
4. S.E. Docherty. “Establishing Microbial Cleaning Limits for Nonsterile Manufacturing Equipment.” Pharmaceutical En­gineering.
Vol. 19 No. 3. May/June 1999. Pp. 36-40.
5. G.L. Fourmen and M.V. Mullen. “Determining Cleaning
Validation Acceptance Limits for Pharmaceutical Manufact­uring
Operations.” Pharmaceutical Technology. Vol. 17 No. 4. 1993.
Pp. 54-60.
6. D.A. LeBlanc. “Establishing Scientifically Justified Ac­ceptance
Criteria of Finished Drug Products.” Pharma­ceutical Technology.
Vol. 19 No. 5. October 1998. Pp. 136-148.
7. R.R. Friedel. “The Application of Water Activity Measurements
to Microbiological Attributes Testing of Raw Materials Used
in the Manufacture of Nonsterile Pharma­ceutical Products.”
Pharmacopoeial Forum. Vol. 25 No. 5. September-October
1999. pp. 8974-8981.
8. <1111> Microbial Attributes of Nonsterile Pharmacopoeial
Articles (proposed). Pharmacopoeial Forum. Vol. 25 No. 2.
March-April 1999. Pp. 77857791.
9. FDA. “Guide to Inspections of Validation of Cleaning Pro­
cesses.” 1993.
12
Institute of Validation Technology
CFU:
FDA:
PQ:
QC:
USP:
WFI:
Article Acronym Listing
Colony Forming Units
Food and Drug Ad­mini­stra­tion
Performance Qualification
Quality Control
United States Pharmacopeia
Water-For-Injection
Cleaning Validation:
Maximum Allowable Residue
Question and Answer
W
e are involved in the pro­
duction of soft gel­atin
capsules and tablets in
our newly built facility. Our prod­
ucts consist of at least 17 minerals
and multivitamins in a single pro­
duct, while other products consist of
the same ingredients having some
quantity (in MG) varying with the
previous one. In some products,
some vitamins are not present. I want
to know how to conduct a cleaning
validation study of each product.
Again, I want to know which ingre­
dients I have to check after cleaning
of the equipment to determine the
residues?
The choice of which ingredient in
a multi-ingredient product should
}…sometimes
serve as the focus of the cleaning
the many
validation is often a difficult one for
vitamin and mineral products. For
possible
classical pharmaceutical products,
combinations
the choice is usually based on choos­
the most potent ingredient, or the
of products and ing
least water soluble ingredient, or a
equipment would combination of these two factors.
vitamins and minerals the choice
result in so many For
may be more difficult because of
studies that the the many ingredients present in the
and the relatively small
company would formulation
amounts present. Coup­led with these
never be able to difficulties is often the difficulty in
assaying the very small amounts of
complete them
active re­sidues that might be pres­
during a
• What will the limit be for the micro­­
ent after cleaning. My suggestion
bial contamination for the cleaning
would be to identify an ingredient for
reasonable
validation studies, and what will be
which there is a good sensitive assay
period of time.~ available. For example, if one of the
the rationale for the same?
• If I’m using some cleaning agent,
in­gredients hap­pens to show good
then what rationale is used for
de­tectable levels of fluorescence
keeping the limit the same?
(e.g., riboflavin, folic acid, and certain B vitamins
show good fluorescence) in water, then this material
could be selected as the “marker” material, and could
Thank you for your question. It is a very good
one because it represents cleaning from the serve as the ingredient to focus on during the analysis
of the rinse samples. In the case of vitamins and min­
point of view of a manufacturer of vitamins and min­
erals, it may be necessary, and even highly desirable,
erals, which in some countries, are considered drugs,
to take this ap­proach because of the extremely low
and in other countries, are considered as “nutraceuti­
levels of residues present after cleaning. It may also
cals,” an important and emerging part of our business.
The first specific question you asked related to be possible to examine equipment in a dark room with
the use of an ultraviolet light to identify areas of equip­
how to conduct a cleaning validation for each prod­
ment that are not cleaned sufficiently (an enhanced
uct, and how to select which ingredient to check
visual examination), again utilizing the known fluo­
after cleaning to verify that the cleaning is adequate.
A:
Special Edition: Cleaning Validation III
13
William E. Hall, Ph.D.
rescent behavior of certain vitamins. A brief study will
need to be carried out to determine if this approach is
appropriate and adequate for your particular situation.
I would suggest that you not try to con­duct cleaning
validation for every product. The reason I say that
is be­cause sometimes the many possible combina­
tions of products and equipment would result in so
many studies that the company would never be able
to complete them during a reasonable period of time.
If, for example, you have 50 products, and each could
be run on ten (10) different pieces of equipment, then
you would need 500 studies to cover all the possible
combinations and permutations. That is simply too
much of a re­source and cost issue for the average
company to face. It would be much better to divide
your products into groups or families, and choose one
or two representatives from each group to conduct full
cleaning validation. The assumption is that you can
pick some “worst-case,” most difficult to clean, potent
products from each group. The first step is to divide
the products into groups. I don’t know the names and
ingredients of the products your company manufactur­
ers; however, you did mention that some products are
vitamin products and others are mineral products. So I
think there would be two major groups – vitamins and
minerals. Then each of these groups might be further
divided, if necessary. For example, in the vitamin cat­
egory you may have some products that contain water
sol­uble vitamins, and some that contain fat soluble
vitamins. So now we have three (3) major groups
(water soluble vitamins, fat soluble vitamins, and
mineral pro­­ducts). So you begin to see our approach.
It might be that if you have vastly different types of
mineral products you might want to also further divide
that group into smaller groups. In any event, you want
to have pro­bably four (4) to ten (10) products in each
group, and then pick a worst-case representative from
each group. So by choosing this “grouping approach,”
you have re­duced the work from a very large resource
requirement to a doable or achievable project.
The choice of the worst-case representative should
be based on a combination of aqueous solubility and
po­tency. The potency can be determined for some
pro­ducts by determining the amount present in the
product from the label or package insert. Sometimes
this may be a little confusing for vitamin products
because the amounts are listed in units instead of
quantitative amounts, such as milligrams. In these
14
Institute of Validation Technology
cases, I would sug­gest that you refer to the Internet,
and conduct a search on the toxicity or potency of these
materials. You may be surprised to find that a vita­
min, such as folic acid, is quite potent in terms of its
medical effect and dosage.
The limits for these products can be calculated
by allowing a certain small fraction of vitamins or
minerals to carry over to each dose of the following
product. Again, you will need basic information, such
as the medical dosage of the initial product, the batch
size and dosage of the next or subsequently manufac­
tured product. In terms of the safety factor, i.e., the
factor that is used to reduce the allowable dosage, I
suggest that you use a factor of 1/100th for vitamin
and mineral products. A factor of 1/1000th is often
used for pharmaceuticals, but I feel a more generous
factor of 1/100th is appropriate for vitamin and min­
eral products. You could refer to some of the articles
published in the Journal of Validation Technology for
the details of how to calculate specific limits.
Your last question related to what rationale should
be used for the cleaning agent itself. The basic
re­quirement is that you be able to provide data that
de­monstrates that the cleaning agent itself is re­moved
during the cleaning process, usually by the final rinse.
You will need to go through the same rationale for
the product residue limits, i.e., establish a scientific
basis or justification that shows that the most potent
ingredient in the cleaning agent is reduced to a medi­
cally insignificant level. It is beyond the scope of this
answer to go into the mathematical details of how to
calculate this data, but again the details can be found
in the various articles published in the Journal of
Validation Technology. You will need to know about
the ingredients in your cleaning agent, as they are
typically multi-ingredient formulations, just like our
pharmaceutical products, and you will need to get that
information from your supplier of cleaning agents.
The good news is that if you use the same cleaning
agent and cleaning procedure for many products, then
you only have to do a single cleaning validation study
(three runs) for the cleaning agent. o
This answer was provided by an Editorial Advisory
Board Member, William E. Hall, Ph.D. Dr. Hall be
reached by phone at 910-458-5068, or by fax at 910458-1087, and by e-mail at cleandoct@aol.com.
Development of Total Organic Carbon
(TOC) Analysis for Detergent
Residue Verification
By James G. Jin
and Cheryl Woodward
Boehringer Ingelheim Pharmaceuticals, Inc.
T
v
he 1993 FDA Guideline for
concluded that the visual detection
cleaning validation states
of foam was the best method for the
}…the
that the removal of deter­
detergents they tested.4 The method
gent residues should be evaluated biotechnology and of visual detection of foam is only
effective for foaming detergents,
and there should be no or very low
pharmaceuti1
but is invalid for low foaming deter­
detergent levels left after cleaning.
cal industry has gents. From a user’s point of view,
Currently, the pharmaceutical in­dus­
try employs varieties of detergents
this paper documents that TOC is an
become
for cleaning and different clean­­ing
effective and quantitative method
increasingly
validation programs. Many com­
for detergent residue verification.
panies have not included detergent
interested in
Total Organic Carbon
residue evaluation as part of their
the use of
Methodology
cleaning validation programs main­
ly due to unavailability of ef­fective
TOC [Total
TOC is a non-specific method for
methodologies or lack of aware­ness
Organic
Carbon]
the compound analyzed. How­ever,
of the requirement by man­agement.
TOC analysis is sensitive to very
In the late 1970s, To­tal Organic
as
an
analytical
low levels of 0.002-0.8 ppm carbon,
Carbon (TOC) analysis had been
tool in cleaning depending on whether the sample is
used for monitoring water quality in
a water sample or a swab sample.
pharmaceuticals and en­viron­mental
validation
Cur­rent­ly, two major oxidation tech­
controls. More re­cent­ly, the biotech­
programs.~
nologies dominate the TOC market:
nology and pharmaceutical industry
combustion and Ultra Violet (UV)/
has be­come in­creasingly interested
persulfate. There has been debate
in the use of TOC as an analytical
about which technique is better suited for TOC testing
tool in cleaning validation programs. TOC analy­
since the late 1980s. The major differences for each
sis has been used as an analytical tool for cleaning
technique5 are described in Figure 1, and give the user
validation in the biotechnology industry for years.2,3
Westman and Karlson recently conducted a compari­
appropriate information to make an informed deci­
son study for different analytical methods – visual
sion as to which technique better serves their needs.
detection of foam, pH, conductivity measurements,
The best TOC oxidation technology is the one
and TOC for detergent residue evaluation. They that meets the application and analytical needs of the
Special Edition: Cleaning Validation III
15
James G. Jin
Figure 1
Types of Total Organic Carbon Techniques
Oxidation
Combustion
Combustion
UV/Persulfate
Heated Persulfate
Combustion
UV/Persulfate
UV
Detection TechniqueAnalytical Range (TOC)Official Methods
Thermal Conductivity Detector (TCD) 0.5 – 100%
AOAC 955.07
Coulometric
1 – 100%
ASTM D4129
Non-Dispersive Infrared Detector (NDIR) 0.002 – 10,000 mg/L
USP 643
NDIR
0.002 to 1,000 mg/L
USP 643
NDIR
0.004 – 25,000 mg/L
USP 643
Membrane/Conductivity
0.0005 – 50 mg/L
USP 643
Conductivity or NDIR
0.0005 – 0.5 mg/L
USP 643
user’s situation. The UV/Persulfate method meets
precision and accuracy requirements for low-level
cal­ibration check standards such as 0.5 ppm carbon
in detergent residue evaluation. However, if captur­
ing the particulate organic matter in the TOC value
is important, then combustion would be the better
oxidation technology. The instrument we chose is a
Tekmar-Dohrmann Phoenix 8000 with the UV/Per­
sul­fate oxidation technique.
degradation of all carbon species to carbon dioxide,
water, and other oxides of heteroelements. The UV
light alone induces breakdown of many carbon spe­
cies with the persulfate providing additional help to
attack compounds difficult to oxidize. The radical
reactions are aggressive and indiscriminate in their
attack.
Chemistry of Oxidation and Total Organic
Carbon Analysis of UV/Persulfate
The NDIR is constructed in such a way as to be
sensitive and selective for carbon dioxide present
in the gas flow. An infrared beam from the source
is passed through a chopper and down the sample
chamber to a dual chamber detector. Each chamber is
filled with carbon dioxide and is separated by a thin
membrane. Varying intensity of the light hitting the
cell causes fluctuation in temperature and thus the
pressure of the gas inside the detector. This causes
the membrane to deflect, which is ultimately read as
a millivolt output signal from the detector.
Wet chemistry oxidation of carbon compounds
utilizes two chemical reactions to complete the
analysis. A 21 percent solution of phosphoric acid
is utilized in converting inorganic carbon species.
Acid­ification of the sample allows for attack on inor­
ganic species such as carbonates and bicarbonates
to convert them to carbon dioxide. This, along with
any dissolved carbon dioxide in the sample is then
sparged out, and either exhausted to vent or routed
to the Non-­Dispersive Infrared detection (NDIR) for
quantification when analyzing for Inorganic Carbon
(IC) or TOC by difference (TC-IC).
H+ + CO -2 → H O + CO
3
2
2
Persulfate is used to do the rest of the oxidation
chemistry that is required for analysis. Sodium persul­
fate, at a concentration of 10 percent, and phosphoric
acid, five percent are added to the UV chamber for
analysis. The persulfate species in the presence of
UV light breaks down at a weak oxygen-oxygen
bond yielding two radicals per molecule. These radi­
cals start chain reactions that ultimately lead to the
16
Institute of Validation Technology
S O -2 → SO -1 + R → H O + CO
2
8
4
2
2
Detergent Evaluation
Three detergents (CIP-100, CIP-200, and Sparquat
256) were tested both in-house using the Tekmar
Dohrmann Phoenix 8000 TOC Analyzer and at a
contract lab, Quantitative Technologies Inc. (QTI),
to ver­ify the total amount of organic carbon in each
de­tergent at its original concentration. The method
and instrument used at QTI was a Perkin-Elmer CHN
Analyzer 2400. This experiment was performed to
make a comparison between our instrument and the
instrument in a qualified contract laboratory for infor­
mation purposes only. One detergent (Chlor-Mate)
was tested in-house and compared with the available
James G. Jin
vendor’s specification. The TOC results for all the
detergents are shown in Figure 2.
The differences between the in-house and QTI
results with respect to the TOC assay for CIP-100 and
CIP-200 are 5.0 percent and 9.6 percent, respectively.
These differences are relatively low compared to the
20 percent recovery criteria during recovery studies.
The difference between the in-house and QTI results
with respect to the TOC assay for Sparquat 256 is
28.4 percent. The in-house result was reviewed and
no error was noted in the performance of the test­
ing procedure. The major differences may be due to
in­strument and testing method variations. The result
for Chlor-Mate is within the vendor’s specification.
Swab Selection
It has been known for years that polyester is a
suitable material for TOC swabbing analysis. Over
20 different kinds of polyester swab samples were
received from The Texwipe Company LLC. Five
of them were chosen for TOC evaluation based on
sample design and the convenience for use. The
purpose of this experiment was to select a type of
swab that has little TOC background interference and
with consistent TOC results over time. Ultra purified
water with 0.05 to 0.08 ppm carbon was used for
swab analysis. The TOC results obtained from our
TOC analyzer are shown in Figure 3.
Swabs TX761 and TX741A showed increasing
TOC results from 0.0813 to 0.9692 ppm carbon and
from 0.1724 to 1.1246 ppm carbon over five days,
re­spectively. Swab TX700 showed an unacceptably
high TOC result of 46.1991 ppm carbon at the begin­
ning of the experiment, and was therefore not tested
further. None of these swabs are suitable for our
TOC analysis.
Both polyester wipers AlphaSorb® HC TX2412
Figure 2
and TX2418 show acceptable results with respect to
result consistency. The average of the seven TOC
results from TX2412 and TX2418 found in Figure
3 is 0.8327 ± 0.1860 ppm carbon. The variation is
acceptable compared to the acceptance criterion of
three ppm carbon. These two swabs with the same
material were selected to be our TOC swabs (cut to
5x5 cm2) for detergent residue verification.
The TX3340 TOC cleaning validation kit including
Eagle EP Picher 03464-40mL clear vials, Tex­wipe®
TX714L-large SnapSwabsTM, and blank vial labels
may be chosen since it is specially de­signed for TOC
swabbing purposes.
Detergent Recovery Evaluation from Stainless
Steel Surface
Ten stainless steel templates were spiked with
detergent solution and swabbed using the polyester
wipers AlphaSorb® HC TX2418 (5x5 cm2) for the
detergent recovery study. The spiking and swabbing
procedures were the same as those used for drug
substance recovery studies. Forty mL of ultra puri­
fied water was added to each test tube as the extrac­
tion solution, vortexed about one minute, and then
sonicated for five minutes for testing. The results are
shown in Figure 4.
The recoveries for CIP-100, CIP-200, and ChlorMate are over 80 percent and no correction factor is
necessary.
For Sparquat 256, a correction factor of 0.61 will
be used. For example, if a result of 0.5 ppm carbon
is obtained from the TOC analyzer, the final reported
result would be 0.82 (0.5 ÷ 0.61) ppm carbon.
Detergent Recovery Evaluation from Non-Stain­
less Steel Surfaces
The aforementioned study was repeated using
non-stainless steel templates. Two or three non-stain­
Total Organic Carbon Results for Detergent Evaluation
Detergent Manufacturer/LotTotal Organic Carbon Result TOC Results
Identification
From BIPI*
From QTI/Vendor
CIP-100
Vestal Convac lot 211097
4.0208 ± 0.0139%
4.22%
CIP-200
Convac lot 213915
2.4986 ± 0.0114%
2.26%
Sparquat 256
ISSA (lot: n/a)
14.0232 ± 0.9336%
18.0%
Chlor-Mate
WestAgro® lot J8G0489AR
1.29% ± 0.0086%
1 – 1.5%
*Boehringer Ingelheim Pharmaceuticals, Inc.
Special Edition: Cleaning Validation III
17
James G. Jin
Figure 3
Total Organic Carbon Results (ppm C) for Swab Selection
Swab TOC/Two HoursTOC/Four HoursTOC/One DayTOC/Two DaysTOC/Five Days
Description
in H O
in H O
in H O
in H O
in H O
Polyester Alpha 0.0813
0.3221
0.3926
0.9410
0.9692
swab TX761
± 0.0041
± 0.0853
± 0.0166
± 0.0288
± 0.0299
Polyester Alpha 0.1724
0.2509
0.5330
0.8091
1.1246
swab TX741 A
± 0.0144
± 0.0068
± 0.0250
± 0.0200
± 0.0394
Polyester wipers 1.1665
0.6091
0.8602
0.7535
0.9723
AlphaSorb® ± 0.0406
± 0.0490
± 0.0264
± 0.0328
± 0.0668
HC TX2412
Polyester wipers 0.7406
0.7269
N/A(1)
N/A(1)
N/A(1)
®
AlphaSorb ± 0.0056
± 0.0297
HC TX2418
Polyester Alpha 46.1991
N/A
N/A
N/A
N/A
swab TX700
± 8.0761
2
2
2
2
2
1. Polyester wipers AlphaSorb ® HC TX2412 and polyester wipers AlphaSorb ® HC. TX2418 is same material cut to different sizes.
less steel templates were spiked with each detergent
solution and swabbed using the polyester wipers
AlphaSorb® HC TX2418 (5x5 cm2). The results are
shown in Figure 5.
For CIP-100 and CIP-200, the recoveries from
each non-metal surface are over 80 percent. There­
fore, no correction factor is needed with respect to
the TOC recovery. For Sparquat 256, the recoveries
vary with different surfaces. The correction factors
are as follows:
For Delrin surface: correction factor = 0.74
For Glass surface: correction factor = 0.75
For Nylon surface: correction factor = 0.43
For Lexan surface: correction factor = 1.0
Evaluation of Detergent Residue After Rinsing
The purpose of this experiment was to evaluate:
∂ The suitability of the Acceptance Criterion
(AC) of three ppm carbon
∑ The effect of detergent concentration on deter­
gent residue after rinsing
∏ Recovery of detergent from different surfaces
with and without rinsing
π Rinsing efficiency and rinse time
Four detergents (CIP-100, CIP-200, Sparquat
256, and Chlor-Mate) were used in both a concen­
trated form and at a working concentration of 0.5
oz/gal. Approximately one mL of detergent solution
18
Institute of Validation Technology
Figure 4
Total Organic Carbon Recovery
Results from a Stainless
Steel Surface
Detergent
PercentNumber Percent
Recovery of Relative
SamplesStandard
Deviation
CIP-100
CIP-200
Sparquat 256
Chlor-Mate
111.7 92.4 61.0 99.1 30
10
20
10
5.92
4.10
8.47
2.76
Note: R
esults were automatically corrected for the
instrument blank effect.
Figure 5
Total Organic Carbon Recovery
Results from a Non-Stainless
Steel Surface
DetergentLexan
Delrin GlassNylon
Surface Percent Percent Percent Percent
RecoveryRecoveryRecoveryRecovery
CIP-100 106.9
CIP-200 90.3
Sparquat 83.3
256
113.8
92.3
74.0
107.6
97.4
75.1
127.0
93.2
42.5
was pipetted and spiked onto the templates with
different materials of construction and dried with
ventilation under a hood in the research and devel­
James G. Jin
opment manufacturing area for a minimum of four
hours. The templates were swabbed per standard
swabbing procedure either before or after rinsing,
using the polyester wipers AlphaSorb® HC TX2412
cut to 5x5 cm2. The rinse was first conducted using
tap water and then purified water United States
Pharmacopoeia (USP), both at room temperature
and with a slow flow rate of approximately 2.7 L/
min. Two different rinse times (30 seconds and 60
seconds) were evaluated for different detergents on
different templates to simulate the final rinse step in
our manual cleaning process. The recovery results
are reported in Figure 6.
The Tekmar Dohrmann Phoenix 8000 TOC ana­
lyzer was easily able to detect the non-rinse samples
with the results of 3.911 ppm carbon, 2.0928 ppm
carbon, and 10.0868 ppm carbon for CIP-100, CIP200, and Sparquat 256, respectively. The results
indicate that the AC of three ppm carbon is still high
for detergents CIP-100, CIP-200, and Sparquat 256.
The AC of one ppm carbon is acceptable. There
were no differences in detectable residue for all four
detergents (both concentrated and at 0.5 oz/gal) on
stainless steel after a 30-second tap water rinse fol­
lowed by a 30-second purified water, USP rinse.
Delrin was chosen for a typical material of construc­
Figure 6
Total Organic Carbon Results on Detergent Residue by Rinsing
Sample ConcentrationTemplatesRinse TimeAreaTOC Results
IdentificationSwabbed
(ppm C)d
CIP-100
0.5 oz/gal
SS a
No rinse
100 cm2
3.9111
a
b
0.5 oz/gal
SS 30”/30” 100 cm2
Less than blank
CIP-100
CIP-100
Concentrated
SS a
30”/30” b
100 cm2
Less than blank
b
2
CIP-100
0.5 oz/gal
Delrin
30”/30” 100 cm Less than blank
0.5 oz/gal
Delrin
60”/60” b
100 cm2
Less than blank
CIP-100
0.5 oz/gal
Nylon
30”/30” b
100 cm2
0.6682
CIP-100
CIP-100
0.5 oz/gal
Glass
30”/30” b
100 cm2
0.0001
b
2
CIP-100
0.5 oz/gal
Lexan
30”/30” 100 cm Less than blank
CIP-200
CIP-200
CIP-200
CIP-200
CIP-200
CIP-200
CIP-200
CIP-200
Sparquat
Sparquat
Sparquat
Sparquat
Sparquat
Sparquat
Sparquat
Sparquat
256
256
256
256
256
256
256
256
Chlor-Mate
Chlor-Mate
Notes:
0.5 oz/gal
0.5 oz/gal
Concentrated
0.5 oz/gal
0.5 oz/gal
0.5 oz/gal
0.5 oz/gal
0.5 oz/gal
SS a
SS a
SS a
Delrin
Delrin
Nylon
Glass
Lexan
No rinse
30”/30” b
30”/30” b
30”/30” b
60”/60” b
30”/30” b
30”/30” b
30”/30” b
100
100
100
100
100
100
100
100
cm2
cm2
cm2
cm2
cm2
cm2
cm2
cm2
2.0928
Less than
Less than
Less than
Less than
0.7720
0.0133
Less than
0.5 oz/gal
0.5 oz/gal
Concentrated
0.5 oz/gal
0.5 oz/gal
0.5 oz/gal
0.5 oz/gal
0.5 oz/gal
SS a
SS a
SS a
Delrin
Delrin
Nylon
Glass
Lexan
No rinse
30”/30” b
30”/30” b
30”/30” b
60”/60” b
30”/30” b
30”/30” b
30”/30” b
100
100
100
100
100
100
100
100
cm2
cm2
cm2
cm2
cm2
cm2
cm2
cm2
10.0868 c
0.2693 c
Less than
Less than
Less than
0.3866 c
Less than
Less than
0.5 oz/gal
Concentrated
SS a
SS a
30”/30” b
30”/30” b
100 cm2
100 cm2
blank
blank
blank
blank
blank
blank
blank
blank
blank
blank
Less than blank
Less than blank
a. Stainless steel.
b. 30”/30” or 60”/60” – rinse time in seconds, tap water/purified water United States Pharmacopoeia (USP).
c. Result without correction factor.
Special Edition: Cleaning Validation III
19
James G. Jin
tion and 30/60 seconds were chosen for evaluation of
the rinse time. There was no difference in detectable
residue for CIP-100, CIP-200, and Sparquat 256 on
the Delrin surface after 30-second and 60-second
rinse times. The results also show that it is more dif­
ficult to remove residues of CIP-100, CIP-200, and
Sparquat 256 from a Nylon surface than from other
materials.
Acceptance Criterion for Detergent Residue
There is no universal AC for detergent residue
allowed to be left on GMP equipment surfaces. In
our detergent residue verification program, the AC
for each detergent residue left on equipment surfaces
depends on the sensitivity of the instrument used for
analysis. This means we must set a low AC that is still
quantifiable and applicable. Toxicity of the detergent
is not a concern at these trace amounts de­tergent
level. Effects on human health from re­sidue left on
equipment surfaces should be insignificant at a low
concentration such as 0.5 oz/gal and with a routine
rinse procedure. Our objective in this program is to
demonstrate that we are able to verify whether or not
the detergent residues are removed to an acceptable
low-level we can achieve.
Therefore, the AC should be established as close
to the instrument’s level of detection as possible. We
tighten the initial limit of three ppm carbon to AC =
1.0 ppm carbon (net reading automatically corrected
with blank by the instrument in a 40 mL solution),
which is less than two times the blank baseline. The
AC can also be expressed as AC ≤ 10 ppb carbon/
cm2. This AC is practical and verifiable.
The significance of the 1.0 ppm carbon AC for
each detergent can be explained in Figure 7.
We can see from the above calculations that AC
= 1.0 ppm carbon means, for all detergents at 0.5
oz/gal, that we allow the maximum of 1 ÷ 3.92 =
0.26 mL of CIP-100, 1 ÷ 2.44 = 0.41 mL of CIP-200,
1 ÷ 13.68 = 0.07 mL of Sparquat 256, and 1 ÷ 1.26
= 0.79 mL of Chlor-Mate to be left on 100 cm2 of
equipment surface after cleaning, respectively.
Detergent Residue Verification Program
Our detergent verification program is designed
to be a one-time verification for each detergent
used. This was based on the rinse experiment and
the assumption that our routine rinsing procedures
performed by well trained operators are sufficient to
remove detergent residues to the level of less than
the AC. This assumption has been verified from the
results shown in Figure 6 that all the residues are eas­
ily removed by a 30-second tap water rinse followed
by a 30-second purified water, USP rinse with very
low spray rate. Verification rather than validation is
currently required by the 1993 FDA, Guide to In­spec­­
tions of Validation of Cleaning Procedures due to
the fact that detergent residue is less significant than
drug substance residue left after cleaning.
Summary
The detergent residue verification program has
been successfully established using the Tekmar
Dohrmann Phoenix 8000 TOC analyzer. This paper
has shown the program development, and presents
critical data to support the detergent verification
reports for each detergent used.
The instrument Installation Qualification (IQ),
Operational Qualification (OQ), system calibration,
and the TOC analysis method development were
performed but not discussed in this paper. The poly­
ester wipers AlphaSorb® HC TX2412 and TX2418
cut to 5x5 cm2 have been selected as the swabs for
sampling detergent residue from equipment surface
for TOC analysis. The AC for the detergents CIP100, CIP-200, Sparquat 256, and Chlor-Mate with
respect to TOC has been established as AC ≤ 10 ppb
car­bon/cm2. Two different rinse times, 30 seconds
and 60 seconds, were evaluated. The results show
Figure 7
Significance of Total Organic Carbon Results for Detergent at 0.5 oz/gal
CIP-100CIP-200Sparquat 256Chlor-mate
1 mL at 0.5 oz/gal
3.92 ppm
2.44 ppm
13.68 ppm
1.26 ppm
diluted to 40 mL
1.0 ppm C per 100 cm2 0.26 mL
0.41 mL
0.07 mL
0.79 mL
corresponding to
20
Institute of Validation Technology
James G. Jin
that 30-second/30-second rinse time (30-second rinse
with tap water and then 30-second rinse with puri­
fied water, USP) is sufficient to remove the detergent
re­sidues from different material templates including
stainless steel, Delrin, Glass, Nylon, and Lexan to a
level below the AC. The correction factors were de­ter­
mined based on the results of the recovery studies and
will be used by analytical sciences to report the final
TOC results for the detergent residue verification. o
About the Authors
James G. Jin is Chairman of the Cleaning Validation
Committee for Boehringer Ingelheim Pharma­ceuti­
cals, Inc., which is responsible for clean­ing validation program development and implementation. He
has more than ten years experience in pharmaceutical science and business arenas. He can be reach­
ed by phone at 203-798-5309.
Cheryl Woodward is Associate Director of Research
and Development (R&D) Manufacturing, for Boeh­
ringer Ingelheim Pharmaceuticals, Inc. She is
re­sponsible for all aspects of GMP manufacturing
for clinical supplies and has over 18 years experience in the pharmaceutical and related industries.
She can be reached by phone at 203-798-5367.
References
1. FDA. Guide to Inspections of Validation of Cleaning Pro­ce­
dures. July, 1993.
2. Jenkins K.M., Vanderwielen A.J, Armstrong J.A, Leonard L.M,
Murphy G.P, Piros N.A. 1996. “Application of Total Organic
Carbon Analysis to Cleaning Validation.” PDA. Journal of
Pharma­ceutical Science and Technology. 50. Pp 6-15.
3. Guazzaroni M., Yiin B., Yu J., 1998. “Application of Total
Or­ganic Carbon Analysis for Cleaning Validation in Pharma­ceuti­
cal Manufacturing.” American Biotechnology Laboratory. Septem­
ber. Pp. 66-67.
4. Westman L., Karlsson G., 2000. “Methods for Detecting Re­si­
dues of Cleaning Agents During Cleaning Validation.” Re­search
Article, Vol. 54, No. 5. September/October.
5. Furlong J., Booth B., Wallace B. 1999. “Selection of a TOC
Analyzer: Analytical Considerations.” Tekmar-Dohrmann
Ap­pli­cation Note. Vol. 9.20.
Special Edition: Cleaning Validation III
21
Total Organic Carbon Analysis
for Cleaning Validation in
Pharmaceutical Manufacturing
By Karen A. Clark
Anatel Corporation
v
I
n the pharmaceutical industry,
specific methods like TOC is that
Good Manufacturing Practice
they cannot identify exactly what
}TOC analysis
(GMP) requires that the clean­
the residue material is. Depending
can be adapted on the chosen cleaning process and
ing of drug manufacturing equip­
ment be validated.1 Many different
established acceptance limits, a
to any drug
validation techniques can demon­
non-­specific method may be all that
strate that the manufacturing equip­
is needed to validate the process.
compound or
ment is cleaned and essentially free
TOC analysis can be adapted
cleaning
agent
from residual active drug substanc­
to any drug compound or clean­
es and all cleaning agents.
ing agent that contains carbon and
that contains
Common analytical techniques
is “adequately” soluble in water.
carbon and is
in the validation process include
Studies have been conducted to
High Performance Liquid Chrom­
demonstrate that TOC methods can
‘adequately’
atography (HPLC), spectrophotom­
also be applied to carbon containing
soluble
in
water.~
etry Ultraviolet/Visible (UV/Vis)
compounds that have limited water
and Total Organic Carbon (TOC).
solubility, and recovery results are
HPLC and UV/Vis are classified as
equal to those achieved by HPLC.6
specific methods that identify and measure appropri­ TOC methods are sensitive to the parts per billion
ate active substances. TOC is classified as a non- (ppb) range and are less time consuming than HPLC
specific method and is ideal for detecting all carbon- or UV/Vis. United States Pharmacopoeia (USP)
containing compounds, including active species, TOC methods are standard for Water-for-Injection
and Purified Water,7 and simple modifications of
excipients, and cleaning agent(s).2,3,4,5
The disadvantage of specific methods, particular­
these methods can be used for cleaning validation.
ly HPLC, is that a new procedure must be developed
Methodology
for every manufactured active drug substance. This
development process can be very time consuming
TOC analysis involves the oxidation of carbon and
and tedious, plus important sampling issues must
also be considered. In addition, HPLC analyses must the detection of the resulting carbon dioxide. A num­
be performed in a relatively short time period after ber of different oxidation techniques exist, including
photocatalytic oxidation, chemical oxidation, and
sampling to avoid any chemical deterioration of the
active substance. Finally, the sensitivity of HPLC high-temperature combustion. In this study, an Anatel
methods can be limited by the presence of degrada­ A-2000 Wide-Range TOC Analyzer, equipped with
an autosampler, was used. The Anatel A-2000 Widetion products. Of course the disadvantage to non22
Institute of Validation Technology
Karen A. Clark
2
2
Figure 1
Measured TOC (ppb)
Range Analyzer measures TOC in accordance with
American Society for Testing and Materials (ASTM)
methods D 4779-88 and D 4839-88. It measures
TOC directly by adding phosphoric acid to the water
sample to reduce the pH from approximately two to
three. At this low pH any inorganic carbon that is
present is liberated as CO into a nitrogen carrier gas
and is directly measured by a non-dispersive infrared
(NDIR) detector. Any remaining carbon in the sample
is assumed to be TOC. A sodium persulfate oxidant is
then added to the sample, and in the presence of UV
radiation, the remaining carbon is oxidized to CO .
The amount of CO generated is then measured by
the NDIR to determine the amount of TOC originally
present in the water.
For equipment cleaning validation there are two
types of TOC sampling techniques. One is the direct
surface sampling of the equipment using a swab.
The second consists of a final rinse of the equipment
with high-purity water (typically <500 ppb TOC)
and collecting a sample of the rinse for analysis. In
general, direct surface sampling indicates how clean
the actual surface is. This study demonstrates how
to develop and validate a TOC method to measure
a variety of different organic residues on stain­
less steel surfaces. Performance parameters tested
include linearity, method detection limit (MDL),
limit of quantitation (LOQ), accuracy, precision, and
swab recovery.
2
Measured TOC (ppb)
Figure 2
Linearity
∂ CIP-100 (alkaline)
∑ CIP-200 ® (acidic)
∏ Alconox ® (emulsifier)
π Triton-X 100 (wetting agent)
®
Results are shown in Figures 1-4. Correlation
coefficients ranged from 0.9787 to 0.9998. Alconox
and Triton-X 100 have a tendency to foam, depend­
ing on the concentrations that are analyzed and this
foaming phenomena can have a negative effect on
the accuracy of the TOC result (reduced R2). Three
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
Figure 3
Measured TOC (ppm)
TOC analysis should provide a linear relationship
between the measured compound concentration and
the TOC response of the analyzer. We evaluated
four different types of cleaning agents for linearity:
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
45
40
35
30
25
20
15
10
5
0
Linearity of CIP-100
y=39.254x + 1.462
R2=0.9997
0
50
100
150
200
250
CIP 100 Concentration (ppm)
Linearity of CIP- 200
y=19.132x + 51.042
R2=0.9998
0
100
200
300
400
500
CIP 200 Concentration (ppm)
Linearity of Alconox
y=0.0355x + 1.1983
R2=0.9787
0
200
400
600
800 1000
Alconox Concentration (ppm)
Special Edition: Cleaning Validation III
23
Karen A. Clark
Linearity of Triton-X 100
Measured TOC (ppb)
12500
y=415.76x + 16.997
R2=0.9982
10000
7500
5000
2500
0
Figure 5
0
Linearity of Sucrose
12000
Measured TOC (ppb)
5
10
15
20
25
Triton-X 100 Concentration (ppm)
y=1.003x + 45.185
R2=0.9996
10000
8000
4000
2000
0
0 2000 4000 6000 8000 10000 12000
Sucrose Concentration (ppb)
Figure 6
Linearity of Vancomycin
Measured TOC (ppb)
8000
y=0.8758x + 62.133
R2=0.9998
6000
4000
2000
24
0
Linearity of Endotoxin
8000
y=0.9287x + 30.8
R2=0.9998
7000
6000
5000
4000
3000
2000
1000
0
0
2000
4000
6000
8000
Endotoxin Concentration (ppb)
representative examples of active substances were
also tested for linearity: an excipient (sucrose), an
antibiotic (vancomycin), and endotoxin. Results
are shown in Figures 5-7. All three compounds
demonstrated excellent linearity with correlation
coefficients (R2) ranging from 0.9996 to 0.9998.
Method Detection Limit and
Limit of Quantitation
6000
Figure 7
Measured TOC (ppb)
Figure 4
0
2000
4000
6000
8000
Vancomycin Concentration (ppb)
Institute of Validation Technology
We determined the Method Detection Limit
(MDL) by measuring the TOC response of the meth­
od blank.
A method blank consists of the sampling vial,
swab, and recovery solution. In this study, the
recovery solution was low TOC (< 25 ppb) water.
Ten pre-cleaned vials were filled with the low TOC
water. One swab was placed in each vial (Texwipe
Alpha Swab TX761; tips cut off). Solutions were
vortexed and allowed to stand for one hour prior to
analysis. Four replicates from each vial were ana­
lyzed. The four replicates from each of the ten blank
vials were averaged. These ten values were averaged
again and a standard deviation was calculated. The
standard deviation was multiplied by the Student t
number for n-1 degrees of freedom (3.25 for n=10),
at 99% confidence levels to determine the method
detection limit. The MDL was calculated to be 50
ppb. The Limit of Quantitation (LOQ) was calcu­
lated by multiplying the MDL by three. A value of
150 ppb was obtained (see Figure 8).
Precision and Accuracy
Karen A. Clark
Figure 8
To demonstrate the precision and accuracy for this
TOC method, a representative solution of CIP-100 as
1000 ppb, or one ppm as carbon, was analyzed sequen­
tially ten times. This carbon concentration was chosen
to evaluate these method parameters because, in gen­
eral, TOC residual limits are typically around one ppm.
Results are listed in Figure 9. At this TOC level, the
precision was ± 1% and the accuracy was ± 5%.
Calculated TOC Averages
from 10 Blank Vials
Vial NumberAverage TOC (ppb)
1
58
2
72
3
75
4
93
5
79
6
102
7
60
8
83
9
67
10
54
Average
74.3
Standard Deviation
15.5
50 ppb
MDL (Student t, n=10)
LOQ
151 ppb
Figure 9
Swab Recovery
Calculated Accuracy and Precision
from 10 Replicates of a 1ppm CIP100 Solution as Carbon
Vial NumberMeasured TOC (ppb)
1
1041
1
1025
1
1039
1
1057
1
1054
2
1034
2
1042
2
1048
2
1054
2
1055
Average
1045
Standard Deviation
10.5
% CV (precision)
1.0%
% Recovery based on
105%
1 ppm C (accuracy)
Figure 10
Stainless steel plates were used in the swab recov­
ery test to simulate manufacturing equipment. One
side of each plate was spiked with a solution of active
substance or cleaning agent. The plates were allowed
to completely dry overnight at room temperature. A
Texwipe alpha swab TX761 was moistened with low
TOC (< 25 ppb) water and the spiked plate surface was
swabbed both vertically and horizontally. The swab
end was cut off, placed into a vial to which we added
40-mL of low TOC water. The vial was capped tight,
vortexed, and allowed to stand for one hour prior to
analysis. The same volume of each solution that was
spiked onto the plates was separately spiked directly
into 40-mL of low TOC water and analyzed. The per­
cent recoveries of the different substances are listed in
Figure 10. Reported values are the average of three
individual swab samples for each substance. The swab
recoveries varied between 79.3% to 95.9%
Conclusion
This study demonstrates that TOC analysis is
suitable for measuring organic residues on stain­
less steel surfaces, and that it is a reliable method
for cleaning validation as demonstrated by surface
residue recoveries of 79%-96%. This methodology
Representative Examples of Swab Recoveries from Cleaning Agents
and Active Substances
Substance
ppm C of Spike
Standard Solution
CIP-100
1810
Sucrose
2663
Vancomycin
661
Endotoxin
902
ppm C of Spiked
Plate
1710
2112
634
736
% Recovery
% RSD
94.5
79.3
95.9
80.0
1.8
4.9
3.0
2.8
Special Edition: Cleaning Validation III
25
Karen A. Clark
shows that low limits of detection, excellent linear­
ity, precision, and accuracy can be obtained. All of
these TOC results, with the exception of Alconox
and Triton-X 100, were generated using the same
TOC method, making TOC analysis a low cost and
less time consuming alternative for cleaning valida­
tion. o
About the Author
Karen A. Clark is a Product Manager at Anatel
Corporation. She has over 15 years experience in
the pharmaceutical/biotechnology industry focusing on drug formulations, analytical methods development and validation, and GLP/GMP laboratory
management. Clark holds a B.S. in Biochemistry
from Millersville University and an M.S. in Chemical
Engineering from the University of Colorado. She
can be reached by e-mail at kclark@anatel.com or at
Anatel Corporation, 2200 Central Avenue, Boulder,
CO 80301.
References
1. FDA. Current Good Manufacturing Practice Regulations, 21
CFR 211.220.
2. Baffi, R. et al. 1991. “A Total Organic Carbon Analysis Method
for Validating Cleaning Between Products in Bio­pharmaceutical
Manufacturing.” Journal of Parenteral Science and Technology
45, no. 1: 13-9.
3. Jenkins, K. M. et al. 1996. “Application of Total Organic Carbon
Analysis to Cleaning Validation.” PDA Journal of Pharm­
aceutical Science and Technology 50, no. 1: 6-15.
4. Strege, M. A. et al. 1996. “Total Organic Carbon Analysis of Swab
Samples for the Cleaning Validation of Bioprocess Fer­men­tation
Equipment.” BioPharm (April).
5. Guazzaroni, M. et al. 1998. “Application of Total Organic Car­
bon Analysis for Cleaning Validation in Pharmaceutical Man­
ufacturing.” American Biotechnology Laboratory 16, no. 10
(September).
6. Walsh, A. 1999. “Using TOC Analysis for Cleaning Val­idation.”
Presented at The Validation Council’s Conference on Cleaning
Validation, 27 October, Princeton, New Jersey.
7. USP 23, Fifth Supplement, 15 November 1996.
26
Institute of Validation Technology
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Special Edition: Cleaning Validation III
27
Detergent Selection – A First
Critical Step in Developing a
Validated Cleaning Program
By Mark Altier
Ecolab, Inc.
v
T
he FDA recognizes the
im­portance of effective
cleaning and sanitizing pro­
tocols as a proactive measure in
preventing cross-contamination in
the pharma­ceutical and cosmetic
in­dus­tries:
detergent for a given ap­plication
}This article will should be based on sound, scientific
discuss the key reasoning.
A sound rationale for detergent
factors that
selection begins at the man­­ufactur­
ing site, where the process and
must be
clean­­ing program will take place. A
ad­dres­sed when full evaluation of the pro­cess, clean­
21CFR 211.67: “Equip­ment
ing strategies, potential contaminant
selecting a
and utensils shall be cleaned, main­
and available utilities is a
detergent. Each levels,
tained, and sanitized at appropriate
good first step. Follow­ing this step,
factor will be
intervals to prevent malfunctions
laboratory testing is re­quired to
or contamination that would alter
de­termine the exact nature of the
discussed
in
the safety, identity, strength, qual­
po­tential contaminant. Next, ident­
detail and
ity, or purity of the drug product
ifi­ca­tion and testing of various clean­
beyond the official or other estab­
ing chem­istries against the potential
examples are
lished requirements.”
contaminant is performed to deter­
given when
mine which de­tergent type is best
In order to comply with this reg­
suit­
ed for con­taminant re­moval. The
appropriate.
ulatory requirement, sound clean­
next step is to return to the manufac­
The roles of
ing and sanitizing protocols must
turing site, test the cleaning chem­
laboratory testing istry, and optimize the program.
be developed and followed. One
of the most critical components of
This ap­proach provides a sound,
and
plant
any cleaning program is detergent
scientific rationale for the detergent
optimization are selection and lays a firm foundation
se­lection. Different pro­­cesses and
po­tential contaminants may require
also addressed.~ to the formal cleaning protocol, once
different de­tergents that are appro­
de­veloped.
priate for the application. In certain
This article will discuss the key
cleaning ap­plications, a neutral foaming de­tergent
factors that must be ad­dres­sed when selecting a
might be appropriate, where­as in others, a non-foam­­
detergent. Each factor will be discussed in detail
ing alkaline detergent is de­sirable. The choice of
and examples are given when appropriate. The roles
28
Institute of Validation Technology
Mark Altier
of laboratory testing and plant optimization are also
addressed.
The Five Factors for Determining
Detergent Suitability
There are five key factors that must be ad­dres­sed
when determining which detergent is most suitable
for a cleaning application. These are:
∂ Nature of the residue (or potential contami­
nant)
∑ Surface to be cleaned
∏ Method of application
π Role of water
∫ Environmental factors
All five of these factors must be addressed when
developing a cleaning program. Failure to address
any of these issues in sufficient detail can result in a
less than desirable cleaning program and could place
the successful completion of the cleaning validation
at serious risk.
The Nature of the Residue
A residue can be defined as any unwanted matter
or potential contaminant on the surface of the ob­ject
or equipment being cleaned. Oftentimes, what is
re­ferred to as a “residue,” is in fact a finished prod­
uct, drug active, or other component that is produced
us­ing the process equipment that is being cleaned.
The terms “residue,” “contaminant,” and “potential
con­taminant” will be used interchangeably through­
out this article.
Determination of the nature of a residue is a funda­
mental component in the development of any clean­­ing
program. In some cases, the exact nature and com­
position of a residue is known. For example, if the
residue is a finished product, the exact composition
and physical properties are almost always known.
However, the identity and nature of the re­sidue may
be completely unknown if the re­sidue is composed
of an intermediate, byproduct, or result of thermal,
chemical, or other degradation of a previously known
substance.
The nature of the potential contaminant plays a
central role in determining what type of detergent
is most appropriate for the application. Individual
re­si­dues require different detergent chemistries. All
residue types will fall into one of the following three
categories: organic, inorganic, or a combination of
these. Most potential contaminants are a combina­
Figure 1
Common Residue Types in the
Pharmaceutical Industry
Organic ResiduesInorganic Residues
Eudragit
Titanium Dioxide
Acetaminophen
Zinc Oxide
Carbopols
Iron Oxide
Albuterol Sulfate
Calcium Carbonate
Neomycin Sulfate
Inorganic Salts
Water/Oil – Oil/Water Emulsions
Silicon Dioxide
Glyburide
tion of organic and inorganic components. Com­mon
residue types in the pharmaceutical industry are
given in Figure 1.
A number of powerful analytical instruments are
available that can provide tremendous insight into
the nature and composition of almost any unknown
potential contaminant type. Some of the more useful
tools include:
• Fourier Transform Infrared Spectroscopy
(FTIR)
• Energy Dispersive X-Ray Spectroscopy (EDS)
• Scanning Electron Microscopy (SEM)
• Compound microscopic imaging
• Nuclear Magnetic Resonance imaging (NMR)
• Inductively Coupled Plasma detector (ICP)
• Atomic Absorption Analyzer (AA)
Often, a combination of two or more of these tools
is required to provide a full picture of a potential
contaminant in question. For example, Fig­ure 2 and
Fig­ure 3 are typical images generated to help char­
acterize unknown potential contaminant samples.
This type of analysis is invaluable in determining the
ex­act residue type and breakdown of the organic and
inorganic portions of a residue.
Figure 2 is an FTIR image of an unknown re­sidue.
This characterizes and gives a general breakdown of
Special Edition: Cleaning Validation III
29
Mark Altier
Figure 2
FTIR Scan of Unknown Sample. This Analysis Indicates the Presence of
Alkyl and Amide Protein Components and Possible Inorganic Content
1631.22
1545.75
1454.66
1396.07
1311.04
1251.26
1077.51
1044.64
980.456
870.204
694.924
.35
.30
.15
.10
.05
2287.55
2257.66
2211.86
2165.99
2155.88
2033.97
2013.07
2958.86
2926.38
2854.86
.20
3933.71
Absorbance
.25
0
3500
Figure 3
3000
2500
2000
Wave Number (cm-1)
1500
1000
EDS Scan of Unknown Sample
Counts
This Analysis Confirms the Presence of Inorganic Components Such as Silicon,
Aluminum, and Iron, in Addition to Organic Compounds
600
580
560
540
520
500
480
460
440
420
400
380
360
340
320
300
280
260
240
220
200
180
160
140
120
100
80
60
40
20
0
C
0.000
30
Fe
Si
Al
O
Fe
1.000
Mg
Fe
2.000
Institute of Validation Technology
3.000
4.000
Key
5.000
6.000
7.000
8.000
Mark Altier
the organic portion of the residue. FTIR imaging
gives valuable insight into the functional groups that
may be present in the organic component of a re­si­due.
Figure 3 confirms the presence of inorganic ma­terial
and identifies the specific inorganic components pres­
ent in an unknown sample. This information is useful
when determining which chelant or surfactant family
is most suitable for re­moving or tying up the free
metal ions and other inorganic material.
Combined, FTIR and EDS imaging can give a
com­plete picture of most unknown residues. These
analyses provide the information needed to select a
group of detergent chemistries that are formulated
and known to be effective against the residue type.
Surfaces To Be Cleaned
Different substrates (i.e., product contact surfaces,
such as stainless steel, glass, or plastic) will interact
differently with the contaminant and the de­tergent
system. Some materials, such as glass, and alumi­num,
are not tolerant to high pH systems. Other substrates
may tolerate high pH, but may not tolerate chlorine or
chlorides. It is important to have a clear understand­
ing of how the substrate being cleaned will interact
with the detergent system, otherwise serious dam­
age to equipment surfaces can result. A SEM image,
shown in Figure 4, is a stainless steel surface that has
been pitted by using an in­compatible detergent. The
prospective customer in this case felt that the residue
was becoming more tenacious with time and was
using higher detergent concentrations to remove the
residue.
A close look at the surface revealed that the surface
was actually being pitted by the detergent, providing
microscopic crevices where the residue was able to
harbor during the cleaning cycle. This problem was
aggravated by the fact that the customer continued to
increase the detergent concentration, which acceler­
ated the rate and degree of corrosion, and provided
the residue with even more locations to harbor during
the cleaning cycle.
These images clearly demonstrate the problems
Figure 4
Microscopic Corrosion of a Stainless Steel Surface Caused by Improper
Detergent Selection. Inset Shows Boxed Region at 1000 Times Magnification
Special Edition: Cleaning Validation III
31
Mark Altier
that can be caused by improper detergent selection.
In this case, the customer was advised to discontinue
the use of the incompatible detergent, and a compat­
ible detergent chemistry was identified and tested.
The customer was also required to re­place or re­pair
damaged equipment.
When developing a cleaning protocol, it is neces­
sary to identify all components of the process that will
be exposed to the cleaning chemical(s). This includes
equipment surfaces, gasket materials, nozzles, piping,
pumps, etc. It is also important to consider surfaces
that will be exposed to the vapor phase of the cleaning
solution, such as overhead spaces in enclosed vessels
and pipes. A common mistake is to concentrate only
on items that will have direct contact with the liquid
solution, neglecting the vapor phase.
Method of Application
There are several common methods of applying
a detergent to equipment surfaces. Some are more
common than others in the pharmaceutical industry.
Some of the more common methods of application
in the pharmaceutical industry include:
• Clean-in-Place (CIP)
• Clean Out-of-Place (COP)
• Manual scrubbing/wiping
• High and low pressure spray
• Soaking/immersion
Each of these application methods dictate cer­
tain desirable or undesirable detergent properties.
For example, a high pH detergent is ideal in a CIP
application where little, or no direct contact is made
be­tween the detergent and the operator. In a manual
application, however, a high pH detergent creates
a significant safety risk to an operator handling the
de­tergent concentrate and use solutions. In a manual
application, a neutral or mildly alkaline de­tergent
(pH 7.0 – 10.0) is much more desirable as it sig­
nificantly reduces the risk for accidental chemical
burn to the operator’s eyes, skin, and mucous mem­
branes.
Other detergent characteristics, such as foam
properties, are important considerations in light of the
method by which the cleaning solution will be applied
to a surface. A moderate-to-high foaming detergent is
not desirable when used in an agitated immersion or
32
Institute of Validation Technology
CIP application, as both create high-shear and thus
are prone to foam formation. The result of this is a
detergent solution that foams out-of-process or CIP
vessels, cavitates pumps, and pro­vides inefficient
surface coverage when sprayed on the inside of a ves­
sel through a spray ball. Con­versely, a high foaming
detergent is desirable in a manual application, as this
gives the operator a visual indication of where the
detergent solution has been applied to the surface.
Some cleaning application technologies exist
that are widely used in other industries, but have
not taken hold in the pharmaceutical industry. These
application methods include:
• Thin film cleaning
• Stabilized foam generators
• Built, solvated detergents (Generally Recog­
nized As Safe [GRAS])
Discussion regarding these application methods
are outside of this article and will not be addressed.
The Role of Water
In general, 95-99% of a cleaning solution is
com­­posed of water. It is important to know the
purity level of the water being used for cleaning and
sanitizing. In many pharmaceutical applications, the
water being used for cleaning and sanitizing is high
purity water. However, this is not the case in every
application and in these cases, know­ing and under­
standing how the purity level of the process water
affects the cleaning process is critical. Some of the
factors that can affect the cleaning process include
water hardness, pH, metals, salts, and microbial con­
tamination. Refer to Figure 5.
Of the factors listed above, water hardness has the
most significant impact on cleaning and sanitizing
solutions. Water hardness can be classified as tem­
porary or permanent hardness. Temporary hard­ness
indicates the presence of bicarbonates of mag­nesium
or calcium. Both of these compounds are readily
water soluble and can be present at high levels. When
heated, these compounds react to form the carbonate
salts, which are water insoluble. Permanent hardness
refers to a condition where the chloride or sulfate
salts of magnesium and/or calcium are present in the
water. These compounds are also very water soluble,
but are unaffected by temperature.
Mark Altier
Figure 5
Typical Water Impurities That Can
Impact a Cleaning Process
ComponentChemical Problem
FormulaCaused
BaSO Barium Sulfate
Carbon Dioxide
CO Calcium Bicarbonate
Ca(HCO ) Calcium Sulfate
CaSO Iron
Fe
Mn
Manganese
Magnesium Bicarbonate Mg(HCO ) Magnesium Chloride
MgCl Magnesium Sulfate
MgSO O
Oxygen
Sodium Chloride
NaCl
Si
Silica
Suspended Solids
r
4
2
3
2
4
3
2
4
2
2
Scale
Corrosion
Scale and
Corrosion
Scale and
Corrosion
Scale
Scale
Scale
Scale and
Corrosion
Scale and
Corrosion
Corrosion
Corrosion
Scale
Deposit and
Corrosion
Both temporary and permanent hardness cause
problems in alkaline solutions, as they both pre­
cipitate in high pH solutions and cause scaling on
equipment surfaces. Water hardness is responsible
for scaling, film formation, excessive detergent con­
sumption, and formation of precipitate. Water hard­
ness can be addressed by installing a water softening
system, or by using a detergent that is formulated to
handle hard water.
Environmental Factors
Many pharmaceutical plants have some type of
effluent restrictions mandated by local municipalities,
or by the plant’s internal effluent treatment facility.
Common factors that must be considered are pH,
phosphate levels, Biological Oxygen De­mand (BOD)
or Chemical Oxygen Demand (COD) loading, Total
Organic Carbon (TOC) levels, and solids levels. In
many cases, the correct choice of detergent can help
reduce the impact on components of the effluent
stream that are a concern. For example, if phos­phates
are a concern, a detergent that contains low levels of
phosphate can be used. Another example is a situation
where the pH of the effluent must not exceed 10 and
must not fall below four. If a strong acid or alkaline
detergent is used, the pH restrictions could be violated.
In this case, choosing a neutral, mildly alkaline or
mildly acidic detergent may be the solution. However,
in some cases, a strongly acidic or alkaline detergent
might be re­quired to effectively re­move the potential
contaminant from equipment surfaces. If a strong
alkaline detergent is re­quired, the cleaning cycle could
be designed to include an acid rinse. The acid rinse
will help reduce the amount of rinse water re­quired to
neutralize residual alkalinity in the system, will help
remove any inorganic residues, and can be captured
and mixed with the alkaline wash water to neutralize.
In general, detergents will have the greatest
im­pact on pH and phosphate levels. Relative to the
residue load, detergents generally have little im­pact
on BOD, COD, TOC, or solids levels.
If effluent restrictions exist, these should be
ad­dressed in the early stages of the development of
a cleaning program to avoid compounded problems
later on when the cleaning protocol is implemented.
At this point, five key factors that should be
considered when selecting a detergent to be used
as a part of a validated cleaning program have been
discussed. Once these factors are addressed and an
appropriate detergent chemistry is identified, labora­
tory testing should be done to verify that the chem­
istry is effective against the potential contaminant.
Other cleaning parameters such as cleaning time,
temperature, and concentration can be evaluated in
the laboratory as well.
Laboratory Testing
Cleaning studies conducted in the laboratory can
be designed to closely mimic the actual applica­
tion method, such as a CIP system, or they can be
Figure 6
Water Hardness
(Reported as CaCO ) Rating
3
Hardness
Soft
Moderately Hard
Hard
Very Hard
Grains Parts Per
Per GallonMillion (PPM)
0 – 3.5
3.5 – 7.0
7.0 – 10.5
>10.5
0 – 60
60 – 120
120 – 180
>180
Special Edition: Cleaning Validation III
33
Mark Altier
de­signed to stress the system to differentiate between
similar cleaning chemistries. An example of the lat­
ter is a designed study that removes all mechanical
action from the system, forcing the chemistry type
and concentration, thermal energy, and contact
time to act on the residue. This ap­proach is espe­
cially effective in differentiating between similar
chemi­stries that appear to be equally effective when
ap­plied using some type of mechanical action.
An important component of designed clean­
ing studies is the preparation of the residue being
tested.
Typically, the residue is applied to a 304 or 316
stainless steel coupon and the treated coupon is then
subjected to the cleaning solution. The application of
the residue to the coupon is critical to obtain results
that can be directly applied to the actual system in
the plant. For example, a manufacturing process
may involve a heating step that causes some of the
finished product to “bake” onto a vessel side wall.
To obtain re­sults that are ap­plicable to this situation,
the residue should be applied to the coupon surface,
heated, and then allowed to bake for an equivalent
amount of time as is experienced in the actual pro­
cess. If this is not done, the results of the study will
have little relevance to the development of a cleaning
program aimed at removing a baked on residue from
equipment surfaces.
Prior to implementing any cleaning studies, a
set of success criteria must be established. Once the
cleaning studies have been completed, quantitative
measurements against the success criteria should be
made. Based on the results of this work, a final deter­
gent chemistry recommendation is derived. Ideally,
the detergent chemistry should meet or exceed all
established success criteria.
The end result of the laboratory work will be a
scientifically sound recommendation of detergent
chemistry and other important cleaning parameters
such as cleaning time, temperature, and concentra­
tion. This is the basis for the overall cleaning pro­
gram that will be tested at the production facility.
The importance of performing preliminary labora­
tory testing is that it provides a sound, scientific
rationale of why the selected chemistry is appropri­
ate for the cleaning application.
Plant Optimization
34
Institute of Validation Technology
Once a cleaning chemistry has been identified
and verified in the laboratory and other cleaning
parameters such as cleaning time, temperature and
concentration have been established, testing and
optimization must be carried out at the production
plant. Initial optimization and testing is usually done
on a pilot scale, prior to scaling up. The re­sults of the
laboratory cleaning studies should be used as a guide
or a starting point for the optimization process at the
plant site.
Conclusion
Process cleaning is an integral component of any
pharmaceutical process. The five key factors that
must be addressed to help identify a detergent when
developing a cleaning program have been defined
and discussed. The interaction of these factors with
each other and with the development of a cleaning
program must be understood. Lab­oratory testing
is critical for documenting the ap­propriateness of
the detergent selection for the cleaning applica­
tion. Plant optimization is a final critical step prior
to starting the validation process at the production
facility. When these steps are taken, a complete,
scientifically sound approach to the development of
a cleaning program can be documented. o
About the Author
Mark Altier is a Principal Chemical Engineer for
Ecolab Inc., where he manages their pharmaceutical and cosmetic programs. Mark has worked
for Ecolab for seven years and has held positions
in quality assurance, process engineering, and
re­search and development. He can be reached at
651-306-5876, by fax at 651-552-4899, or by e-mail
at mark.altier@ecolab.com.
Analyzing Cleaning
Validation Samples:
What Method?
By Herbert J. Kaiser, Ph.D.
and
Maria Minowitz, M.L.S.
Steris Corporation
C
v
leaning validations are very
and cons of each technique will be
difficult to perform. They
ex­am­ined. Validating the methods
can be made easier if an
}This article will will be discussed, as well. The ref­
ap­propriate method for analyzing
erences included with this paper can
describe
various
the samples is used. The method
be used to provide more in-depth
used should be based on the previ­
information to the reader and act as
analytical
ously established residue limits of
guides to the available literature.
the active and cleaning agents. There
Choosing the appropriate ana­
technologies
are many choices of an­alyt­i­cal tech­
lytical tool depends on a variety of
niques that can potentially be used. available for use, factors.5,6 The most important fac­
is determining what species or
This article will de­scribe various
particularly for tor
parameter is being measured.7 Is it
analytical technologies avail­able for
use, particularly for cleaning agent cleaning agent res- an or­ganic compound or inorganic
compound? The next question is
residues. Ref­­­er­ences are provided to
idues.
References
mea­sure­ment. How is this com­
guide the reader to more in-depth
information.
are provided to pound going to be measured? Is it
going to be swabbed from a surface
Cleaning validation in the phar­
maceutical industry is of critical guide the reader to or determined from a rinse water
sample? If it is going to be swabbed
im­portance.1, 2, 3 There are many
more in-depth
analytical techniques available that
from a surface, where will this swab­
4
can be used in cleaning validations.
bing oc­cur? Another im­portant fac­
information.~
The choice of the technique used
tor in choosing an analytical tool is
in analyzing a particular sample is
establishing the limits of the resi­
very important in cleaning valida­
due. The limit should always be
tion. The technique must be appropriate for measur­
established prior to selecting the analytical tool.8, 9
ing the analyte at and below the acceptance residue The limits should not be established solely based on
limit. Today’s analytical chemist has a wide vari­ detection limits of a particular method. Yet, another
ety of techniques available for use. These choices important factor in choosing an analytical tool is
in­clude specific and nonspecific methods. Many whether or not the method can be validated. If the
methods are complementary to each other. The pros method can’t be validated, then another technique
Special Edition: Cleaning Validation III
35
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.
needs to be chosen.
Sampling Technique
The sampling technique plays a large role in
determining which analytical technique to use. Some
techniques are more applicable for swab samples,
and other techniques are more applicable for rinse
water sampling. The acceptable sampling techniques
include direct surface sampling (swab) and rinse
water samples.10 The rinse water sample is a direct
measure of potential contaminants, but the analysis
should not just be a compendial test for water. Rinse
water analyses should be directed toward responses
peculiar to the possible contaminants. A questionable
form of sampling is placebo sampling. The placebo
method sampling is when the product, not contain­
ing the active ingredient, is processed in the specific
piece of equipment. This is analyzed for any active
that may have been picked up from the equipment.
A problem with placebos is the potential lack of uni­
formity. The contaminant may not be evenly distrib­
uted throughout the placebo. Another problem is the
analytical power of the tools that are used to analyze
the samples. The residue levels may be extremely
low if in fact the contaminant is evenly distributed
throughout the sample. The use of placebos is only
acceptable if used with swab or rinse water data.
Therefore, placebos are generally not used because
of the additional work involved.
Another important factor to consider in choosing
an analytical method is the type of residue being
analyzed. Residues can be drug actives, formulation
components, cleaning agents, organic, inorganic,
water soluble, water insoluble, particulate, microbial,
and/or endotoxins. If the residue being detected is a
drug active, and the method used for detection is the
same method that is used for quality control purposes
of the final formula, it must be established that the
active has not changed its chemical nature during the
cleaning process. That is, it must be established that
the active is still detectable and quantifiable using
the analytical method. This can easily be established
by performing forced degradation studies. Exposing
the active to the cleaning compound at an elevated
temperature and then analyzing that sample will help
determine the compatibility of the cleaner with the
active. If the active has indeed changed its chemical
nature during the cleaning process, a new technique
36
Institute of Validation Technology
will need to be established for its analysis.
Limit of Detection and Quantitation
Before choosing a method, some definitions need
to be established. The Limit of Detection (LOD) is
the lowest amount of a compound that can be detect­
ed. The Limit of Quantitation (LOQ) is defined
as the lowest amount of a compound that can be
quantified. The LOD is usually lower than the LOQ,
but is never higher. The LOD should never be used
to establish residue acceptance limits. The residue
ac­cep­tance limit should be well above the LOQ so
that it can be accurately quantitated.
Specific and Nonspecific Methods
A specific method is a method that detects a
unique compound in the presence of potential con­
taminants. Some examples of specific methods are
High Performance Liquid Chromatography (HPLC),
ion chromatography, atomic absorption, inductively
coupled plasma, capillary electrophoresis, and other
chromatographic methods. It should be noted that
HPLC is not inherently specific. What is meant is
that the conditions in an HPLC measurement can
usually be adjusted to separate out known potential
contaminants.
Nonspecific methods are those methods that detect
any compound that produces a certain response. Some
examples of nonspecific methods are Total Organic
Carbon (TOC), pH, titrations, and conductivity. A
very interesting and sensitive nonspecific technique
is dynamic contact angle.11 Titrations may be specific
for acids or bases, but they are not specific for par­
ticular acids or bases. There are, however, specific
titrations for classes of surfactants.12
Interferences
A good nonspecific strategy that could be fol­
lowed is to first identify possible interferences. These
interferences can be either positive or negative. The
nonspecific property is then measured, and the resi­
due is calculated as if all of the measured property
is due to that residue. For example, if the cleaning
agent was the analyte and TOC was the method used,
all of the TOC would be assumed to have come from
the cleaning agent and calculated as such. This would
then provide a worst-case upper-limit value.
There are many possible sources of interferences.
Cleaning agents and compounds can be a source of
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.
interferences, for example. Active agents and their
byproducts, water system components, maintenance
materials, and the atmosphere can all be sources, as
well as people, if samples are not handled properly.
The materials used to perform the analytical method
can also be a source of interference. For example, if
a swab that has a high TOC value is used to sample,
it could increase the level of TOC detected.
For specific methods, there should be no interfer­
ence if the method is properly designed. Again, it
should be stressed that the method must be able to fol­
low the analyte after exposure to the cleaning en­viron­
ment. It is necessary to establish that the cleaning
environment or the cleaning process does not change
the analyte. For nonspecific methods (which measure a
nonspecific property), any compound with the prop­
erty that is introduced into the sample will interfere.
For example, if the method being used is TOC, atmo­
spheric carbon that may enter the sample could cause
interference. With all nonspecific methods, there is a
need to identify potential sources of interference.
n High Performance Liquid Chromatography
The first technique that will be discussed is HPLC.
Almost every pharmaceutical company has an HPLC
instrument. HPLCs utilize a variety of detectors.
These include ultraviolet (UV), fluorescence, elec­
trochemical, refractive index, conductivity, evapo­
rative light scattering, and many others. The ultra­
violet de­tector is by far the most common. However,
Evaporative Light Scattering Detection (ELSD) may
be the most appropriate detector for cleaning agents.
We will discuss the use of both UV and ELSD detec­
tors in depth.
• Ultraviolet Detectors
There are many advantages of using UV detec­
tors. Many compounds have chromophores and
therefore, they can be easily detected by UV. Many
in­struments are equipped with diode array spec­
tral capabilities. This allows for easy detection of
impurities or potential contaminants within peaks.
Ultraviolet detection usually requires no additional
reagents or post column or pre-column reactions.
UV detectors are not harmful to the sample, if that is
important. They are generally inexpensive and read­
ily available. Also, molar absorptivities are gener­
ally not affected by temperature and therefore, there
is no need for heating or cooling the detector.
While there are many advantages of UV detec­
tors, there are also some significant disadvantages.
UV detectors cannot detect all types of compounds
and therefore are not considered to be universal.
All compounds do not have chromophores. This is
particularly true of surfactants that are used in the
pharmaceutical industry. Dirty cells, air bubbles,
and the use of gradients can affect baseline drift and
detection capability. The limits of detection can be
higher than other detector types due to background
interferences.
• Evaporative Light Scattering Detection
In ELSD, the compound is separated on an HPLC
column as usual, and then enters a nebulizer that is
combined with a gas stream and passed through a
heated column. The heated column evaporates the
mobile phase leaving the solid analyte in the column.
The solid analyte then passes through a detector that
consists of a laser or light source. The laser or light
source is scattered when it hits the solid analyte. The
detector then picks up this scattering.
There are many advantages associated with evap­
orative light scattering detectors. ELSD is claimed
to be universal. It is called universal because it can
detect any type of compound. ELSDs are simple,
versatile, and rugged in use. Since it is a mass
detector, all compounds produce similar responses.
Additionally, there is no baseline drift due to mobile
phase effects.
There are two primary disadvantages of ELSD.
First, there is a very limited choice of buffer salts
that can be used. Recall that the mobile phase is
evaporated or removed, leaving the analyte. Any
buffers that will not evaporate will also produce
solid particles that will then be detected and cause
interferences. The second disadvantage is that the
nebulizer and detector must produce consistent par­
ticle sizes. This requires careful cleaning and moni­
toring of the nebulizer.
Actives and Detergent
There are many types of residues that can be ana­
lyzed using HPLC techniques. These include both
actives and detergent residues. When dealing with
detergent residues, it is important to identify what
is being analyzed: surfactant, builder components,
Special Edition: Cleaning Validation III
37
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.
chelating agents, etc. The separation and quantita­
tion of surfactants at low levels is difficult, at best.
Industry literature is full of references for surfactant
analyses using HPLC. The vast majority of tech­
niques described in the literature are for the deter­
mination of surfactants in concentrated products.13,14
There­fore, the limits of quantitation and the limits of
detection are rather high. There are also references
for the analysis of surfactants related to the environ­
ment.15,16 In environmental analysis, the sample is
pre-concentrated so that the limits of quantitation
are very low. The pre-concentration
can be up to one thousand fold.
coating simply to make it more rugged. All common
detection techniques (UV, fluorescence, etc.) can
be used in capillary electrophoresis detection. The
capillary itself serves as the detector cell. A small
portion of the polyimide coating is scraped off prior
to use, and the bare portion of the capillary is placed
in the light path. This detection is different from that
seen in HPLC because the detection occurs while the
separation is taking place, rather than after separation
has been completed. Using a Z-cell can increase the
sensitivity of the technique. This is accomplished by
}The TOC is then computed by
subtracting the inorganic carbon
concentration from the total
carbon concentration
of the sample.~
Suggested Reading
Authors Lin, et. al., compared
the analysis of anionic, cationic,
and amphoteric surfactants con­
taining n-dodecyl groups using
HPLC and capillary electrophore­
sis.17 They found that HPLC was
best for all classes of surfactants, especially for for­
mulated surfactants. Authors Carrer, et. al., utilized
ELSD for amphoteric type surfactants.18 Amphoteric
surfactants are a class of surfactants that display
cationic behavior in an acidic solution and anionic
behavior in an alkaline solution. The lowest cali­
bration standard that they utilized was 50 ppm, but
they probably could have gone much lower. Authors
Guerro, et. al., obtained a limit of quantitation of
0.49 ppm for alkyl polyethylene glycol ethers using
ELSD.19
n Capillary Electrophoresis
An interesting method of analysis is Capillary
Electrophoresis (CE). There are many different types
of CE. Capillary Zone Electrophoresis (CZE) is by
far the most common. CE instrumentation is fairly
simple, consisting of a high voltage source, a capil­
lary, and a detector. The high voltage source is used
to apply a potential across two solutions. One of
the solutions contains the analyte, and the potential
ap­plied to the solutions causes the analyte to migrate
through the capillary, through the detector, and
into the other solution. The column or capillary is
typically composed of fused silica with a polyimide
coating. The diameter of the capillary is typically
25-75µm in diameter. The capillary has a polyimide
38
Institute of Validation Technology
using a special accessory that bends the capillary,
causing the source radiation to penetrate lengthwise
through the capillary rather than a cross-sectional
sampling. This, in effect, increases the path length
of the cell. The Z-cell can be used in all types of CE
where UV detection is used.
CE can be used for many different types of analy­
ses. Surfactants can be determined quite readily using
this technique.20,21 However, detection limits typically
are higher than with HPLC. This can be overcome by
pre-concentrating the samples on the capillary itself.
A voltage is applied to the capillary in a manner that
allows the compounds to collect at one end of the
capillary without flowing through to the detector. An
advantage that capillary electrophoresis holds over
HPLC is the ease with which indirect detection can
take place. Indirect detection is where a highly UVabsorbing material is included in the mobile phase.
As the analyte is eluted or travels along the capillary
through the detector, a negative peak is seen for the
analyte. This typically is done for compounds that dis­
play low UV absorption. In addition to being useful
for the analysis of surfactants, capillary electrophore­
sis can be used to analyze organic acids, inorganics,22
and trace drug residues.23
Suggested Reading
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.
Vogt, et. al., provided a good overview of the
separation of cationic, anionic, and nonionic surfac­
tants using capillary electrophoresis.24 They indicated
that one can easily adjust the parameters of the sepa­
ration to coelute or separate oligomers. Coelution of
the oligomers increased the sensitivity at the ex­pense
of increasing the potential for coeluting positive
interferences. Direct UV detection could be used
for UV-absorbing materials and indirect or non­-UV
absorbing materials.
Heinig, et. al., utilized micellar electrokinetic cap­
illary chromatography for the separation of non-ionic
alkylphenol polyoxyethylene type surfactants.25 How­
ever, the use of this method was limited because of
insufficient peak resolution and relatively low detec­
tion sensitivity. Heinig, et. al., also compared HPLC
and CE analyses of surfactants.26 The surfactant types
they studied were linear alkylbenzenesulfonates,
nonylphenolpolyethoxylates, cetylpyridinium chlo­
ride, and alkylsulfonates. For the CE analyses, they
utilized UV detection either in the direct or indirect
modes, depending on the nature of the surfactant.
For the HPLC analyses, they utilized either direct
UV detection or conductivity detection. An­ionic sur­
factant samples were pre-concentrated one thousand
fold through the use of solid phase extraction. This
allowed for detection limits in the parts per billion
range to be obtained.
Kelly, et. al., utilized CE with indirect detec­
tion to determine sodium dodecylsulfate concentra­
tions.27 They also indicated that it is important to
look at the absorption of the surfactants onto filters
if the samples are indeed filtered prior to analysis.
This is most important in dilute solutions. Filtering
large volumes of sample can minimize this. Again,
appropriate studies need to be done to determine if
this indeed is a problem.
Altria, et. al., examined the use of CE in the analy­
sis of sodium dodecylbenzenesulphonate.28 They
ob­tained a limit of quantitation of 0.6 ppm and a
0.3 ppm limit of detection. They utilized direct UV
detection. Shamsi, et. al., utilized CE with indirect
de­tection for the determination of cationic and anion­
ic surfactants.29 The authors obtained limits of detec­
tion of 0.25 and 0.5 ppm, respectively. Heinig, et. al.,
also utilized CE in the analysis of cationic surfactants
using indirect UV detection.30 They compared this
with HPLC. They obtained a limit of quantitation for
CE of 4.0 ppm; and for HPLC, they obtained a limit
of quantitation of 5.0 ppm.
n Total Organic Carbon
TOC is used widely in the pharmaceutical indus­
try.31, 32, 33 The TOC is determined by the oxidation
of an organic compound into carbon dioxide. This
oxidation can occur through a number of mecha­
nisms depending on the instrument being used.
Some typical methods are persulfate, persulfate/UV
oxidation, and direct combustion. The carbon diox­
ide that is produced from these oxidations is either
measured using conductivity or infrared techniques.
In­stru­ments generally measure the inorganic carbon
content of a sample. The inorganic carbon consists
of carbon dioxide, bicarbonate, and carbonate. They
then determine the total carbon content of the sam­
ple. The TOC is then computed by subtracting the
inorganic carbon concentration from the total carbon
concentration of the sample.
There are two primary advantages associated
with TOC. The first is that it does not take long to
develop a method. There are not a lot of variables in
the actual analysis. The second advantage is that it is
relatively quick. A third potential advantage (which
can also be a disadvantage) is that it will detect and
analyze any compound containing carbon.
As with most techniques, there are disadvantages
in using TOC. A significant disadvantage is that the
compound or the analyte must be water soluble. This
does not mean that the compound must be soluble in
the hundreds of parts per million range but soluble in
the low parts per million range. Another disadvantage
is that organic solvents cannot be used. If organic
solvents were used, the TOC of the solvents would be
measured instead of the residue. There are also many
sources of contamination that can occur using TOC.
These sources can include the atmosphere, the swab
it­self, personnel, and many other sources. Methods
de­veloped using TOC should be written to include
controls and blanks to identify or account for possible
contamination. For example, a common source of con­
tamination is the technique used to cut the handles of
the swabs so that they fit into the TOC vials. Many
times, the scissors or utensils are not clean enough
for TOC use. This introduces contamination into the
sampling vial when the swab is cut.
Excipients
Special Edition: Cleaning Validation III
39
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.
Some methods/techniques can be used in certain
situations to complement each other. Examples
in­clude TOC and HPLC. Consider the case of a drug
in the presence of excipients. The excipients are very
soluble in water while the drug active has ex­tremely
low solubility in water. The excipients con­tribute
to the TOC values because they are very soluble in
water; however, the drug active does not show up in
the TOC analysis. An HPLC analysis is performed
to monitor the loss of the drug. The ex­ci­pients are
removed much faster from a surface during cleaning
than the drug active is removed. In this case, TOC
analysis is not a good stand-alone method. It is,
however, a good complement for the HPLC assay.
The TOC analysis enables the analyst to see what
water soluble matter is left behind, if any.
Suggested Reading
Guazzaroni, et. al., examined the use of total
or­ganic carbon for the analysis of detergents, endo­
toxins, biological media, and polyethylene glycol.34
For detergents, they were able to obtain a 0.7 ppm
limit of quantitation. Endotoxins were found to have
a 0.2 ppm limit of quantitation. The biological media
produced a total organic carbon limit of quantitation
of 20.3 ppm; and the polyethylene glycol produced a
0.5 ppm limit of quantitation. They examined swab
and rinse water recoveries. They were able to obtain
78-101 percent recoveries utilizing swabs, and 93
percent or better for rinse water recoveries.
There are many examples in the literature that uti­
lize ion chromatography as the method for analysis
of surfactants.35 The surfactants have to be charged
in order to be analyzed using ion chromatography,
that is, only anionic or cationic surfactants can be
detected. Pan, et. al., recorded limits of quantitation
down to 0.5 ppm for linear alkane sulfates and sulfo­
nates.36 Takeda, et. al., recorded a limit of quantita­
tion of 0.1 ppm for dodecyl alkyl sulfates.37 Nair, et.
al., separated different sulfate, sulfonate, and cat­
ionic type surfactants using ion chromatography with
suppressed conductivity detection.38 They reported
detection limits at less than 1.0 ppm.
n Ion Chromatography
In addition to its use for surfactants, ion chro­
matography can be used for the analysis of inor­
ganics and other organic compounds present in
40
Institute of Validation Technology
cleaners.39, 40, 41 Most cleaners contain sodium and/or
potassium. The ion chromatography detection tech­
nique of suppressed conductivity is more sensitive
to potassium than it is to sodium. Very low levels of
cleaning agent can be detected using this technique.
This assumes that the rinse water used contains no
potassium. Ionizable organic acids are also readily
quantitated using ion chromatography. This includes
chelating agents that are often found in cleaning
compounds.
Suggested Reading
In determining surfactants, an excellent review
concerning their analysis was done by Vogt, et. al..42
They compared the use of HPLC, CE, ion chro­
matography, Liquid Chromatography-Mass Spectro­
scopy (LC-MS) and Gas Chromatography-Mass
Spectro­scopy (GC-MS). They also discussed pre-con­
centration of the samples. They compared the use of
solid phase extraction, super critical fluid extraction,
Soxhlet extraction, and steam distillation as means
of pre-concentrating samples. They found, by far,
that the best method was solid phase extractions
for the pre-concentration of surfactants. They also
examined the use of titrimetric methods of analysis
for surfactants. For detecting anionics, substances
like methylene blue, pyridinium azo, and triphenyl­
methane dye was used to complex the surfactants
prior to photometric determination. Non­ionics were
determined indirectly by forming a cationic complex
with barium. This complex was then precipitated by
bismuth tetraiodide ion in acidic acid. The bismuth
was then quantified by potentiometric titrations.
Cationics were complexed with anionic dyes such as
disulfine blue.
Theile, et. al., brought up an excellent point that
surfactants tend to concentrate at interfaces.43 This
can be a problem in extremely dilute solutions of
surfactants. The surfactants can collect at the surface
of the containers that they are stored in. This may
cause errors in analysis. Proper controls in studies
should be done to determine if this is a problem.
The authors indicated that pre-concentration was
re­quired to determine very low levels of surfactant.
Solid phase extraction was the best method for this.
They were also able to obtain detection limits for
linear alkylbenzenesulfonates of 2.0 ppb with fluo­
rescence detection and 10.0 ppb using HPLC with
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.
UV detection after pre-concentration.
n Thin-Layer Chromatography
There are many examples in the literature for the
use of Thin-Layer Chromatography (TLC) for the
qualitative determination of surfactants.44, 45 Henrich
described the TLC of over 150 surfactants in six
different TLC systems.46 This was excellent for iden­
tification of the surfactants, but the author did not
attempt to quantify the surfactants. Buschmann and
Kruse combined diffuse reflection infrared spectros­
copy and TLC, along with SIMS and TLC for sur­
factant identification.47 Although these techniques
are tedious and time-consuming, there is no doubt
that these methods could be developed into quantita­
tive analyses. Novakovic has used high performance
TLC for two generic drugs.48
Other Techniques
Other excellent techniques for inorganic con­
taminants, and in some cases actives, are Atomic
Absorption (AA)49 and Inductively Coupled Plasma
(ICP) atomic emission. These techniques can detect
inorganic contaminants down to extremely low lev­
els. Inorganic contaminants in a system are often
ignored. These can come from rouge that forms in
Water for Injection (WFI) systems. They can also
come from the detergent utilized in cleaning the
equipment.
n Fourier-Transform Infrared Spectroscopy
Fourier-Transform Infrared (FTIR) spectroscopy
is never used as a stand-alone method for analyzing
residues on equipment. This is because of the lack of
portability of FTIR equipment and the semi-quanti­
tative nature of the reflectance techniques used for
these types of analyses. However, it is very useful
in performing screening studies and in evaluating
po­tential cleaning agents. This is done by soiling
standard coupons with the cleaning agent, allowing
them to dry, and performing static rinsing studies.
These types of studies can indicate whether or not
the cleaning agent is readily removed from surfaces.
The height or area of a particular peak is measured
versus the concentration of the standard coupon.
n Bioluminescence
Bioluminescence is quite useful for biologicals.
This type of analysis usually uses Adenosine Tri­
phosphate (ATP) bioluminescence.50 This is based on
the reaction of ATP with Luciferin/Luciferase. This
technique is often used in biopharmaceutical facilities.
It has extremely high sensitivity and a very high repro­
ducibility. In many cases, the instruments can be used
at the equipment site. This technique utilizes swabs for
surface analyses.
n Optically Stimulated Electron Emission
In some cases, a company’s established limits of
residue are so low that they cannot be detected by
conventional methods. A very sensitive method that
may be applicable is Optically Stimulated Electron
Emission (OSEE).51 The instrumentation for OSEE
is fairly portable, and can be readily taken to tank
side for analysis. The technique uses a probe that is
placed against a surface, and a UV source illuminates
and activates the surface. When some surfaces are
exposed to UV light at certain wavelengths, electrons
are emitted from the surface. The instrument measures
the current that is produced. If even small amounts of
residues are present on the surface, the current will be
affected. The current can be affected either in a posi­
tive or negative way depending on the nature of the
residue. This is an extremely sensitive technique.
It can be used in either a qualitative or quantitative
manner.
n Portable Mass Spectrometer
For those companies that require ultrasensitive
measurements and identification of the residues, a
technique has been developed – Lawrence Liver­
more National Laboratories has developed a port­
able mass spectrometer.52 The unit consists of a gun
portion of the instrument that is connected with
cables to vacuum pumps. The gun portion is held
against the surface to be analyzed. A seal is formed,
and the surface is heated to volatilize any com­
pounds that are present. This instrument is used not
only to measure how much of something is present,
but also what that something is. This piece of equip­
ment has been utilized in the aerospace industry.
One drawback of the portable mass spectrometer
is that it requires relatively flat surfaces. However,
they are currently working on adaptors to be used on
non-flat surfaces.
Additional Techniques
Special Edition: Cleaning Validation III
41
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.
In the biopharmaceutical industry, a wide vari­
A minimum validation requires two different
53
ety of techniques are utilized. These include the analysts, instruments, columns (if chromatographic
Enzyme-Linked Immunosorbent Assay (ELISA),54
method), days, and prepared standards and sam­
the Limulus Amoebocyte Lysate (LAL), and a wide
ples.60 These are the “twos” of method validation.
variety of protein determinations. These are all con­ The point of any method validation is to show that
taminant specific assays. For example, the LAL test the method can be utilized by different analysts and/
measures the level of endotoxins present. There is or laboratories, along with the ability to produce the
same results. If a validated method is given to a lab­
also the anthrone assay that can be used to monitor
the levels of carbohydrates on sur­
faces. These techniques are usually
}For those companies that require ultraused in combination with TOC.
The nonspecific techniques of
sensitive measurements and
pH, conductivity, and titrations
identification of the residues, a
can be used throughout all areas
of pharmaceutical manufacturing.
technique has been developed
Ob­viously, these techniques are
– Lawrence Livermore National
most often utilized in rinse water
monitoring. The conductivity and
Laboratories has developed a
pH of rinse water is typically moni­
portable mass spectrometer .~
tored and compared to the conduc­
tivity and pH of the water prior
to introduction to the equipment.
If acidic or alkaline materials are being measured, oratory, that laboratory must revalidate the method
for their laboratory. It is not sufficient or accurate
titration is a very useful technique. In some cases,
to assume that another laboratory’s validation will
titration can be more sensitive than performing
TOC analyses. The sample size can be adjusted, apply in all laboratories. For example, if a surfactant
and/or the normality of the titrant can be adjusted to is being quantitated, typically a low wavelength
is used in a UV detector for HPLC. UV detectors
increase the sensitivity of the titration.
vary in their noise levels at these low wavelengths.
Method Validation
A detector used in one laboratory may have sig­
nificantly less noise than a detector in another lab­
It is very important to scientifically establish the
oratory. The second laboratory may not be able to
residue limit prior to choosing the method of analy­ detect at the same low level as the first laboratory.
sis. This includes the limit in the analytical sample
and the limit in the next product. This will ensure Coupons and Swabs
that the method chosen will be able to detect and
Coupons can be prepared for recovery studies
quantitate the limit chosen. Once the technique for
through the use of aerosol bottles available through
analysis has been chosen, it is very important to vali­ laboratory supply companies. A known weight per­
date the method used.55- 60 The validation of a meth­od cent of a solution containing the analyte can be
is very different than the validation of recovery. A
sprayed fairly evenly over the surface of a coupon.
validated method is one that is rugged and robust The coupon can be swabbed using a standard tech­
enough to measure the residue limit established.
nique. It does not matter how you swab the coupon,
The validation of a recovery is to determine the
as long as the complete surface is covered and that the
amount that can be recovered from a surface. Again,
coupons are swabbed the same way – each and every
it should be stressed that these are two completely time. The type of swabs used in recovery studies must
different validations.
be the same as those used in the validation protocol. If
this is a simulated rinse procedure, then the coupons
“Twos” of Validation
are rinsed and the rinse water is analyzed.
42
Institute of Validation Technology
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.
For swabs, a desorption process is carried out.
This can consist of simply shaking the sample vial
or using an ultrasonic bath. The samples are then
analyzed. Recovery studies are always done below
ac­ceptance limits in the test solution. This ensures
that the limit will be (or can be) measured in the anal­
ysis. A recovery of greater than 80 percent is good.
If the recovery is greater than 50 percent, it may be
acceptable. However, if the recovery is less than 50
percent, questions arise and the source of the poor
recovery should be investigated. A possible cause of
a poor re­covery can be that the residue is being too
tightly held by the swab. This can be investigated
by spiking a swab with a known amount of residue,
allowing it to dry, trying to desorb the res­idue, and
following up with analysis. If the analyte is held too
tightly by the swab, another type of swab material
should be investigated. The recovery factor should
be included in analytical calculations or in the accep­
tance limit calculation. It should not be included in
both of the calculations.
Containers
The choice of containers used in the analysis of
samples is very important. It has been shown that,
in very dilute solutions, surfactants can adsorb onto
the surfaces of sample vials. This will produce arti­
ficially low results in the analysis. This, however,
typically only occurs in static systems. There is no
need to worry about the adsorption of the surfactant
on the walls of manufacturing equipment. This is
because the agitation that is involved in cleaning
removes the surfactants from the surfaces. This is
another matter in sample vials. Appropriate spiking
studies should be performed to ensure that this phe­
nomenon is not occurring and will not interfere with
the analytical method. This includes both HPLC or
ion chromatography sample vials, as well as TOC
sample vials. This phenomenon is not limited to sur­
factants. Proteins have been shown to adsorb readily
onto glass surfaces. These proteins are much more
difficult to remove from surfaces than surfactants.
Specific Versus Nonspecific
The choice of using a specific or nonspecific
method can be difficult. If a drug active is highly
toxic, a specific method is always recommended.61
Detergents can be quantitated either using spe­
cific or nonspecific methods; however, care must
be taken in choosing which component is measured.
For example, a detergent may contain five percent of
a surfactant and 20 percent of another organic ingre­
dient. Assuming equal sensitivities of the analyti­
cal methods, the limit of quantitation of the whole
detergent system will be lowered by a factor of four
if the ingredient present in the greater amount is
determined.
If a nonspecific method (i.e., TOC) is used for
the same system, a much lower limit of quantitation
could be determined simply because there would be
a tremendous amount of carbon present in the sam­
ple. In addition, if detergent systems are combined,
such as in the case of adding a detergent additive
to another product, the choice of a specific method
would be made even more difficult. The question
would be, “Which detergent do I determine?” A
disadvantage of using a nonspecific method for the
entire cleaning validation analysis is that, if there is
a failure in the future, it would not be known where
the failure originally occurred. The failure could be
due to the active, excipients, detergent system, or
even an unknown source.
Conclusion
There are many different analytical techniques
available that can be used to detect residues. These
range from simple titrations to more complex LCMS. The choice of technique should be based on
what equipment is available, the type of residue,
and the scientifically established residue limit. It is
important for an analytical chemist to keep abreast
of the literature and what techniques are available.
There are techniques available that will analyze any
residue at any level. At the end of the day, however,
it is always wise to choose the simplest technique
that can be used to reach the desired goal. o
About the Authors
Herbert J. Kaiser, Ph.D. is Manager – Hard Surface
Products at STERIS Corporation. He has 18 years of
experience in cleaning and surface technologies,
which includes developing products and methods
for the cleaning and analyzing of a wide variety of
Special Edition: Cleaning Validation III
43
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.
surfaces. Dr. Kaiser has developed a wide variety of
products for the healthcare, industrial, and pharmaceutical markets. He is the sole inventor listed in
five United States Patents for various industrial treatment schemes. Dr. Kaiser received his B.A. degree
from St. Mary’s University in San Antonio, Texas,
his M.S.(R) from St. Louis University, and his Ph.D.
from the Un­iversity of Missouri. He is a member of
the American Chemical Society and the Association
for the Ad­vancement of Medical Instrumentation. Dr.
Kaiser can be reached by phone at 314-290-4725, by
fax at 314-290-4650, or e-mail at herb_kaiser@steris.
com.
Maria Minowitz, M.L.S., Information Associate at
STERIS Corporation, has 10 years of experience in
corporate research and development librarianship.
She has been responsible for information management in the disciplines of chemistry, medicine, and
engineering. Minowitz received her A.B. degree
from St. Louis University and an M.L.S. from the
University of Missouri-Columbia. She is a member
of the Special Libraries Association, Midcontinental
Chapter of the Medical Library Association, and the
St. Louis Medical Librarians.
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Surfactants in Environmental Matrices.” Tenside Surfactants and
Detergents. Vol. 36(1). 1999. pp. 8-12, 14-18.
44. Bosdorf, V., Krüßmann, H. “Analysis of Detergents and
Cleaning Agents with Thin-Layer Chromatography.” Fourth
World Surfactants Congressional Asociacion Espanola de
Productores de Sustancias para Aplicaciones Tensioactivas.
Barcelona, Spain. Vol. 4. 1996. pp. 92-95.
45. Read, H. “Surfactant Analysis Using HPTLC and the Latroscan.”
Proceedings of the International Symposium on In­strumental
High Performance Thin-Layer Chromatography. Third Edition.
Ed. Kaiser, R. Institute of Chromatography. Bad Duerkheim.
Federal Republic of Germany. 1985. pp. 157-171.
46. Henrich, L. H. “Separation and Identification of Surfactants in
Commercial Cleaners.” Journal of Planar Chromatography —
Mod. TLC. Vol. 5(2). 1992. pp. 103-117.
47. Buschmann, N., Kruse, A. “In-Situ TLC-IR and TLC-SIMS:
Powerful Tools for the Analysis of Surfactants.” Comunicaciones
Presentadias a las Jornadas del Comite Espanolde la
Detergenteia. Vol. 24. 1993. pp. 457-468.
48. Novakovic, J. “Validation of a High Performance Thin-Layer
Chromatographic Method for Trace Analysis for Some Generic
Drugs Affecting Gastrointestinal Function.” Journal of AOAC
International. Vol. 83(6). 2000. pp. 1507-1516.
49. Raghavan, R., Mulligan, J. A. “Low-Level (PPB) De­ter­min­a­tion
of Cisplatin in Cleaning Validation (Rinse Water) Samples. I.
An Atomic Absorption Spectrophotometric Technique.” Drug
Device Industry Pharmaceutical. Vol. 26(4). 2000. pp. 423-428.
50. Davidson, C. A., Griffith, C. J., Peters, A. C., Fielding, L. M.
“Evaluation of Two Methods for Evaluating Surface Cleanliness
– ATP Bioluminescence and Traditional Hygiene Swabbing.”
Luminescence. Vol. 14. 1999. pp. 33-38.
51. Chawla, M. K. “Is It Clean?” Precision Cleaning. Vol. 8(6).
2000. pp. 36,38.
52. Meltzer, M., Koester, C., Steffani, C. “Criteria Evaluation for
Cleanliness Phase 0.” Lawrence Livermore National Lab­oratory.
UCRL-CR-133199, PPG99-003. 1999.
53. Inampudi, P., Lombardo, S., Ruezinsky, G., et. al. “An
Integrated Approach for Validating Cleaning Procedures in
Biopharmaceutical Manufacturing Facilities.” Annals of the
New York Academy of Sciences. Vol. 782. 1996. pp. 363-374.
54. Rowell, F. J., Miao, Z. F., Neeve, R. N. “Pharmaceutical Analysis
Nearer the Sampling Point, Use of Simple, Rapid On-Site
Immunoassays for Cleaning Validation, Health and Safety, and
Environmental Release Applications.” Journal of Pharma­cy and
Pharmacology. Vol. 50. 1998. p. 47.
55. Seno, S., Ohtake, S., Kohno, H. “Analytical Validation in Prac­tice
at a Quality Control Laboratory in the Japanese Pharma­ceuti­cal
Market.” Accreditation and Quality Assur­ance. 1997, 2(3), 140145.
56. Kirsch, R. B. “Validation of Analytical Methods Used in
Pharmaceutical Cleaning Assessment and Validation.” Pharma­
ceutical Technology. 1998 (Analytical Validation Supplement).
pp. 40-46.
57. Brittain, H. G. “Validation of Nonchromatographic Analytical
Methodology.” Pharmaceutical Technology. Vol. 22(3). 1998.
pp. 82-90.
58. Ciurczak, E. “Validation of Spectroscopic Methods in
Pharmaceutical Analyses.” Pharmaceutical Technology. Vol.
22(3). 1998. pp. 92-102.
59. Swartz, M. E., Krull, I. S. “Validation of Chromatographic
Methods.” Pharmaceutical Technology. Vol. 22(3). 1998. pp.
104-120.
60. USP 23, United States Pharmacopoeia Convention. Rock­ville,
Maryland. 1995.
61. Segretario, J., Cook, S. C., Umbles, C. L., et. al. “Validation of
Cleaning Procedures for Highly Potent Drugs. II. Bisnafide.”
Pharmaceutical Device Technology. Vol. 3(4). 1998. pp. 471-476.
Special Edition: Cleaning Validation III
45
Control and Monitoring
of Bioburden in Biotech/
Pharmaceutical
Cleanrooms
By Raj Jaisinghani
Technovation
&
Greg Smith
Encelle, Inc.
&
Gerald Macedo
Med-Pharmex, Inc.
v
T
his paper describes results
HEPA fan filter units (FFUs) was
of monitoring biotech clean­­­­­­­­
possible. The results showed that at
}The FDA
rooms and a pharmaceutical
the same flow rate the EEF resulted
has
specific
cleanroom equipped with an Elec­
in significantly lower bioburden as
trically Enhanced Fil­tration (EEF) requirements and compared to the FFUs.
system that significantly reduces
1
Background
airborne bioburden in cleanrooms. guidelines for bioThe EEF High Ef­ficiency Part­
burden for
The fundamental purpose of
iculate Air (HEPA) system traps
various
cleanrooms in the pharmaceutical,
and kills bacteria and also im­proves
the filtration performance of a filter
pharmaceutical medical device, biotechnology, and
hospital applications is to control
me­dia by two to three orders of
operations and
the amount of bioburden due to
magnitude. In laboratory tests the
both internal operations and trans­
EEF tech­nology has been shown
processes.~
port from the air. From a particu­
to kill Staphyl­o­coccus epidermidis
late point of view, cleanrooms in
and Escherichia coli. These field
these industries are classified and specified accord­
test results support laboratory testing and show that
basically there is no airborne bioburden in both a ing to the same cleanroom standards (e.g., Federal
Class 10 room, with terminal HEPA in addition to the Stand­ard 209E) as in other industries. It is often
EEF, and in a Class 1000 room that utilizes only the assumed that the particle (total) concentrations
will generally correlate to concentration of viable
EEF without any terminal HEPA filters. In the case
of an old laboratory converted to a cleanroom, direct microorganisms. This may not always be valid.
comparison of the EEF with respect to conventional Hence, the concentration of viable organisms is also
46
Institute of Validation Technology
Raj Jaisinghani, Greg Smith, & Gerald Macedo
directly measured – both at the work surfaces (or at
the process) and in the air.
Cleanrooms in these industries must meet separate
standards for bioburden. The FDA has specific require­
ments and guidelines1 for bioburden for various phar­
maceutical operations and processes. Similarly, the
United States Pharmacopoeia (USP) and the European
Union’s GMP 2 guidelines give specific recommended
limits for microbial contamination for each class of
room. A cleanroom that meets the particle concentra­
tion requirements, but does not result in the desired
level of bioburden, will clearly be inadequate.
One of the main obstacles in achieving the required
bioburden levels is that the measurement of bioburden
is time consuming. Typically, bioburden measure­
ment involves sampling, incubation, and counting of
colonies. This is a time consuming pro­cess and thus
“real” time monitoring is not possible. Thus, it is not
always possible to relate higher incidents of bioburden
to operating events. Recently, however, ultraviolet
(UV) fluorescence (cf. Seaver and Eversole3, Pinnick
et al.4) technology has made it possible to achieve real
time monitoring of particles of biological origin. This
technology will find increasing use in the real time
monitoring of air in hospitals, cleanrooms, and mili­
tary nuclear, biological and chemical (NBC) warfare
protection systems – as a real time supplement to the
standard methods of determining bioburden. As this
happens, more attention will be focused on cleanroom
contamination control systems – currently mainly
mechanical filtration.
One problem with mechanical filters is that under
certain conditions common bacteria caught on the
filter can start growing on the filters, grow through
the media, and start shedding into the room.5,6,7 The
well-known case of the Legionnaire’s outbreak at the
veterans convention in Philadelphia has been attributed
to this phenomena. In that case the filters were sup­
posedly in a wet state. Generally, it is accepted that
bacteria is difficult to grow on clean glass fiber filter
media, used in HEPA filters, under normal humidity
conditions. However, since the function of these fil­
ters is to capture all particulate contamination, filters
eventually get dirty. The experiments conducted by
Jaisinghani et al.8 show that very little contaminant is
needed for growth of Staphylococcus epidermidis and
Escherichia coli on HEPA glass filters. In their experi­
ments Jaisinghani et al.8 found that very little of the
applied E. coli survived on the clean glass filter after
four hours of airflow, keeping in mind that E. coli is
not a hardy organism. Next about one gm of colloidal
kaolin was added to the E. coli solution that was to be
aerosolized. This time the recovery of E. coli was about
104 – 105 CFU/square inch of the filter media. Similar
tests with S. epidermidis recovered a little more S.
epidermidisthan with E. coli even without the colloidal
kaolin, due to the hardier nature of S. epidermidis. With
1 gm of colloidal kaolin in the 25 ml S. epidermidis
solution (in tryptic soy broth) the recovery of S. epider­
midis was about 105 – 106 CFU/square inch of filter
media. Tests with airflow continuing for seven hours
(following the aerosol) did not result in any significant
reduction in bacteria recovery. This result suggests
that, even in normal environments, bacteria can sur­
vive or grow on the filters. As the trend towards using
HEPA cleanroom filters for longer periods continues,
the possibility of bacterial growth on the filter, and thus
the rise in the airborne bioburden, also increases.
EEF Technology
Jaisinghani7 has played a significant role in the
development and commercialization of EEF tech­
nology. The most recent version (see Figure 1) of
this technology7 maintains the filter under an ion­
izing (as opposed to a simple electrostatic field)
Figure 1
Technovation’s Ionizing
EEF Technology
Ionizing
Wires
Filter
Flow
Downstream
Ground
Electrode
Control
Electrode
Ionizer
Electrode
H.V.
Supply
Special Edition: Cleaning Validation III
47
Raj Jaisinghani, Greg Smith, & Gerald Macedo
field. Another higher intensity ionizing field charges
incoming particles, stabilizes the electrical fields,
and increases the safety and reliability by ensuring
that no spark over can occur towards the filter. This
method provides two fundamental benefits:
1. Bacteria are killed as they pass through a first
high intensity ionizing field and then killed
as they are subjected to continuous ionizing
radiation when they are trapped on the filter.
This inhibits growth of bacteria on the filter.
2. The same ionizing fields enable penetration
re­duction by about two to three orders of mag­
nitude.
Since the cost of the additional electrical compo­
nents is partially offset by the increase in filtration
performance (either higher flow at the same pres­
sure drop and filtration efficiency or lower pressure
drop at the same flow and efficiency, as compared to
mechanical filtration of the same size) this is a highly
cost effective method for the control of bioburden.
Laboratory Evaluation of the EEF
Jaisinghani et al.8 have demonstrated the bacteri­
cidal properties of the EEF under laboratory condi­
tions. This study, conducted at Virginia Polytechnic
Institute, is summarized in this section.
Experimental Methods
S. epidermidis was grown in Tryptic Soy Broth
(TSB) to a concentration of 3 x 109 colony form­
ing units (CFU)/ml. The culture was lyophilized
in Wheaton vials in 5 ml aliquots – 1.5 x 1010 CFU
per vial. All vials were stored in a desiccator at 4
– 6ºC.
Pleated 15.24cm by 15.24cm by 5cm (6” x 6” x
2”) deep filters were first coated with colloidal kaolin
and TSB using an Aztek airbrush. The airbrush cup
was filled with 1g kaolin suspended in 25ml TSB
and sprayed onto a filter inside a laminar flow hood
and allowed to air dry before being used. The pleated
filters were placed in a miniature version of the EEF.
One vial of lyophilized S. epidermidis was resuspend­
ed with 1 ml of sterile distilled water. A small aliquot
of this suspension was serially diluted ten-fold to 10-8
and plated on Columbia Blood Agar (CBA) plates to
48
Institute of Validation Technology
confirm the initial viable concentration of bacteria.
The rehydrated culture was then sprayed onto the filter
using a Meinhard nebulizer, which was placed eight
inches from the center of the filter.
A control assay was performed to determine the
amount of viable S. epidermidis on the filter, without
application of high voltage. The bacteria were sprayed
onto the filter as previously described, and the temper­
ature and humidity were monitored every 15 minutes
for four hours or seven hours during which the airflow
was on. The relative humidity was held between 4555% using a Kaz steam vaporizer. At the end of each
control run three pieces of the filter were extracted
using a sterilized scalpel and forceps. The pieces of
filter were approximately one square inch on the face
of the filter, which when unfolded measured approxi­
mately 28 square inches of filter material. Filter pieces
were removed from the center of the filter, directly
above the center, and directly below the center.
The samples were cut into small pieces and placed
into 10 ml of sterile phosphate buffered saline (PBS)
pH 7.4 in a Nasco Whirl-Pak bag. The bags were
then processed in a Tek Mar Stomacher Lab-Blender
80 for one minute. Each sample was then serially
diluted ten-fold to 10-2, 10-3, and 10-4, then spread on
CBA plates to determine the number of viable bacte­
ria per sample filter piece.
Similar tests were then conducted by applying high
voltage to the EEF. In addition to monitoring the tem­
perature and humidity, the current was also monitored
at fifteen minute intervals during the four or seven hour
period of airflow with the applied high voltage on.
Results and Discussion
The results are summarized in Figure 2. In the
absence of any voltage applied to the EEF unit (i.e.,
control tests), viable bacteria were recovered from
one square inch of filter in the range of 1 x 105 CFU
to 2 x 106 CFU. Counts greater than about 3 x 106
CFU were too crowded to be accurately counted and
were considered to be too numerous to count. When
high voltage was applied for four hours, the majority
of the bacteria were killed. The kill rate increased
with increased voltage or with the first applied field
strength (applied voltage divided by the distance of
the ionizer wires from the control ground electrode –
see Figure 1), V/d1. At a field strength (V/d1) of 4.2
kV/cm, there was no growth after 24 hours of incu­
Raj Jaisinghani, Greg Smith, & Gerald Macedo
Figure 2
EEF Bactericidal Test Summary Using S. epidermidis
FilterIncubationEEF ExposureEEF FieldAverageComment
TimeTimeStrengthColonies
Control orHoursHours
EEF
Control
24.00
(v/d1) kv/cm
0.00
0.00
#/Sq. inch
1.00E+06
No additional growth
00.0E+00
100% Killed
Control
24.00
0.00
0.00
1.02E+05
24.00
4.00
3.99
3.44E+02
99.93% Killed
0.00E+00
Some growth
EEF
EEF
EEF
EEF
EEF
EEF
EEF
EEF
EEF
24.00
24.00
24.00
24.00
24.00
48.00
48.00
48.00
4.00
4.00
4.00
4.00
4.00
7.00 4.00
4.00
bation. After 48 hours, there was either no growth or
small (in size and in number) colonies grown. These
small colonies were identified as S. epidermidis, and
were identical in biochemical profile as the isolate
used in the tests. It was concluded that four hours
at 4.2 kV/cm (V/d1) did not completely kill the S.
epidermidis. If the bacteria were not all killed, some
of them were damaged sufficiently so that no growth
or very limited growth could occur after 24 hours
incubation. When the ionizing time was increased to
seven hours, over 99% of the bacteria (as compared
to the control) were killed.
When the applied field strength, V/d1, was
increased to 4.5 kV/cm or higher, no growth occurred
on any of the filter pieces except for one experiment.
This exception may have occurred because the start­
ing dose of bacteria for this experiment was three
times higher than for the control and up to 10 times
higher than for any other experiment. Nonetheless,
there were still three to four logs of killing using an
applied field strength, V/d1 of 4.5 kV/cm or higher,
as compared to the control experiments. It should be
noted that, in practice, bacteria caught on the filter
are held within the ionizing field for an almost infi­
nite amount of time, thus receiving an almost infinite
radiation dosage. Hence, in practice, the killing effi­
ciency should be higher even at lower field strengths.
Similar results were obtained using E. coli in a previ­
4.64
4.24
0.00E+00
4.50
4.20
4.20
4.80
After 48 Hours
5.44E+02
99.9% Killed
2.16E+02
4.20
3.51E+03
Figure 3
100% Killed
0.00E+00
6.26E+03
4.20
After 24 Hours
98.75% Killed
99.95% Killed
99.3% Killed
Model 3001 EEF Filter
ous study conducted with the EEF at the University
of Wisconsin.
Field Results in Cleanrooms
Model 3001B or Model 1001B BIO PLUS® EEFs
(Figure 3) manufactured by Technovation Systems,
Inc., were used in the cleanroom discussed here.
Special Edition: Cleaning Validation III
49
Raj Jaisinghani, Greg Smith, & Gerald Macedo
Both models have a flow rating of about 3000 scfm
without attached ductwork.
Comparison to FFU in a Converted Cleanroom
Jaisinghani et al.8 have conducted a field study com­
paring FFUs to an EEF in a small laboratory converted
to a cleanroom. This will be referred to as the “older
Encelle cleanroom.” (Encelle, Inc., Greenville, NC.)
Encelle had four conventional HEPA fan filter units
(FFUs) installed in this tissue culture laboratory, prior
to replacing these with one Model 1001 EEF. One
model 1001 provides HEPA filtered air at about the
same total flow (approx. 4250 m3/h (2500 scfm) in this
case) as the four FFUs. This allowed direct evaluation
of the effect of EEF on the bioburden in the room,
under field conditions. Airborne bioburden in the room
was reduced by as much as 75% after switching to the
EEF system. The airborne bioburden in the Class 10K
room was 0.021 cfu/ft3 (no process) and 0.392 cfu/ft3
(in process) after installation of the EEF filter.
Figure 4 shows the Federal Standard 209E, USP,
and European Union recommended airborne biobur­
den and particulate concentration for various class
cleanrooms. Clearly, from a bioburden perspective
Encelle’s older Class 10K room performs (at rest) at a
level equivalent to a Class 100 room – without incur­
ring the higher cost associated with building a Class
100 room. Most of this benefit should be attributed to
the EEF filter system.
New Class 1000 Biotech Tissue
Cul­ture Cleanroom
Facility Description
A 1,300 square foot Class 1,000 cleanroom and
900 square feet of Class 10,000 surrounding space was
constructed at Encelle, Inc., Greenville, NC facility. It
utilized eight 3001B BIO PLUS® EEF filters. The total
flow rate utilized here (22 fpm average air velocity)
was on the lower end of flow normally used in Class
1,000 cleanrooms. This room will simply be referred
to, henceforth, as “the Encelle cleanroom.”
All processing and manufacturing conducted within
the cleanroom areas are done aseptically. Workers are
gowned in sterile coveralls, shoe covers, goggles, or
face-­masks with shields, hair nets, and sterile gloves.
The Class 1K cleanroom and clean Class 10K sur­
rounding zone are cleaned daily with a monthly rotation
of sterile chemicals using cleanroom equipment and
trained personnel. Disinfectants include Hypochlor®,
Process LpH®, Process Vesphene®, and as needed, treat­
ments with a spore-killing agent called SporKlenz®.
Sampling Methods
An environmental monitoring program has been
designed to establish the standards and limits that
are acceptable to the facility management and to
regulatory agencies that will audit the manufactur­
ing within the cleanroom environments.
Daily activities for monitoring include tempera­
ture and pressure readings. Relative humidity is also
reported on sampling days.
Surface samples are collected on a routine basis
using only sterile supplies. Surfaces monitored in­clude
floors, equipment, walls, and ceilings. A five per­
cent sheep’s blood agar plate (three inch diameter)
is swabbed with a sterile, moist, cotton swab after
sampling various surface areas. Plates are labeled and
incubated at 37ºC, with five percent carbon dioxide for
72 hours. Colony forming units that grow are counted
and identified using standard microbial techniques.
Particle counting is conducted biweekly or as
needed for monitoring during critical processes. A
Biotest® APC Plus particle counter measures concen­
tration at particle sizes of 0.3, 0.5, 1.0, and 5.0µm.
Figure 4
Recommendations for Airborne Bioburden for Various Cleanroom Classes
Class
Parameter
10K
10K
1K
1K
100
100
0.5 Um particles #/ft3
<10K
CFU/ft3
0.5 Um particles #/ft3
<1K
CFU/ft3
0.5 Um particles #/ft3
<100
3
CFU/ft 50
209EEUUSP
Institute of Validation Technology
<10K
<2.83
<1K
NA
<100
<0.028 at rest; <0.283 Process
<2.5
NA
<0.1 Process
Raj Jaisinghani, Greg Smith, & Gerald Macedo
Data is collected in nearly 200 areas within the
filtered-air areas. These areas are categorized by pro­
cess and particle counts are reported to the facilities
manager for evaluation and disposition if warranted.
Data is downloaded via an RS-232 port for digital
documentation of these counts.
Microbial air sampling is performed in parallel
with particle counting to provide data on airborne
viable particulate counts and comply with Federal
Standard 209E Cleanliness classes for cleanrooms
and clean zones. A Biotest® Centrifugal Air Sampler
collects 500 liters of air in each location on sterile
tryptic soy agar strips that are designed for this type
of sampler. Strips are labeled and incubated similarly
to the surface agar plates. Classification and identifi­
cation are performed using the standards described in
the current edition of the USP.
USP standards9 for microbial growth follow in
Figure 5.
Results – Particle Concentration
The particle concentration measurements are
shown in Figure 6.
From the perspective of particle counts alone, the
design Class 1,000 cleanroom is functioning very close
Figure 5
USP Standard for Bioburden
in Cleanrooms
Class 100
Requirements
Air
Surface
Gowns
0.1 cfu/ft3
3 cfu/plate*
5 cfu/plate
Class 10,000
Requirements
Air
Surface
Gowns
0.5 cfu/ft3
5 cfu/plate (10 from floor)
10 cfu/plate
Class 100,000
Requirements
Air
Surface
Gowns
* 2 in2 surface
2.5 cfu/ft3
20/plate
30/plate
to a Class 100 cleanroom in operation. It should be
noted that the cleanroom certified as Class 100 at rest
(i.e., without personnel). Similarly, the design Class
10,000 area is functioning as a borderline Class 1,000
in operation. It should be noted again, that the airflow
rate used in this Class 1,000 cleanroom (22 fpm) was on
the low end for a normal Class 1,000 cleanroom. The
high performance of this room can be attributed to the
high degree of ceiling coverage (i.e., Airflow is highly
distributed throughout the room) which is an inherent
feature of the Technovation BIO PLUS® EEF system.
Results – Airborne Microorganisms
Figure 7 shows the results of airborne microor­
ganism monitoring in the Class 1,000 cleanroom.
The results clearly indicate that, from the perspec­
tive of airborne bioburden, the Class 1,000 area is
performing at a level that is to be expected for a Class
100 to Class 10 level. Note that (by comparing to the
particulate data in Figure 8) the airborne bioburden
does not correlate to the particulate concentration.
During the months of December and January, the
sub-micron particulate concentration actually went up
while the airborne bioburden was reduced.
Figure 8 shows the results of airborne microor­
ganism monitoring in the Class 10,000 cleanroom.
Note again that the bioburden does not relate to
the particulate concentration. One probable reason
for this is that the bacterium are larger than the size
of the sub-micron particles being monitored. Also
note that on the average the Class 10K area performs
between a USP Class 100 and a USP Class 10K from
a bioburden perspective.
Results – Surface Microorganisms
Figure 9 shows the results of surface microorgan­
ism monitoring in the Class 1,000 cleanroom.
The surface concentrations of bacteria are almost
zero throughout the cleanroom suite. This can be
attributed to:
n The cleaning protocols instituted at Encelle
n The use of the disinfecting compounds
n The low airborne bioburden
Observations from the Encelle Cleanroom Mon­
itoring
1. The Encelle cleanroom operates significantly
Special Edition: Cleaning Validation III
51
Raj Jaisinghani, Greg Smith, & Gerald Macedo
Figure 6
Particle Concentration Measurements in the Encelle
Tissue Culture Laboratory
Air Particle Concentration (per ft3)
11/14/99 11/14/99 12/1/99 12/1/99 12/31/99 12/13/99 1/6/00 1/6/00 1/28/00 12/28/00
Size – > 0.3 uM 0.5 uM 0.3 uM 0.5 uM 0.3 uM 0.5 uM 0.3 uM 0.5 uM 0.3 uM 0.5 uM
Class 10,000 Design AreasApprox.
Sq. Ft.
Mechanical Corridor
235
1959
506
786
602
1163
903
801
899
852
512
Side Corridor
555
2240
642
4559
1035
3598
762
4058
687
7989 1422
Water Filtration Area
171
3649
2598
4657
2429 11406 4312
8350 4368 6378 1295
Labware Processing
212
343
102
981
719
1233
1798
243
326
618
530
Gowning Room
139
236
87
853
1063
1506
913
2874 2808
957
235
Materials Pass Through
40
257
88
450
539
2461
2463
3887 6064 1451
595
482
671
683
1065
1187
1859
1123 2525 1014
765
Total Square Feet – > 1312
Actual Area Classification
Class 1,000 Design Areas
Specialized Cleanrooom
152
158
66
223
145
350
337
313
379
754
237
Formulations Mfg. Area
142
208
24
84
52
415
228
322
307
360
126
Product Testing Area
132
28
5
9
9
43
49
19
74
19
76
Product Finishing Area
127
264
26
129
49
474
139
400
179
472
184
Product Mfg. Area
145
293
83
250
87
534
180
639
698
829
731
Refrigerator/Freezer Storage
212
262
55
233
157
832
692
427
335
659
381
Total Square Feet – >
910
67
43
52
83
147
271
118
329
172
289
Actual Area Classification
better than the design classification although
the flow rate (average room velocity = 22 fpm)
used is at the low end of what is normally used
in such a cleanroom. This is due to the higher
distribution of flow rate – an inherent feature
of the BIO PLUS® filter system.
2. The airborne bioburden in both the Class 1K
and 10K areas is lower than what would be
expected for such rooms based on USP rec­
ommendations. The Class 1K room has the
bioburden of what would be expected (based
on USP) for a Class 100 room. Coupling this
observation to the laboratory studies on the
bactericidal properties of the EEF technology
and the direct comparison with respect to con­
ventional FFU HEPAs, it may be inferred that
52
Institute of Validation Technology
the low airborne bioburden is due to the BIO
PLUS® EEF filters.
3. The surface bio contamination is almost non
existent in the Class 1K cleanroom. This may
be attributed to the good cleaning practices
used at Encelle and due to the low airborne
bioburden in the suite.
4. The airborne bioburden seems to be lower in
the winter months, although the room tempera­
ture is held constant at 66ºF. This may be due
to lower humidity in the winter months.
New Class 10 Pharmaceutical Cleanroom
Facility Description
A 12’x20’ Class 10 cleanroom (including a
Raj Jaisinghani, Greg Smith, & Gerald Macedo
Figure 7
Airborne Bioburden in the Encelle Class 1,000 Cleanroom
New Facility’s Microbial Air Sample Results
Collected with Biotest Plus Centrifugal Air Sampler
Design Class 1,000
11/14/99
11/30/99
Average cfuAv. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*Average cfuAv. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*
Device Testing
0
0
0
0
0
0
0
0
Coating
0
0
0
0
1
0.028
0.028
0.028
Formulations
0
0
0
0
0
0
0
0
Device Manufacturing
3
0.085
0.085
0.085
2
0.057
0.057
0.057
Refrigeration
0
0
0
0
0
0
0
0
Isolation
3
0.085
0.085
0.085
15
0.425
0.425
0.425
Class 1,000 Average
1
0.028
0.028
0.028
3
0.085
0.085
0.085
Design Class 1,000
12/13/99
1/28/00
Average cfuAv. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*Average cfuAv. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*
Device Testing
0
0.000
0
0
0
0.000
0
0
Coating
0
0.000
0
0
0
0.000
0
0
Formulations
0
0.000
0
0
0
0.000
0
0
Device Manufacturing
0
0.000
0
0
0
0.000
0
0
Refrigeration
0
0.000
0
0
0
0.000
0
0
Isolation
0
0.000
0
0
0
0.000
0
0
Class 1,000 Average
0
0.000
0
0
0
0.000
0
0
The time period refers to the incubation time in hours.
4’x12’ Class 10K gowning room) was constructed at
MedPharmex in Pomona, CA using two BIO PLUS®
Model 3001B filters with eight terminal 2’x4’ HEPA
filters. The Model 3001Bs were used for the 12’x16’
Class 10 inner room. The resultant average room ve­loc­
ity in the Class 10 area was 24 fpm (4500 scfm). The
design specification for the room was Class 100. This
airflow was much lower than used in a Class 100 clean­
room – normally, with conventional single terminal
HEPAs, at least 40 fpm average room velocity is used
in a Class 100 room. However, due to the double HEPA
filter system (each Model 3001B powered Airflow
through four terminal HEPAs) the cleanroom easily
classified as Class 10 as per Federal Standard 209E.
This resulted in significant energy savings. The room
was validated for bioburden initially and then has been
shut down since the facility is now being moved to
a new location. The facility was cleaned with 0.25%
hypochlorite solution.
Sampling Methods
Air sampling was done using Tryptic Soy Agar
(TSA) and Sabouraud Dextrose Agar (SDA). The
TSA values reflect total bacterial counts while the
SDA reflects molds and yeast, although it contains
no bacterial inhibitors. In some cases Rose Bengal
Agar (RBA) was used. This reflects a better value for
molds/yeast since the RBA contains bacterial growth
inhibitors. Surface monitoring was done using 24-30
cm2 RODAC plates with TSA and SDA. The TSA
plates were incubated for a minimum of 48 hours at
32.5 +/- 2.5ºC while the SDA plates were incubated
for a minimum of 72 hours at 22.5 +/- 2.5ºC.
Results – Airborne Microorganisms
The gowning room was sampled in two zones
Special Edition: Cleaning Validation III
53
Raj Jaisinghani, Greg Smith, & Gerald Macedo
Figure 8
Airborne Bioburden in the Encelle Class 10K Area
New Facility’s Microbial Air Sample Results
Collected with Biotest Plus Centrifugal Air Sampler
Design Class 10,000
11/14/99
Average cfuAv. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*Average cfu cfu/ft3
11/30/99Average
cfu/ft3/24hr* cfu/ft3/72hr*
Water Filtration Area
30
0.850
0.850
0.850
2
0.057
0.057
0.057
Side Corridor
3
0.085
0.085
0.850
3
0.058
0.085
0.085
Manufacturing Corridor
19
0.538
0.538
0.538
11
0.312
0.312
0.312
Labware Processing
5
0.142
0.142
0.142
4
0.113
0.113
0.113
Gowning Room
0
0
0
0
3
0.085
0.085
0.085
Materials Pass Through
3
0.085
0.085
0.085
11
0.312
0.312
0.312
Class 10,000 Average
10
0.283
0.283
0.283
5.67
0.160
0.160
0.172
12/13/99
1/28/00
Design Class 10,000 Average cfuAv. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*Average cfuAv. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*
Water Filtration Area
2
0.057
0.057
0.057
2
0.057
0.057
0.057
Side Corridor
2
0.057
0.057
0.057
0
0
0
0
Manufacturing Corridor
1
0.028
0.028
0.028
0
0
0
0
Labware Processing
0
0
0
0
0
0
0
0
Gowning Room
0
0
0
0
0
0
0
0
Materials Pass Through
2
0.057
0.057
0.057
0
0
0
0
1.17
0.033
0.033
0.033
0.333
0.009
0.009
0.009
Class 1,000 Average
The time period refers to the incubation time in hours.
while the Class 10 cleanroom was sampled in five
zones. All plates (TSA and SDA) were negative
(i.e., zero counts) in all the areas. The Class 10 area
was also sampled using RBA and once again the
results were negative – zero counts.
Results – Surface Microorganisms
The surface measurements were made before and
after cleaning the newly constructed cleanroom. The
results are shown in Figure 10. The 0.25% Hypo­
chlorite cleaning is obviously very effective in elimi­
nating surface bacteria.
Observations From the Medpharmex Clean­room
Validation
1. The new Encelle Class 1000 and the Med­Pharmex
Class 10 room have about the same airflow aver­
age velocity. From the particulate point of view the
MedPharmex room operates at Class 10 simply
54
Institute of Validation Technology
because of the double HEPA filter system used.
The MedPharmex cleanroom validates as a Class
10 cleanroom, although the airflow used was lower
than what is normally used in a Class 100 room.
2. It should be noted that the MedPharmex room was
simply validated and then shut down in order to
move it to an adjacent facility, while the Encelle
room is being continuously monitored and is
operational. How­ever, from the point of view
of airborne bioburden, after the first month of
operation the Encelle Class 1000 room operates
at an equivalent level as the MedPharmex Class
10 room – with essentially zero airborne bacte­
rial counts. The low bioburden benefit to Encelle
(this Class 1000 room is operating at essentially
zero airborne bioburden) may be attributed to the
bactericidal properties of the EEF system. o
Raj Jaisinghani, Greg Smith, & Gerald Macedo
Figure 9
New Facility Surface
Contamination Summary
Design Classification
10,000
Average number of cfu/plate grown in 72 hours
Date – > 11/14/99 11/30/99 12/13/99 01/28/00
Water
Filtration Area
0
0
2
0
1
0
0
0
Side Corridor
Manufacturing Corridor 0
0
0
0
Labware Processing
1
0
0
0
Gowning Room
0
0
0
0
Materials Pass
Through
0
0
0
0
Design Classification
1,000
Average number of cfu/plate grown in 72 hours
Date – > 11/14/99 11/30/99 12/13/99 01/28/00
Device Testing
0
0
0
0
0
0
0
0
Coating
Formulations
0
0
0
0
Device Manufacturing
0
0
0
0
0
0
0
0
Refrigeration
Isolation
0
0
0
0
Control
255
210
134
104
Figure 10
Surface Bioburden in the
Class 10/10K Suite
(Counts per 25 cm RODAC Plates)
2
Area
Before CleaningAfter Cleaning
TSA SDA TSA SDA
CountsCountsCountsCounts
Gowning
Table-gowning 22
5
0
0
Wall-gowning
3
0
0
0
Class 10
Tank
0
1
0
0
Fill
21
12
0
0
Filter table
9
4
0
0
Wall
1
0
0
0
About the Authors
Rajan (Raj) Jaisinghani is a chemical engineer
with thirty years of research, product development,
and business development experience. Jaisinghani
holds a B.S. from Banaras Hindu University, India,
and an M.S., with additional graduate work, from
the Un­iver­sity of Wisconsin. Jaisinghani has extensive re­search experience in air and liquid filtration,
colloid and aerosol science, fluid mechanics, heat
transfer, and physical surface chemistry. He holds
10 patents and has many publications in technical
journals and handbooks. He can be reached by
phone at 804-744-0604, by fax at 804-744-0677, or
by e-mail at technova@sprynet.com.
Greg Smith is facilities manager at Encelle, Inc.
He holds a B.A. in Psychology from West Virginia
University and a B.S. in Chemistry from East Carolina
University. Smith has assisted in the development of
medical devices and has five years experience as
a hospital pharmacy aseptic compounding technician. He can be reached by phone at 252-355-4405
or by e-mail at gsmith@Encelle.com.
Gerald Macedo has a B.S. degree in Pharm­acy and
an M.S. in Pharmaceutical Sciences. He has over
30 years experience in pharmaceutical manufacturing, with extensive experience in the manufacture
of sterile injectables. He has ser­ved as head of
man­ufactur­ing, quality control, quality assurance,
research and development, and regulatory affairs.
Macedo currently heads Med-Pharmex, Inc., a
pharmaceutical manufacturing company. He can
be reached by phone at 909-593-7875 or by fax at
909-593-7862.
References
1. FDA. “Guideline on Sterile Drug Products by Aseptic Pro­
cessing.” Rockville, MD.
2. EU. 1998. “The Rules Governing Medicinal Products in the
EU.” Good Manufacturing Processes 4. Luxembourg.
3. Seaver, M. and Eversole, J.D. 1996. “Monitoring Biological
Aerosols Using UV Fluorescence.” Proceedings 15th Annual
Meeting AAAR. October. Orlando, FL: 270.
4. Pinnick, R.G., Chen, G., and Chang, R.K. 1996. “Aerosol
Analyzer for Rapid Measurements of the Fluorescence Species
of Airborne Bacteria Excited with a Conditionally Fired Pulsed
266 nm Laser.” Proceedings 15th Annual Meeting AAAR.
October. Orlando, FL.
5. Rhodes, W.W., Rinaldi, M.G., and Gorman, G.W. 1995.
“Reduction and Growth Inhibition of Microorganisms in
Commercial and Institutional Environments.” Environmental
Health 12 (October).
6. Tolliver, D.I. 1988. “Domestic and International Issues in
Contamination Control Technologies.” Microcontamination 6,
no. 2 (February).
7. Jaisinghani, R. A. U.S. Patent 543,383. 4 April 1995.
8. Jaisinghani, R.A., Inzana, T.J., and Glindemann, G. 1998.
“New Bactericidal Electrically Enhanced Filtration System
for Cleanrooms.” Paper presented at the IEST 44th Annual
Technical Meeting. April. Phoenix, AZ.
9. “Microbial Evaluation of Cleanrooms and Other Controlled
Environments.” United States Pharmacopoeia, <1116>, p. 20992106.
Special Edition: Cleaning Validation III
55
A Cleaning Validation Program
for the ELIFA System
By LeeAnne Macaulay, Jeff Morier, Patti Hosler,
& Danuta Kierek-Jaszczuk, Ph.D.
Cangene Corporation
v
T
he Enzyme-Linked Immuno­
filtration Assay (ELIFA)
provides high sensitivity of
de­tection with rapid results. For this
reason we developed a very sensitive,
semi-quantitative ELIFA to determine
IgA in therapeutic Win­Rho SDF™
im­munoglobulin. In the course of the
development we no­ticed that non-uni­
form and unusually high background
(blank) re­sponses, that occurred infre­
quently, greatly interfered with the
test results obtained. We hypothe­
sized that such background responses
resulted from inadequate cleaning of
the ELIFA apparatus. Accordingly,
a cleaning program for the apparatus
has been devised and validated. In this
paper the results supporting the hypo­
thesis will be presented, and the ratio­
nale and core aspects of the developed
program delineated.
port individual CVPs and enforce
compliance. The guidelines and pro­
}The
grams may cover a plethora of dif­
establishment
ferent types of equipment but they
usually refer to equipment used in
of Cleaning
the manufacture, processing, hold­
Validation
ing, filling, and packaging of raw
Programs (CVP) materials, inter­mediate/­final prod­
ucts, and associated components. The
in the
guidelines do not refer to equipment
pharmaceutical used in Research and Development
(R&D), and to our understanding,
industry is
there is no regulatory requirement
dictated by the for the development of CVPs for
equipment used in these areas. The
regulatory
Easy-Titer™ ELIFA system2 is a
small, micro­titer format compatible
requirements
apparatus developed and manufac­
to develop
tured by Pierce Chemical Company.
As shown in Figure 1 (adapted from
and observe,
Product Instructions, Pierce Chemical
in a fully
Company) the apparatus utilizes a
documented way, nitrocellulose mem­brane (NC) sand­
Cleaning Validation
wiched be­tween the sample appli­
effective
cleaning
Programs for Research
cation plate and vacuum collection
and Development?
chamber. Similar to the widely used
procedures.~
Enzyme-Linked Immunosorbent
The establishment of Cleaning
Assay (ELISA), the ELIFA is an
Validation Pro­grams (CVP) in the pharmaceutical immunoassay well suited for testing of multiple sam­
industry is dictated by the regulatory requirements ples over a range of serial dilutions.3 In the ELIFA,
the immunological reaction between NC immobilized
to develop and observe, in a fully documented way,
effective cleaning procedures. Regulatory guidelines ligand and ligand-specific analyte in the test sample
followed by an enzymatic reaction with a chromo­
for validation of cleaning pro­cesses1 are meant to sup­
56
Institute of Validation Technology
LeeAnne Macaulay, Jeff Morier, Patti Hosler, & Danuta Kierek-Jaszczuk, Ph.D.
Figure 1
Exploded View of the Easy-Titer™ Elifa System
Thumb
Screws
Sample
Application
Plate
Nitrocellulose
Clamp
Microtiter
Plate
Vacuum
Relief Valve
Pump
Tubing
Port
Gaskets
Tubing
Position Stops
(Acrylic Balls)
Transfer
Cannula
Collection
Chamber
Membrane
Support
Plate
Guide Pins
genic substrate gives rise to colored dots. The color
intensity of the individual dots varies proportionally
to the amount of the analyte in the samples and dots
produced by the samples devoid of analyte (blanks or
background) are very pale or even colorless. We used
the ELIFA system to research and develop a screen­
ing assay for human IgA.4 The developed IgA ELIFA
will be used for testing of the licensed WinRho SDF™
therapeutic, hence, its performance characteristics need
to be established and validated. In pre-validation stud­
ies, however, we observed that the developed ELIFA
lacked reproducibility. The color of the blank dots var­
ied sometimes from experiment to experiment or, even
within the same experiment, from well to well. We also
observed that the color of dots produced by the repli­
cated test samples occasionally varied. We postulated
that the observed variability is a result of external con­
tamination carried over from previous experiment(s).
An inadequately cleaned ELIFA apparatus would then
be the cause of obscured test results. We, therefore,
decided to develop a CVP for the ELIFA apparatus
before proceeding to assay validation.
ELIFA CVP – Approaches and Hallmarks
A body of experience at Cangene with valida­
tion5,6 or cleaning7 programs, as well as manufac­
turer’s cleaning instructions for the ELIFA system
(Figure 2, adapted from Product Instructions, Pierce
Chemical Company) was the foundation when devel­
oping the ELIFA CVP. Among others, the devel­
oped program addressed the following:
nS
pecific design of apparatus, its individual parts
and accessories that require cleaning
n Disassembling and re-assembling the unit
before and after cleaning
Figure 2
Cleaning of the Easy-Titer™
ELIFA System
Clean all of the pieces to the Easy-Titer™ ELIFA
System unit in a two percent PCC-54 solution and
then rinse with distilled water. The unit may also
be soaked in the PCC-54 solution to remove stains
from the unit caused by the substrate solution.
Special Edition: Cleaning Validation III
57
LeeAnne Macaulay, Jeff Morier, Patti Hosler, & Danuta Kierek-Jaszczuk, Ph.D.
nC
leaning operations
n Cleaning procedures including cleaning ves­
sels, agents and utensils
n Compatibility of cleaning agents with equip­
ment and assay
n Decontaminating abilities of cleaning agents
n Sampling on cleaned equipment
n Analytical methods for monitoring of cleaning
processes
n Storage of cleaned parts
n Inspection of apparatus for cleanliness before use
n Recording and documenting the cleaning pro­
cedures
n Establishing acceptance criteria, and
n Maintaining cleaning records
Strategy for Validation of
Cleaning Procedures
Two cleaning procedures (procedure 1 and 2 in
Figure 3) utilizing either enzyme or detergent-based
cleaning agents were developed and tested in conjunc­
Figure 3
Procedure 1
tion with two ELIFA experiments; the standard IgA
ELIFA and the mock ELIFA. Such a combination of
analytical methods allowed for instantaneous moni­
toring of the effectiveness of the cleaning pro­cess.
Standard IgA ELIFA method4 involved testing 96
replicates of a sample at a high (worst-case condition)
IgA concentration, which were applied into 96 wells of
the ELIFA apparatus. As expected, these experiments
invariably produced highly colored dots (Figure 4). A
tested cleaning regimen (procedure 1 or 2 in Figure
3) followed by the second mock experiment was then
executed. The mock ex­periment involved the use of
the diluting buffer in lieu of a sample with high IgA
concentration that was also applied into 96 wells of
the ELIFA apparatus. Providing that the cleaning regi­
men was effective, the mock experiment should not
produce colored dots, as there was no specific analyte
that could attach to the immobilized ligand to facilitate
subsequent enzymatic and color reactions. The results
ob­tained show that whereas Procedure 1 did not
re­move the contaminants from preceding experiments
well enough (Figure 5), procedure 2 was fully effec­
Cleaning Procedures 1 and 2
Procedure 2
Disassemble the unit by first removing the thumb
Disassemble the unit by first removing the thumb screws
screws on the top of the sample application plate, then located on the top of the sample application plate, then
removing the application plate and top gasket, and removing the application plate and top gasket, and
finally unclamping the membrane support plate from finally unclamping the membrane support plate from
the collection chamber.
the collection chamber.
Rinse all parts for two minutes under running Reverse Rinse all parts for two minutes under running RO water.
Osmosis (RO) water.
Immerse them into a vessel with two percent TERG-A- Immerse them into a vessel with five percent RBS10 solution
ZYME (Alconox Inc., New York, NY, U.S.A.) solution and at 50ºC and wash for five minutes by agitating the vessel.
wash for five minutes by agitating the vessel.
Rinse all parts for two minutes under RO water.
Rinse all parts for two minutes under RO water.
Clean all 96 wells of the sample application plate with Clean all 96 wells of the sample application plate with
TOC swabs by dipping the swabs into the detergent TOC swabs by dipping the swabs into the detergent
solution, inserting them into wells once from top and solution, inserting them into wells once from the top and
once from the bottom, and swabbing the inner part of once from the bottom, and swabbing the inner part of
each well by turning the swab first to the right and
each well by turning the swab first to the right and
then to the left.
then to the left.
Clean all 96 wells of the top gasket in a similar way. Clean all 96 wells of the top gasket in a similar way.
Raise all 96 cannulas on the membrane support plate Raise all 96 cannulas on the membrane support plate and
and soak the plate for five minutes in the detergent clean them with TOC swabs by dipping the swabs into the
solution.detergent solution and swabbing the surface of individual cannulas and also spaces between cannulas and bottom gaskets.
Rinse each part and the spaces between the bottom Rinse each part and the spaces between the bottom
gasket and the membrane support plate for two
gasket and the membrane support plate for two seconds
seconds under running RO water.
under running RO water.
58
Institute of Validation Technology
LeeAnne Macaulay, Jeff Morier, Patti Hosler, & Danuta Kierek-Jaszczuk, Ph.D.
Figure 4
IgA ELIFA Results Obtained for a
Test Sample Containing Human
IgA at a Concentration of 2µg/mL
Figure 5
IgA ELIFA Results Obtained for a
Replicated Test Sample Deprived
of Human IgA.
The experiment was performed in an apparatus
cleaned with TERG-A-ZYME (Procedure 1).
Figure 6
IgA ELIFA Results Obtained for a
Replicated Test Sample Deprived
of Human IgA.
tive (Figure 6). Procedure 2 was then validated, in two
independent experiments performed by two analysts.
It was shown that it invariably leads to results similar
to those presented in Figure 6.
Assessment of the Effectiveness
of the Validated Procedure
The Total Organic Carbon (TOC) method is
widely utilized in industrial CVPs as it measures
low levels of carbon and is compatible with swab
sampling techniques. The standard IgA ELIFA4 fol­
lowed by the validated cleaning procedure and swab
sampling of the surface of three randomly selected
wells were, therefore, used to assess the cleanliness
of the apparatus by standard TOC. A procedure used
at Cangene8 was followed. The results obtained con­
firm that the validated cleaning procedure was fully
effective as the carbon concentration determined in
The experiment was performed in an apparatus
cleaned with RBS (Procedure 2).
Figure 7
Results From Total Organic Carbon Analysis (in ppb)
SampleSampleReplicaReplicaReplicaAverageSD
Percent
NumberName
1
2
3CV
1
2
3
4
Well B11
Well D8
Well G2
Water
277.8
235.4
228.4
185.2
271.7
234.3
219.1
165.4
268.9
225.9
255.3
167.4
272.8
231.8
234.3
172.7
4.55
5.20
4.73
10.9
Special Edition: Cleaning Validation III
1.67
2.24
8.03
6.29
59
LeeAnne Macaulay, Jeff Morier, Patti Hosler, & Danuta Kierek-Jaszczuk, Ph.D.
water extracts of test samples was only slightly great­
er than that of water used for extraction (Figure 7).
Implementing of the ELIFA CVP
The validated ELIFA cleaning procedure will
become part of a written Standard Operating Procedure
(SOP). Although addressing R&D instrumentation, the
SOP document will detail the activities that were con­
ducted by adhering to industrial standards for cleaning
validation.1,9 The document will also advise on safety
precautions, cleaning schedule, and assignment of
responsibility for cleaning and storage of the cleaned
apparatus. The SOP document will be observed not
only when validating the performance of the IgA
ELIFA, but also during routine use of the ELIFA sys­
tem. It will be the subject of a periodic evaluation and,
if deemed necessary, be updated and/or revised.
Conclusions
nA
comprehensive, credible CVP designed and
developed at Cangene for the Easy-Titer™
ELIFA system has been shown to effectively
remove contaminants and residues entrapped
in the apparatus after the conclusion of the
ex­periment(s) and/or subsequent cleaning.
n The CVP has been demonstrated to vastly
re­duce the analytical background of the IgA
ELIFA, improve its signal to background ratio,
increase the quality of the test results and may,
therefore, be expected to notably support the
upcoming assay validation.
n The CVP, by virtue of anti-viral and anti-bacte­
rial properties of the RBS10, allows for simulta­
neous decontamination and sanitization of the
ELIFA unit, thus facilitating its safe use with
infectious samples.
n The CVPs generated for R&D equipment
that fulfill the standards of industrial cleaning
validation not only improve the quality of the
as­says utilizing this equipment but may become
vital components of assay validation. o
About the Authors
LeeAnne Macaulay is a Technician at Cangene
Corporation. She completed the first year towards
60
Institute of Validation Technology
a B.Sc. degree at the University of Winnipeg and
received a diploma in Chemical and Bioscience
Technology from Red River College in Winnipeg.
She has experience in QC/QA Laboratories in the
areas of microbiology and biochemistry.
Jeff Morier is a Senior Assay Development Tech­
nologist at Cangene Corporation. He received his
B.Sc. degree in Microbiology from the University
of Manitoba. He has seven years experience in the
pharmaceutical industry in the areas of QC microbiology, QA biotechnology, and R&D experience in the
validation of immunoassays of various formats.
Patti Hosler is a Technician at Cangene Corporation.
She completed the first year of a B.Sc. degree program at Brandon University and received a diploma in
Chemical and Bioscience Technology from Red River
College. She has seven years experience as a QA/QC
laboratory technician in the food production industry.
Danuta W. Kierek-Jaszczuk is a Senior Research
Scientist/Assay Development Supervisor at Cangene
Corporation. She obtained her M.S. degree in
Biology from the Nicolaus Copernicus University,
and a Ph.D. degree in Agricultural Sciences from
the Polish Academy of Sciences Institute of Genetics
and Animal Breeding. She can be reached by
phone at 204-275-4263, by fax at 204-269-7003,
and by e-mail at dkjaszcz@cangene.com.
References
1. FDA. 1993. “Guideline to Inspection of Validation of Cleaning Pro­
cesses.” Office of Regulatory Affairs, USFDA, Washington, D.C.
2. Pierce Chemical Company. Product Instructions, Easy-Titer™
ELIFA System. Rockford, IL.
3. Paffard, S.M., Miles, R.J., Clark, C.R., and Price, R.G. 1996.
“A Rapid and Sensitive Enzyme Linked Immunofilter Assay
(ELIFA) for Whole Bacterial Cells.” Journal of Immunological
Methods 192, no. 1–2: 133-6.
4. Morier, J., Macaulay, L., and Kierek-Jaszczuk, D. “Screening for
the Presence of Human IgA in a Hyper Immune Product Using An
Enzyme-Linked ImmunoFiltration Assay.” Poster Presentation at
IBC Conference on Assay Development for Future High-Throughput
Screening. 8 – 9 November 1999. Annapolis, MD.
5. Faurschou, A. 2000. General Procedure for Validation Program.
SOP Document # 11.001.0001.RR. Cangene Corporation.
Winnipeg, MB, Canada.
6. Alejo, M. and Faurschou, A. 1998. Process Validation Qualification.
SOP Document # 11.001.0002.RR. Cangene Corp­oration.
Winnipeg, MB, Canada.
7. Heise, R. and Poschner, E. 1999. Manual Cleaning and Sanitizing
Equipment. SOP Document # 2.010.0017.RR, Cangene Corp­
oration. Winnipeg, MB, Canada.
8. Shinkarik, T. 1998. Surface Sampling for Total Organic Carbon
(TOC). SOP Document # 500602.RR, Cangene Corporation.
Winnipeg, MB, Canada.
9. Chudzik, G.M. 1998. “General Guide to Recovery Studies Using
Swab Sampling Methods For Cleaning Validation.” Journal of
Validation Technology 5, no. 1: 77-81.
10. Pierce Chemical Company. Product Information, RBS. Rockford, IL.
A Cleaning Validation Master
Plan for Oral Solid
Dose Pharmaceutical
Manufacturing Equipment
By Julie A. Thomas
McNeil Consumer Healthcare
W
v
ith the benchmark con­
Validations of Clean­ing Processes
stantly being raised,
– July 1993.” Each of these will be
}Often,
many companies find
discussed in greater detail in the sec­
companies have tions below.
that they are in perpetual valida­
tion mode. Often, companies have
executed
executed validations for equipment,
n Objective
cleaning, and processes, but the
n Scope
validations for
doc­­umentation no longer stands up
n Introduction
to the latest in validation standards.
n Responsibilities
equipment,
Although these validations are gen­
n Philosophy
cleaning,
and
proerally complete and on file, there
n Methodology
are many opportunities to improve
n Schedule
cesses,
but
the
docboth the supporting documenta­
Objective
tion and the execution. One way to
umentation
en­sure that your company’s policies
no longer stands
This section should state the pur­
and procedures regarding cleaning
pose of your cleaning master valida­
validation are state-of-the-art is to
up to the latest tion plan and define whether you will
assemble a multi-disciplined team
be revalidating current procedures or
from the appropriate manufacturing
validation
prospectively validating new ones.
sites that can review and revise all
Often, the plan will have provisions
components associated with clean­
standards.~
for both situations.
ing validation. What follows are
ex­cerpts from a Cleaning Validation
Scope
Master Plan (the Plan) that was painstakingly com­
posed and has now be­come the standard for planning
The scope needs to list exactly which aspects of val­
and executing cleaning validations at several manu­
idation will be covered in the document and to which
facturing sites.
types of products and/or processes the Plan applies. For
An outline of the Plan contains the following seven
elements, the concepts of which are taken directly example, “This document provides steps for planning,
executing, and maintaining equipment cleaning valida­
from the FDA publication, “Guide to Inspections of
Special Edition: Cleaning Validation III
61
Julie A. Thomas
tions for oral solid dose products at Your Company’s
manufacturing facility in Your City, State.”
Introduction
The introduction should let the reader know
what elements will be addressed in the Master
Validation Plan and why a formal plan is necessary.
For in­stance, “This plan is intended to be a roadmap
clarifying the course the Company will take as it
plans and executes the cleaning validations required
by current Good Manufacturing Practices (cGMP).
This program describes and defines the various
categories of cleaning validation, provides the nec­
essary protocol elements, and offers guidance for
un­ex­pected results. Furthermore, it includes provi­
sions for revalidation and monitoring and serves as
a mechanism to organize and store critical informa­
tion that supports the cleaning validation process.”
Responsibilities
There are many departments and disciplines
involved in planning for and executing a cleaning
validation. It is necessary to list each contributing
area and the associated tasks for which it is respon­
sible. This serves to clarify roles and to ensure that
tasks are not overlooked. Typically, representatives
from Validation, Manufacturing, Quality Control,
En­gineer­ing, and Research and Development (R&D)
will be needed. The following are some examples of
departmental responsibilities:
Validation Specialist
• Review cleaning procedures
• Assist the cleaning validation team in iden­
tifying equipment test sites for swab or
rinse samples
• Write cleaning validation protocols
• Coordinate execution of the cleaning pro­
cess with the appropriate departments and
laboratories
• Prepare the sampling schedule
• Assemble the test data into final report
form for approval
Manufacturing
• Provide technical information for the devel­
opment of protocols and reports
• Review and approve protocols and reports
62
Institute of Validation Technology
for accuracy and agreement with operating
practices
• Create and/or revise related SOPs and
cleaning checklists
• Perform cleaning processes per SOP as
referenced in the validation protocol
• Provide documented training for all person­
nel responsible for cleaning the equipment
Quality Assurance
• Review and approve protocols and reports
for conformance with cGMPs and internal
procedures
• Provide analytical technical support
• Provide documented training for all person­
nel responsible for sample collection and
testing
• Collect analytical samples as specified in
the protocol
• Perform analytical testing using validated
procedures
• Label, package, and send out those samples
that need to be analyzed by an external
laboratory
• Review and approve analytical results
• Notify departments of test results
Engineering
• Inform the affected department in advance
of any anticipated change to the facility or
equipment
• Include all utilities and cleaning equipment
in the calibration and maintenance pro­
gram
• Review and approve equipment drawings
and surface area calculations
Research and Development
• Provide swab and surface recovery data for
active ingredients and cleaning agents
• Validate analytical test methods for chemi­
cal and cleaning agent analyses
• Transfer validated methods to the site QC
laboratories and/or contract laboratories
• Provide recommended cleaning procedures
for new active ingredients and/or cleaning
agents
Cleaning Validation Philosophy
This section discusses the considerations you
Julie A. Thomas
have made in developing a comprehensive cleaning
validation program, such as how to define equipment
holding time, equipment storage time, and campaign
length. In general, the philosophy section presents the
Company’s position on what is being achieved by the
cleaning validation and how it will be demonstrated.
For instance, “Cleaning validation is required for all
manufacturing and packaging equipment that comes
into contact with the product or product components
during production. Prior to validation, acceptance
criteria will be developed for active ingredient and
cleaning agent residues. Verification of acceptable
equipment holding time will be included as part of
the validation. Holding time is defined as the time
between the end of the last product manufactured and
the start of the cleaning process. This will demonstrate
that the cleaning procedure effectively removes resi­
due after the equipment has remained idle for a speci­
fied period of time. Additionally, holding time will be
evaluated to ensure storage conditions are adequate
for a predetermined length of time. Storage time is
defined as the time between cleaning completion and
the next batch processed on the equipment. Campaign
length will be determined jointly by Operations and
R&D and validated with at least three iterations using
the maximum number of batches or maximum length
of time. This approach fully challenges the cleaning
procedure by providing worst-case residues.”
Cleaning Validation Methodology
To ensure all of the elements are in place for a
thorough and successful validation, a chronological
methodology should be followed. One such design is
illustrated through the following four phases: devel­
opment, planning, execution, and maintenance. (See
Figure 1) In this section of the Plan, it is appropriate
to include the number of sampling/testing iterations
required for each piece of equipment and/or each
analyte. (See Figure 2.)
If you intend to reduce the number of tests
re­quired to validate cleaning after various products
by using a grouping approach, it should be explained
in this section.1
Development Phase
The initial phase of the cleaning validation plan is
preparatory and includes analytical methods valida­
tion, recovery studies, surface types, degradants, and
methods transfer. There is a considerable amount of
scientific activity that must be completed before the
validation can begin. These steps are explored in the
following sections.
1. Analytical Methods Validation
Describe how the analytical methods will be
developed and validated for active ingredients, deg­
radants (if applicable), and cleaning agent residue.
Validation of the method should assess reproducibil­
ity, linearity, specificity, limit of detection (LOD),
and swab and surface recovery. Other elements for
consideration are the instrumentation, swabbing and
dilution solvents, dilution volume, and sample han­
dling and storage.2,3
2. Recovery Studies
Recovery studies evaluate quantitative recovery
of chemical residue from both the surface to be
sampled and the swab material to be used for sam­
pling. The results confirm the appropriateness of
the sampling method and material used. You should
determine the minimum recovery criteria for each
surface type and state that percentage in this sec­
tion. For instance, you may want recovery values of
at least 70% of actual readily soluble residues, but
may choose a much lower recovery value for rela­
tively insoluble proteins.4 Most important, you must
provide data to justify the chosen value.
3. Surface Types
Since different surface types have different affini­
ties, you may want to choose a few surface materials
to represent the many product contact surfaces used
in manufacturing. For oral solid dose manufactur­
ing, you may determine that stainless steel, silicone,
and polypropylene are the most abundant surfaces
and that they also provide varying degrees of poros­
ity. A matrix of all surface types and the representa­
tive material that will be used in recovery studies is
appropriate. (See Figure 3)
4. Degradants
Many degradant products are more soluble in the
cleaning solvent than are the active ingredients; there­­
fore, you should determine the degree of degradant
Special Edition: Cleaning Validation III
63
Julie A. Thomas
Figure 1
Cleaning Validation Flow Diagram
Development Phase
Analytical Method
Validation
Analytical Method
Development
•D
egradant
identification
• Transfer
• Recovery
• Surface types
Planning Phase
Equipment
•S
ample site
selection
• Surface area
calculation
• Schematic
Analyte Selection
and Acceptance
Criteria
Protocol
Development
rite
•W
• Approve
• Train
ctive ingredient
•A
• Cleaning agent
Cleaning
SOP
rite
•W
• Approve
• Train
Execution Phase
Protocol Execution
•C
lean
• Sample
• Test
Pass?
No
Incident
Investigation
Yes
Validation Report
•W
rite
• Approve
Maintenance Phase
Monitoring
Change Control
Revalidation
64
Institute of Validation Technology
Julie A. Thomas
Figure 2
Cleaning Iteration Summary Requirements
SampleTotal Number of IterationsConditions
Active Residue
3
1 at maximum campaign length or
maximum time period plus holding
time.
2 at maximum campaign length or
time period.
Cleaning Agent Residue
3
3 per cleaning procedure, per piece
of equipment.
testing required based on the toxicity and solubil­
ity of potential degradants. Likewise, active ingre­
dients should be exposed to the selected cleaning
agent under normal usage conditions to determine
if degradants are formed as a result of the cleaning
process.
5. Analytical Methods Transfer
In this section, you can state how sampling and
analytical methods will be transferred from the
R&D laboratories to the site QC laboratories and
how the analysts conducting validation testing will
be qualified. Reference appropriate SOPs and/or
De­velopment Transfer Report.
Planning Phase
The next phase of preparation is the planning phase.
This is a broad category that focuses on equipment
information, analyte selection, acceptance criteria,
cleaning procedures, and protocol development. At this
point, you are starting to think about what equipment
will be included in the validation, which analytes will
be chosen, and how you will determine acceptance cri­
teria. This leads to an in-depth review of the procedures
and, finally, to protocol development.
Figure 3
Recovery Surface:
1. Equipment Information
This section should detail the methodology for
providing specific equipment information. One
option is to prepare a binder containing detailed
surface area calculations, swab sampling sites (with
justification), photos, and schematic diagrams for
each piece of equipment. This binder can be main­
tained separately and used as an attachment to the
cleaning validation protocol as needed.
a) Sample Site Selection
Explain how you will select sampling sites to rep­
resent the product contact surface area of the equip­
ment. One of the best sources of information is the
operator who routinely cleans the equipment. He or
she can certainly point out the areas they find most
difficult to clean. Make the operator part of a larger
team of experts to include representatives from
Validation, QA, and Operations, and let the team
determine the product contact surface areas that are
most difficult to clean and those that are most repre­
sentative of the equipment. Sampling these sites will
represent the entire equipment surface area using
the assumption that residue will be evenly distrib­
uted over the equipment and that the most difficult
to clean locations will represent the worst case for
residue removal. Include the basis for selecting each
Surface Recovery Matrix
316L Stainless Steel
PolyethyleneSilicone
Material Used:
316L Coupon
Plastic Bulk Container
Hose
To Represent:
304 Stainless
Aluminum
Brass
Teflon
Lexan
HDPE
Rubber
EPDM
Neoprene
Special Edition: Cleaning Validation III
65
Julie A. Thomas
site. For example, sampling sites may be deemed to
be the most difficult to clean, most difficult to dry,
or of different material of construction. Swab sites
Figure 4
Kason Separator
Swab Site
can be indicated with either digital photographs or
suitable diagrams. (See Figure 4)
b) Surface Area Calculation
An accurate surface area must be calculated for
each piece or section of equipment. This can be
done with manufacturer’s drawings, but should be
confirmed by field measurements. If drawings are
not available, the equipment must be measured to
determine surface area (see Figure 5). Although not
shown here, it is advisable to include the calcula­
Figure 5
Surface Area
Swab NumberArea Swabbed
1
Screen/ring interface
gasket
2
Discharge port – inside
of top circular area
(top seam)
Total contact S.A. of Kason Separator (in )
2
Total contact S.A. of Filter Socks (in2)
3,171.2
15.6
tions with the schematic diagram in the equipment
information binder mentioned above.
c) Schematic Diagram
To clearly illustrate each piece of equipment, pre­
66
Figure 6
Institute of Validation Technology
pare schematic diagrams labeled with the major sec­
tions of the equipment. (See Figure 6) The drawings
do not have to be to scale, but should appropriately
represent the equipment. If a schematic is not practical
(i.e., a packaging line), a photograph is acceptable. The
intent is to depict the product contact surfaces that are
included in the calculations. This helps to ensure that
the swab samples are taken from the intended location.
2. Analyte Selection
Analyte selection is required for active, excipi­
ent (possibly), and cleaning agent residues. Keep
in mind that you are validating a cleaning proce­
dure, not a manufacturing process. In the situation
where the same cleaning procedure is used for many
product formulas, there is an opportunity to select a
representative analyte to cover multiple active ingre­
dients and reduce the amount of testing.
a) Actives
If several active ingredients are processed in a single
piece of equipment, a marker active, or guiding sub­
stance, can be selected based on the active ingredient
solubility in water, potency, previous production expe­
rience, and R&D studies. This reduces the number of
studies required to validate the cleaning procedure.5
b) Excipients
Julie A. Thomas
The removal of excipients can either be con­
firmed by visual inspection or through analytical
testing. The approach should be stated here along
with training requirements for individuals perform­
ing visual inspection.
c) Cleaning Agents
Testing for cleaning agent residue is essential but
is often an area in which current cleaning validations
are deficient. For most cleaning agents, a marker
compound can be selected for analysis based on the
recommendation of the cleaning agent manufactur­
er. Removal of volatile cleaning agents that do not
leave a residue, such as isopropyl alcohol, may not
need to be validated.
3. Acceptance Criteria
The equipment must pass visual and olfac­
tory inspection, where appropriate, as defined in
the cleaning validation protocol prior to initiation
of swabbing.6 This is a critical step in the validation
process that, if skipped, can lead to failed results.
a) Active Ingredient
Acceptance criteria for active ingredients should
be based on medical and pharmacological properties
and scientific information. Calculations using the
maximum allowable carryover (MAC) and/or 10ppm
formulas can be used.7
To ensure that all active contact surfaces are consid­
ered in the carryover calculation, you may want to iden­
tify equipment trains. Acceptance criteria are calculated
using the surface area from the entire equipment train;
however, protocols are executed per each piece of equip­
ment. Equipment trains could be designated as follows:
n Granulation – granulator system through the
product container
n Compression through printing – compression,
film-coating, and printing phases
n Packaging – product contact surfaces for each
type of packaging line
b) Cleaning Agent
Acceptance criteria for the cleaning agent marker
should be based on toxicity, limit of detection of
validated assay method, and/or data gathered dur­
ing certification studies. Acceptance criteria can be
calculated using a formula such as the No Observed
Effect Limits (NOEL).8
4. Cleaning Procedures
This section should indicate that cleaning procedures
will be developed (or existing procedures reviewed)
prior to the validation. It should also list the required
elements for cleaning procedures, such as temperature,
pressure, water quality, cleaning agent concentration,
spray nozzle location, etc., or it should reference where
these requirements can be found.9 Additionally, you
should describe the process for training the operators
who will be executing the validation studies.10
5. Protocol Development
The next step is to write a cleaning validation pro­
tocol for each cleaning procedure that you intend to
validate. The protocol should describe all documenta­
tion required to complete the cleaning validation. It
should also present the rationale for using a marker
active to cover validation for multiple products. For
ease of review, include a matrix of the products and
equipment that are covered by each validation, or
reference where this information can be found. For
example, if there are three active ingredients processed
in Fluid Bed Granulator #1, indicate which active will
be used to represent the other two. Likewise, indicate
which pieces of equipment will be used to validate
Figure 7
Equipment Cleaning Matrix
Active AActive BActive CCleaning
Agent A
Fluid Bed Gran 1
Fluid Bed Gran 2
Starch Kettle 1
X
–
–
–
–
–
–
–
–
X
–
X
the removal of active ingredient and cleaning agent
residues. (See Figure 7)
Execution Phase
When all of the supporting documentation is com­
plete, it is time to execute the plan. During the execu­
tion phase, you will complete the protocol, investi­
gate any nonconformances that may have occurred,
and write a report to summarize your findings.
1. Protocol Execution
Special Edition: Cleaning Validation III
67
Julie A. Thomas
Typically, three iterations of cleaning, sampling,
and testing using the same procedure are required.
Acceptance criteria for all cleaning iterations must
be met for both the active ingredient and the clean­
ing agent. Be sure to reference the procedure where
a detailed description of the chemical swab prepara­
tion and sampling methods can be found.
2. Incident Investigation
This section explains how the Company will
handle test failures and nonconformances during
execution of the validation. Once the root cause of
the failure has been identified, options are to addend
the protocol or start over with a new protocol. For
any incident that occurs during validation, docu­
ment the investigation along with corrective and
preventive actions. The incident report may contain
elements such as:
n Cleaning validation protocol number
n Incident report number
n Equipment model and location
n Initiator and date
n Incident description
n Root cause analysis
n Corrective actions recommended/taken
n Assessment of effect on product
3. Reports
Describe the report format and content that will
be used to summarize the validation. Reference
appropriate SOPs for detailed report information.
An explanation of all deviations should be included
in the validation report.
Maintenance Phase
The final phase of the Plan should specify how
you will maintain the conditions you have just
validated. This includes periodic monitoring, using
a control of change process, and potentially, revali­
dating.
1. Monitoring
This section details how you will ensure that the
conditions used during validation remain in con­
trol during routine production. This is especially
important for manual cleaning procedures, where
68
Institute of Validation Technology
repeatability is highly dependent on the quality and
consistency of training. Monitoring should include,
at a minimum, a review of changes made to the
cleaning procedure or equipment, visual inspection
of the equipment, and direct observation of employ­
ees executing the cleaning procedure. For some
equipment, swab samples for active ingredients may
be necessary in addition to the visual inspection
and observation. Indicate the frequency that you
intend to monitor the cleaning process. Reference
the appropriate SOP for detailed requirements of the
monitoring program.
2. Change Control
Indicate how changes will be managed to ensure
the validated state is maintained. Any change in
the facility, process equipment, cleaning procedure,
cleaning agent, product formulation, or addition
of new products to the equipment train should be
documented and approved via the Change Control
System. The change should be reviewed by the Val­
idation Group, Operations, and QA, who will decide
if revalidation is necessary. Reference appropriate
SOPs.11
3. Revalidation
Indicate the criteria that will be used to determine
the need for revalidation. Based on the nature of the
change, a determination will be made as to which,
if any, phases of the validation must be repeated.
Ref­erence where documentation of the revalidation
will be filed.12,13
Cleaning Validation Schedule
Prioritization
As is usually the case, all cleaning validations
cannot commence at one time; therefore, it is nec­
essary to set up a priority list. Some situations to
consider are:
n Equipment shared between products contain­
ing different active ingredients
n Equipment in contact with raw material with
high potential for contamination
n Unshared primary equipment currently in use
with outdated validations
n Unshared auxiliary equipment used for pro­
Julie A. Thomas
duction with limited potential for product
contamination
Tactical Schedule
A proposed schedule, including equipment pri­
oritization and target initiation dates, should be pre­
sented in this section. This gives an indication that
you have contemplated the order of execution, and
it also provides a tool that can be used to track your
progress.
Summary
There are many aspects of cleaning validation
that must be carefully planned to guarantee a suc­
cessful validation program. If you begin with a phi­
losophy, this will set the stage for you to develop a
structured approach. By dividing the approach into
sections, such as development, planning, execu­
tion, and maintenance, it breaks down the project
into manageable segments. To complete the Plan,
generate a tactical schedule and begin monitoring
pro­g­ress towards your new and improved cleaning
validation status. o
About the Author
References
1.Jenkins, K.M. and Vanderwielen, A.J. “Cleaning Validation: An
Overall Perspective,” Pharmaceutical Technology, April 1994,
p. 62.
2.McCormick, P.Y. and Cullen, L.F., Pharmaceutical Process
Validation, 2nd ed., edited by I.R. Berry and R.A. Nash, 1993,
p. 334.
3.Kirsch, R.B., “Validation of Analytical Methods Used in
Pharmaceutical Cleaning Assessment and Validation,”
Pharmaceutical Technology, Analytical Validation, 1998.
4.Chudzik, G.M., “General Guide to Recovery Studies Using
Swab Sampling Methods for Cleaning Validation,” Journal of
Validation Technology, Vol. 5, No. 1, pp. 77-81.
5.Hall, W.E., “Your Cleaning Program: Is It Ready for the PreApproval Inspection?” Journal of Validation Technology, Vol.
4, No. 4, August 1998, p. 302.
6.Alvey, A.P. and Carrie, T.R., “Not Seeing is Believing – A
Non-Traditional Approach for Cleaning Validation,” Journal of
Validation Technology, Vol. 4, No. 3, pp. 189-193.
7.Fourman, G.L. and Mullen, M.V., “Determining Cleaning
Validation Acceptance Limits for Pharmaceutical Manufact­ur
ing Operations,” Pharmaceutical Technology, 17 (4), 1993, pp.
54-60.
8.Hall, W.E., “Validation of Cleaning Processes for Bulk
Pharmaceutical Chemical Processes,” Cleaning Validation An
Exclusive Publication, p. 4.
9.Hall, W.E., “Proper Documentation and Written Procedures,”
Journal of Validation Technology, Vol. 4, No. 3, pp. 199-201.
10.Tunner, J., “Manual Cleaning Procedure Design and Validation,”
Cleaning Validation An Exclusive Publication, p. 28.
11.PDA Technical Report No. 29, “Points to Consider for Cleaning
Validation,” March 1998, p.43.
12.Coleman, R.C., “How Clean is Clean?” Journal of Validation
Technology, Vol. 2, No. 4, August 1996, p. 278.
13.Jenkins, K.M. and Vanderwielen, A.J., “Cleaning Validation: An
Overall Perspective,” Pharmaceutical Technology, April 1994,
p. 70.
Julie Thomas is Validation Manager at McNeil
Consumer Healthcare in Round Rock, Texas. She
has 14 years of experience in various aspects of
solid dose pharmaceutical manufacturing. Most
recently, she chaired a company-wide committee to enhance cleaning validation practices and
procedures for all McNeil facilities. She can be
reached by phone at 512-248-4470 or by e-mail at
jthomas1@mccus.jnj.com.
This article presents only one alternative for pre­
paring a Master Validation Plan. The views ex­pressed
in this article are strictly those of the author and in
no way represent the view of McNeil Con­sumer
Healthcare, Johnson & Johnson, or this publication.
© Advanstar Communications Inc. All rights reserved.
Special Edition: Cleaning Validation III
69
PROPOSED VALIDATION STANDARD
VS-3
Cleaning Validation
VALIDATION TECHNOLOGY
Journal of Validation Technology
~
Proposed Validation Standard VS-3
PROPOSED VALIDATION STANDARD VS-3
Cleaning Validation
Introduction
T
his document is the third in a series of new proposed validation standards issued by the Institute of
Validation Technology Standards Committee (IVT/SC). The initial proposed standard (Process
Validation Standard VS-l: Nonaseptic Pharmaceutical Processes) was issued in February 2000, and
is intended to help practitioners worldwide who develop, implement, control, and validate processes that produce Active Pharmaceutical Ingredients (APIs) and drug products. Our second proposed validation standard
VS-2: Computer-Related System Validation was issued in May 2001. The current document (Cleaning
Proposed Validation Standard VS-3) is intended to offer more specific proposed standards for the cleaning
processes for equipment used to manufacture APIs and drug products. These proposed standards, will be used
by reviewers of manuscripts intended for publication in the lournal of Validation Technology (1VI).
Just as with the previous proposed standards, readers are encouraged to offer comments, questions, and recommendations. Such feedback will be useful to the IVT/SC and JVT editors in updating this document and in
developing future proposed standards. Technologies are continually changing, sometimes in ways that can influence the way validation is best conducted. Therefore, the IVT/SC plans to periodically update each proposed validation standard, including its corresponding Preamble and reference list. In order to be dynamically responsive
to changing industrial practices and regulatory requirements, and make it easier for readers to cut and paste the
contents for their own use, all three proposed standards are available on the IVT web site at www.ivthome.com.
A fundamental need the IVT/SC intends to meet with its new proposed standards stems from the fact that most
Good Manufacturing Practice (GMP) regulations today call for numerous written procedures; for example, more
than 100 different kinds of written procedures are required to comply with current GMP regulations in the United
States. Many firms find it helpful to issue written policies in order to coordinate and reduce the number and length
of required Standard Operating Procedures (SOPs). Thus, the IVT proposed validation standards format includes
statements and definitions that can be excised and used directly or with minor editing in a firm's policies and SOPs.
Contents of the Proposed Cleaning Validation Standard
In order to be consistent with the prototype standard (Validation Standard VS-l) the Proposed Cleaning
Validation Standard VS-3 will be divided into the following five sections:
I.
II.
III.
IV.
~
Policy statements - Proposed standards that indicate what is required
Procedural Statements - Proposed standards that describe how to meet requirements
Acronyms - Meaning of each acronym/abbreviation used in the document
Glossary - Definition of key terms, which are highlighted and asterisked (*) when first used in the
proposed validation standard
Institute of Validation Technology
Proposed Validation Standard VS-3
V. Regulatory Excerpts - Regulatory language (United States, Australia, Canada, World Health Organization [WHO], Japan, and European Union) related to each Standard
T
he following proposed standard is intended to reflect desirable contemporary practices, is not binding in
any way, and can be modified to suit a firm's specific needs. This proposed standard incorporates imperative verbs (e.g., shall, will, must) to provide users with unambiguous quality assurance auditing tools,
and is prefaced by a Preamble that provides rationale for several of the more complex concepts. This document
is also directed toward users located at a given plant site that mayor may not be a part of a larger corporation.
Terms that are bold and asterisked (*) the first time they are used are defined in Section IV - Glossary.
I. POLICY STATEMENTS
POL 1.1
The critical cleaning processes associated with the manufacture of Active Pharmaceutical Ingredients
(API)*, critical Intermediates*, Drug Products*, or In-Process Materials* shall be validated or verified.
POL 1.2
The critical cleaning processes associated with products in the development stage of the product lifecycle
shall be verified. The administrative responsibility for such products will reside in either the appropriate
development group or in the Site Validation Steering Committee (SVSC)*. If the company decides that
responsibility for cleaning verification shall reside in the appropriate development group, then the documentation describing the verification procedure and the Cleaning Verification Protocols* must also be approved by the site Quality Authority*.
POL 1.3
During development of the new product, the manufacturing equipment, batch size, and formulation is constantly changing and the cleaning procedure must be appropriate and customized for each manufacturing event.
The lifecycle for the development and validation of a new cleaning procedure consists of the following steps:
l.3.1
1.3.2
1.3.3
1.3.4
l.3.5
1.3.6
1.3.7
1.3.8
l.3.9
1.3.10
1.3.11
1.3.12
Determine what materials need to be cleaned from the equipment or surfaces.
Determine what methods should be used to evaluate the anticipated residues (from Section
1.3.1). Determine the sensitivity and reproducibility of these methods.
Define the Critical Product Cleaning Specifications*.
Define the specific equipment to be used for each development batch.
Define the specific formulation to be used for manufacturing each individual development batch.
Identify the cleaning agents to be used, if appropriate.
Determine what other products are manufactured in the same equipment.
Calculate Cleaning Verification Limits* for the specific equipment taking into account the
critical product cleaning specifications as well as the other products made in the same equipment.
Draft a Cleaning Procedure* for the specific combination of product and manufacturing equipment. Identify Critical Cleaning Process Operating Parameters* and Cleaning Agents*.
Prepare a cleaning verification protocol.
Manufacture a single product batch, clean the equipment; then test the equipment, as specified in the cleaning verification protocol.
Once development is complete, perform Cleaning Validation* on the first three (3) commercial batches.
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1.3.13
1.3.14
1.3.15
1.3.16
1.3.17
1.3.18
1.3.19
1.3.20
Validate analytical methods to be used for cleaning validation samples.
Determine recovery factors of expected residues from representative materials (stainless steel,
glass, plastics).
Prepare and obtain approval of a Cleaning Validation Protocol*.
Train and qualify operational and supervisory laboratory and production personnel in product-specific cleaning procedures, sampling procedures, and analytical procedures.
Ensure that interrelated systems (automated clean-in-place, utilities, Programmable Logic
Controllers [PLCs]) are all validated.
Conduct Cleaning Performance Qualification (CPQ)*.
Assemble and document evidence that the cleaning process is acceptable and consistent.
Provide for retention of archived cleaning validation files for required periods following the
last commercial lot expiration date.
POL 1.4
The cleaning processes associated with products in the marketed stage of the product lifecycle shall be validated for all products manufactured with a normal frequency of production. For rare instances where products are infrequently manufactured (e.g., one batch per year or less frequently), it may be difficult to achieve
fully validated cleaning processes and the principle of cleaning verification should be utilized. The administrative responsibility for cleaning validation and cleaning verification of products will reside in the Site
Validation Steering Committee (SVSC). The SVSC shall adjudicate cleaning validation issues and appoint
project-specific validation teams as needed that include principal(s) having experience in the cleaning
processes involved. Such SVSC responsibilities extend to cleaning processes used by contract vendors and
suppliers of the firm's drug products and/or APIs, as well as to those cleaning processes employed on-site.
POL 1.5
A written Cleaning Verification Policy (CVP) shall be used to define and describe the strategies and
approaches used to verify cleaning procedures associated with drug products, biotechnology products,
medical devices, and APIs during the development stage of the lifecycle.
POL 1.6
A written Cleaning Validation Master Plan (CVMP)* shall be used to define and coordinate validation
activities related to any cleaning process associated with the manufacture of a commercially marketed
drug product, biotechnology product, medical device, and API.
POL 1.7
Cleaning Verification Protocols shall be used to define individual cleaning verification runs.
POL 1.8
Cleaning Validation Protocols shall be used to define individual cleaning validation runs.
POL 1.9
Cleaning Verification Reports* shall be used for documenting and summarizing results of cleaning verification studies. Definite statements must be used, especially in describing the scientific rationale for the
limits chosen and whether the cleaning process was effective in meeting the limits.
POL 2.0
Cleaning Validation Reports* shall be used for documenting and summarizing results of cleaning validation studies. Definitive statements must be used, especially in describing the scientific rationale for the
limits chosen and whether the cleaning process was effective in ensuring that these limits were met.
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POL 2.1
All Cleaning Verification Policies, Cleaning Validation Master Plans, Cleaning Verification Protocols,
Cleaning Validation Protocols, Cleaning Verification Reports and Cleaning Validation Reports must
be approved and available to the SVSc. All such cleaning documents created on-site must be approved by
the site Quality Authority and, when production is involved, also by the site Production Authority*.
POL 2.2
Relevant cleaning process verification and validation information from other divisions, departments
(including Research and Development), production sites, and outside contract services is to be gathered,
evaluated, utilized, and maintained by the SVSC.
POL 2.3
Certain cleaning processes are considered critical manufacturing steps and thus require validation (it
should be noted that not all cleaning procedures are considered critical and thus require validation). Once
the cleaning procedures are validated, they must not be altered without prior review, and any changes
should be subjected to a formal Change Control* review process prior to making the change. The site
Quality Authority must approve all changes to validated cleaning procedures.
II. PROCEDURAL STATEMENTS
PROC - 2.a [ref. POL 1.3.2]
Critical product cleaning specifications are known factors that can influence the development of the
cleaning process. These can be physical in nature such as solubility in a variety of solvents, polymorphic
crystal form, and stability. These factors could also be chemical in nature such as reactivity with water
or other solvents. They could also include medical information such as potency, toxicity, and allergenicity. They could also be safety factors such as toxicity when inhaled and could require personal protection attire to protect the operator. These factors, which are normally determined during pre-formulation,
are vital information that must be known before meaningful cleaning procedures and limits can be developed.
PROC - 2.b [ref. POL 1.3.3]
During development, various types of equipment may be used in an effort to develop an optimum process
or effective product. This means that normally the specific equipment or the scale of the equipment may
vary from batch-to-batch. Because of this variability in the equipment used, the cleaning procedures may
also vary from batch-to-batch even for the same product. Therefore, the cleaning verification results apply
only to the specific cleaning event (i.e., the specific combination of equipment, processes, and materials)
used for the individual study. The cleaning verification report should contain the details of the specific
equipment (size, model number), formulation, and processes used.
PROC - 2.c [ref. POL 1.3.4]
During development, the formulation may vary from batch-to-batch in order to identify the combination
of ingredients that presents the best product performance 'in vitro' and 'in vivo'. Excipients may be varied as well as the concentration of active ingredient. These combinations will present different degrees
of cleaning challenges. A given cleaning procedure may be adequate for one formulation but inadequate
for another formulation of the same active ingredient. This data will be useful for the selection of the
ultimate cleaning procedure that will be used for commercial product. It will be necessary to include the
formulation in the cleaning verification study, either by reproducing in total, or by reference to a formula number.
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PROC - 2.d [ref. POL 1.3.5]
Since it is other products made in the same equipment that will be contaminated due to inadequate cleaning, it is necessary to evaluate the other products made in the same equipment. Some of the factors pertaining to other products that will be needed are:
• Batch sizes
• Normal daily doses
• Route of administration
PROC - 2.e [ref. POL 1.3.6]
In order to develop a scientific basis for cleaning verification limits, information will be needed for both
the product being cleaned as well as other products made in the same equipment. The following information should be assembled:
• For product being cleaned
- Solubility in various solvents
- Potency
- Toxicity
- Stability (wet and dry)
- Allergenicity
- Route of administration
- Daily dosage
- Difficulty of cleaning
- Physical and chemical interaction with cleaning agent
• For other products made in same equipment
- Batch sizes
- Daily doses
- Stability
- Chemical interaction with product being cleaned
- Route of administration
The pharmacological relationships between the potential contaminating product and other products, which
could be possibly cross contaminated, may also be significant and should be considered if known. The contaminating product has the potential to amplify the medical activity of other products resulting in a synergistic effect. The contaminant could also partially negate the medical effect of the other products by having an antagonistic effect.
PROC - 2.f [ref. POL 1.3.7]
Just as there are critical parameters for the manufacturing process, there are critical parameters for the
cleaning process. These factors will lead to either inadequate or inconsistent cleaning if not controlled.
Critical parameters for the cleaning process must be determined and may vary from one cleaning process
to another. Some potential critical cleaning parameters (list is not all inclusive) are:
•
•
•
•
Temperature of wash solutions
Temperature of rinse solutions
Amount of mixing or agitation during cleaning
Mechanical wiping or brushing
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•
•
•
•
•
•
•
•
•
•
•
•
•
•
Flow rates
Concentration of cleaning agent
Time of washing
Time of rinsing
Length of time and environmental conditions (temperature, humidity) between manufacturing and
cleaning
Nature and amounts of excipients
Concentration or amount of residue left on equipment
Physical properties of residues
Chemical properties of residues
Cleaning solvent chosen
Soak times
Contact time with cleaning agent
Rinse volumes
Order of application of cleaning solvents (acid, alkaline, and organic solvents)
PROC - 2.g [ref. 1.3.8]
Each cleaning verification protocol shall include, and is not limited to, the following:
o Statement of objective or purpose
Justification for cleaning verification limits, if applicable
~ Descriptions of sampling procedure(s), and locations, types, and numbers of samples to be taken
o Indications of most difficult-to-clean locations in equipment
o Experimental plan to be executed, including number of samples, and how data will be calculated
<D Descriptions of analytical methodology and sensitivity of analytical method as well as recovery factors
o Descriptions of all testing instruments to be used and specific calibration plans for each
o Complete description of acceptance criteria including visual examination (if possible) and quantitative
analytical data
o Training records of operators and analytical personnel
@
PROC - 2.h [ref. 1.3.11]
Prior to cleaning validation studies, analytical methods must be validated to demonstrate that they are suitably sensitive to detect residues at levels below the allowable limits. Analytical Method Validation* for
cleaning validation shall include, and is not limited to, the following:
o Accuracy
Precision
~ Linearity
o Robustness
o Sensitivity-Limit of Detection (LOD)*, Limit of Quantitation (LOQ)*
<D Specificity
@
The specificity of the analytical method may not be as critical for cleaning validation as for process validation due to the fact that the levels of residue detected is very low, and often non-specific analytical methods are available that may be at least or more sensitive than specific methods. The assumption is often
made that all of the residue detected is composed of the most potent ingredient (usually the active) present and, if this amount is still below the established limits, then the exact nature of the residue is irrelevant, i.e., the 'worst case' assumption was made and limits were met.
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PROC - 2.i [ref. 1.3.12]
Following validation of the analytical method, the analytical method should be challenged concurrently
with the sampling procedure(s) to determine the percentage recovered from representative manufacturing
surfaces. The determination of recovery is important and will differ according to the composition of the surface sampled (e.g., stainless steel, glass, plastics), the nature of the sampling technique, and the nature of
the residues themselves. The recovery factor must be used to correct observed analytical results to account
for portions of residue that remain on equipment even after swab and rinse sampling.
PROC - 2.j[ref. 1.3.13]
Each cleaning validation protocol shall include, and is not limited to, the following:
o Statement of objective or purpose
Justification for cleaning validation limits
Descriptions of sampling procedure(s) and diagrams of locations
o Indications of most difficult-to-clean locations in equipment
o Experimental plan to be executed, including number of cleanings to be evaluated, number of samples
from each cleaning, and how data will be calculated
<D Descriptions of analytical methodology and sensitivity of the analytical method as well as recovery factors
fi Descriptions of all testing instruments to be used and specific calibration plans for each
«l) Complete description of acceptance criteria including visual examination (if possible) and quantitative
analytical data
CD Criteria for determining when the cleaning process may be considered validated, i.e., how many successful consecutive cleanings (normally at least three (3) are required)
@ Training records of operators and analytical personnel
@
@)
PROC - 2.k [ref.1.3.14]
Prior to implementation of the cleaning validation protocol, it is important to verify the training of the production operators who actually conduct the cleaning, sampling personnel (production, analytical, validation) who sample the equipment, analytical personnel who analyze cleaning validation samples, as well
as personnel who implement the protocol and process the documentation. If documentation does not
already exist that demonstrates each of these types of training, then the training should be done before any
actual validation runs are carried out.
PROC - 2.1 [ref. 1.3.15]
Special equipment and critical utilities such as water and steam must be qualified prior to implementation
of the cleaning validation protocol. In addition, any automated cleaning equipment such as Clean-inPlace (CIP)* systems and their associated automated controllers must also be validated or qualified prior
to implementation of the cleaning validation protocol. In the case of CIP, Sprayball Pattern Analysis*
should be carried out to verify that cleaning solutions reach all locations in closed systems. The qualification of equipment and utilities is normally accomplished by means of an Installation Qualification
(IQ) * and an Operational Qualification (OQ) * (see next two sections).
PROC - 2.m [ref. 1.3.15]
An Installation Qualification (IQ) must exist for all equipment that is critical to the cleaning process
including specialized cleaning aids such as Spray Devices (Sprayballs)*, equipment that delivers cleaning solutions, high pressure wands, water heating devices, steam generators, and utilities. The IQ is to include at least the following:
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o List of all equipment, the operation of which has potential bearing on the quality of the cleaning process
As-built drawings of all specialized cleaning equipment such as pumps, high pressure delivery devices,
and hose cleaners
@) Verification that all such equipment and the installation thereof meets original intent, including applicable building, electrical, plumbing, and other such codes
o Preventative maintenance plans and schedules for all such equipment
@
PROC - 2.n [ref. 1.3.15]
An Operational Qualification (OQ) must exist for all equipment that is critical to the cleaning process and
should include at least the following:
o A list identifying each step of the cleaning process that relates to the specific equipment
Process operating parameters for each piece of equipment that is critical to the cleaning process
An OQ protocol that is designed to demonstrate via appropriate tests that the equipment operates as intended throughout the cleaning process
o Report that describes the successful execution of each OQ protocol for each piece of equipment critical to the cleaning process
@
@)
PROC - 2.0 [ref. 1.3.16]
At least three consecutive, successful cleanings shall be completed on the equipment used to produce the
commercial product. Normally, the cleanings follow the production of each of the batches used for the validation of the manufacturing process. A Cleaning Performance Qualification (CPQ) shall be performed
when the following items are complete and commercial production has been authorized.
• The cleaning process is fully defined in writing, including identification of critical cleaning process
operating parameters
• A justification for Cleaning Validation Limits* has been prepared that takes into account the potency
and toxicity of the material, as well as the other products to be made in the same equipment
• IQ and OQ steps are complete for critical utilities and any specialized equipment used in cleaning such
as pumps, sprayballs, high pressure wand cleaners, etc.
• Operating, sampling, and analytical personnel are trained and qualified and the training is documented
• An appropriate change control procedure is in place
PROC - 2.p [ref. 1.3.17]
Once the cleaning validation protocol has been implemented on three cleanings and the sampling and testing has been completed, the data must be assembled and evaluated for each cleaning event. A cleaning
validation report should be prepared that consists of:
• The cleaning validation protocol
• All data assembled in a logical format
• An analysis of the data that addresses any deviations in the protocol, explains any failures, compares
the data to the acceptance criteria, and ultimately states whether the cleaning process mayor may not
be considered validated
PROC - 2.q [ref. POL 1.5]
The Cleaning Verification Policy (CVP) can be considered to be the master plan for cleaning for a product
during the development phase of the lifecycle of the product. Since each cleaning is a unique event because
of the variability in the manufacturing equipment, formulation, and batch size between batches of the same
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product, it is not possible to validate the cleaning process during the development phase. Still it is possible
to prepare a policy describing the testing of development equipment and what criteria will be used to determine if the equipment has been suitably cleaned. The strategy and approach to cleaning in the development
areas must be in writing and clearly explain how equipment will be sampled and tested, and how limits will
be determined, recognizing that only a single set of data will be available. Since only a single set of data is
available, it would be erroneous to refer to this situation as "validation". Thus the term "cleaning verification" is a more appropriate description of this scenario.
PROC - 2.r [ref. POL 1.6]
The Cleaning Validation Master Plan (CVMP) may take different forms in companies around the world.
Some may have a separate independent document. Others may have a Standard Operating Procedure*
that describes in general terms how the cleaning program will operate. Still others will devote a section of
the Validation Master Plan* to cleaning. Regardless of the exact form taken, it is essential to have a written plan describing how the cleaning program will be organized and controlled. The essential elements of
the CVMP are:
• A description of the approach and strategy to be used for controlling, verifying, and/or validating in the
various departments such as Basic Research *, Research and Development *, Scale-Up! Pilot Plant*,
Production*, Packaging*, Contract Manufacturing Facilities *, and Contract Packaging Facilities *.
• A mechanism for defining what is adequate cleaning, based on the potency, toxicity, potential allergenicity, potential teratogenicity, and potential carcinogenicity of the material, as well as other factors
such as route of administration and properties of the other products made in the same equipment.
• Sampling methods to be used to evaluate cleaned equipment. Examples are Swab Sampling* and Rinse
Sampling*, or a combination of these two methods depending on the nature of the equipment or product.
• Selection of sampling locations to include 'worst case' and/or most difficult-to-clean locations.
• For equipment used for manufacturing multiple products, how the Worst Case Product* for cleaning purposes might be selected from a group of very similar products. Typically, a Product Matrix Approach*
is used to compare the critical cleaning properties of the products in the group. Critical cleaning properties are potency/toxicity, solubility, and the inherent difficulty of cleaning.
• Provision for how documentation will be developed, reviewed, and approved. This would include a list of
those responsible for preparing, reviewing, and approving Cleaning Verification Protocols, Cleaning
Verification Reports, Cleaning Validation Protocols, Cleaning Validation Reports, Cleaning Procedures,
Change Control Procedures, and Cleaning Monitoring Programs *.
• Criteria for Revalidation* of cleaning procedures.
• Provision for creation of a Site Validation Steering Committee (SVSC), that would serve as the group
immediately responsible for all cleaning issues. This group would normally select project teams related to cleaning activities, e.g., for a new product.
• Training of development, pilot plant, sampling, and analytical testing personnel.
• Definition of resources required and allocated.
• Schedule of cleaning activities including cleaning validation and assignment of responsibilities.
III. ACRONYMS
API
BPC
CGMPs
CIP
CPQ
Active Pharmaceutical Ingredient
Bulk Pharmaceutical Chemical
Current Good Manufacturing Practice (U.S.)
Clean-in-Place
Cleaning Performance Qualification
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CVMP
CVP
IPEC
IQ
OQ
PIC
SOP
SVSC
Cleaning Validation Master Plan
Cleaning Verification Policy
International Pharmaceutical Excipients Council
Installation Qualification
Operational Qualification
Pharmaceutical Inspection Convention
Standard Operating Procedure
Site Validation Steering Committee
IV. GLOSSARY
Reference Standard
Number
POL 1.1
Active Pharmaceutical Ingredients (API) - (synonymous with drug substance). A substance
that is represented for use in a drug and, when used in the manufacturing, processing, or packaging of a drug, becomes an active ingredient of a finished drug product. Such substances are
intended to furnish pharmacological activity or other direct effects in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure and function of the body
of humans or other animals.
Bulk Pharmaceutical Chemical (BPC) - includes active pharmaceutical ingredients (APIs) as
well as non-active excipients such as starch, lactose, rnicrocellulose, and other materials that
have no direct therapeutic effect but may indirectly affect the performance of drug dosage forms.
PROC·2.h
Analytical Method Validation - documented evidence that an analytical procedure will consistently detect and/or quantitate materials.
PROC-2.r
Basic Research - the segment of the pharmaceutical industry that evaluates new chemical
entities for potential application to treatment of disease. This includes, but is not limited to,
basic disciplines such as biochemistry, molecular biology, toxicology, pharmacology, and
pharmacokinetics.
POL 2.3
Change Control Procedure - A procedure for:
(a.) Identifying all modifications or alterations that are potentially significant to a state of control, qualification, or validation.
(b.) Implementing corrective action, such as repair, readjustment, requalification, and/or
revalidation.
(c.) Implementing interim measures to be taken until effective corrective actions are complete.
(d.) Documenting all of the above.
POL 1.3.9
Cleaning Agents - any chemical or solvent that facilitates the cleaning of equipment by dissolution, hydrolysis, or other chemical or physical action.
PROC-2.r
Cleaning Monitoring Program - a formal, written program describing how cleaning procedures can be monitored on a regular schedule to evaluate the effectiveness and consistency of
the cleaning process.
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POL 1.3.18
Cleaning Performance Qualification (CPQ) - documented evidence that a cleaning procedure is consistent in removing product residue and cleaning agent from equipment.
POL 1.3.9
Cleaning Procedure - a detailed written procedure (SOP) that describes how equipment will
be disassembled, cleaned, examined, and reassembled.
POL 1.3.12
Cleaning Validation - documented evidence that a cleaning procedure is consistent in removing product residue and cleaning agents from equipment (sometimes also referred to as
Cleaning Performance Qualification [CPQ]).
POL 1.6
Cleaning Validation Master Plan (CVMP) - a comprehensive, written plan that describes the
company's strategy in ensuring that all cleaning procedures are effective and in a state of control to ensure that all products are free of contamination and of high qUality. The plan includes
or references all appropriate cleaning procedures, and SOPs describes how protocols, cleaning
validation reports, and other documentation will be assembled, provides for the testing and
analysis of data, identifies resources to be allocated, provides for training of personnel, describes
qualification of equipment, indicates the process for assigning responsibility for the various
activities, provides a criteria for revalidation of cleaning procedures, and describes a mechanism
for controlling changes to validated procedures and equipment.
PROC-2.o
Cleaning Validation Limits - The maximum allowable amounts of material that can remain
on equipment after cleaning without compromising the safety of the consumer or the quality
of the product. These limits are applied during the cleaning validation study and depending on
the manufacturing circumstances, limits may be for:
•
•
•
•
•
•
•
•
POL 1.3.15
Residues of active ingredients
Residues of excipients
Degradation materials
Intermediates
Cleaning agent or by-product residuals
Bioburden
Endotoxin
Other foreign materials
Cleaning Validation Protocol - a product specific plan of sampling and testing of equipment
after at least three consecutive cleanings to establish that equipment is appropriately cleaned
after a specific product is manufactured in a development area by a specific, detailed written
cleaning procedure.
POL 2.0
Cleaning Validation Reports - a written report that summarizes results and conclusions of
the cleaning validation study and includes:
•
•
•
•
•
•
Protocol
Test results
Analyses
Conclusions
Discussions of any deviations from procedures specified in the original protocol
Discussion of any failures
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• Indication as to whether the testing met the acceptance criteria specified in the protocol
POL 1.3.8
Cleaning Verification Limits - Maximum amount of residue that may remain on equipment
during a cleaning verification study. These limits are derived in a similar fashion to those for a
cleaning validation study, but are applied to a single cleaning event, as versus multiple cleaning runs (at least three) for cleaning validation studies. Just as for cleaning validation, the limits may be for any of the following:
•
•
•
•
•
•
•
•
Residues of active ingredients
Residues of excipients
Degradation materials
Intermediates
Cleaning agents or by-product residuals
Bioburden
Endotoxin
Other foreign materials
POL 1.5
Cleaning Verification Policy (CVP) - a written document describing how equipment will be
verified as clean after a single manufacturing event in a development area. This is a general
document that will pertain to all cleaning in development areas.
POL 1.2
Cleaning Verification Protocol- a product specific plan of experimental sampling and testing to verify that equipment is appropriately cleaned after a specific product is manufactured
in a development area.
POL 1.9
Cleaning Verification Reports - a written report that summarizes results and conclusions of
the cleaning verification study and includes:
•
•
•
•
•
•
•
Protocol
Test results
Analyses
Conclusions
Discussions of any deviations in procedures from those specified in the original protocol
Discussion of any failures
Indication as to whether the testing met the acceptance criteria specified in the protocol
PROC-2.1
Clean-in-Place (CIP) - cleaning of equipment that is accomplished without disassembly of
the equipment but rather through the application of cleaning solutions delivered internally by
one or more internal spray devices (sprayballs) or recirculation of cleaning solution throughout
the equipment. CIP may be entirely automated or the cycle parameters may be controlled by
the operator. This type of cleaning is also known as closed system cleaning.
PROC-2.r
Contract Manufacturing Facilities - facilities or companies that manufacture products for
customers on a contractual basis.
PROC-2.r
Contract Packaging Facilities - facilities or companies that package products for customers
on a contractual basis.
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POL 1.3.9
Critical Cleaning Process Operating Parameter - an operating variable that is assigned a
required control range with acceptability limits, outside of which exists potential for product
or process failure. A critical process operating parameter is determined by process development and/or investigational work.
POL 1.3.3
Critical Product Cleaning Specifications - physico-chemical properties as well as therapeutic
or medical information that are used to determine cleaning procedures and set limits for cleaning
processes. Examples are solubility, stability, hydrophobicity, therapeutic potency, and toxicity.
POL 1.1
Drug Products - a finished dosage form (e.g., tablet, capsule) that contains an API, generally in association with excipients. Synonymous with finished drug product.
POL 1.1
In-Process Materials - (as applied to drug product manufacture) - any material manufactured, blended, compacted, coated, granulated, encapsulated, tableted, or otherwise processed
that is produced for and used in the preparation of a drug product. (Corresponding materials
used in the preparation of APIs are referred to as intermediates.)
PROC-2.1
Installation Qualification (IQ) - documented verification that equipment, system, or a subsystem has been properly installed, adheres to applicable codes and approved design intentions, and supplier recommendations have been suitably addressed.
POL 1.1
Intermediate - a material produced during steps in the synthesis of an API that must undergo further molecular change or processing before it becomes an API. The degree to which a
given intermediate should be rated "critical" with respect to cleaning must be determined by
a firm's experts based on such criteria as:
• Potential toxicity or other physiological activity
• Degree to which equipment used is dedicated to the process, as opposed to having multiple uses
• Ease or difficulty of removing process residuals when cleaning equipment
PROC-2.h
Limit of Detection (LOD) - the lowest amount or concentration of a material that can be
detected by an analytical instrument or chemical test. Although detectable, the amount of
material in the sample cannot be determined at this level.
PROC-2.h
Limit of Quantitation (LOQ) - the lowest amount or concentration of a material that can be
quantitatively determined by an analytical instrument or chemical test.
PROC-2.1
Operational Qualification (OQ) - documented verification that equipment, system, or
process performs as specified throughout representative or anticipated operating ranges.
(Note: Overlap between IQ and OQ often occurs and is considered allowable, but should be
addressed in the VMP.)
PROC-2.r
Packaging - The area or department that receives bulk product and incorporates the product
in packaging that will either be sent to the customer or sent to another area for additional packaging and/or labeling.
PROC-2.r
Production - The unit of the company responsible for the manufacture of bulk product. This
mayor may not include the packaging function depending on the size and organization.
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POL-2.1
Production Authority - counterpart of quality authority, sometimes referred to as production
head or, in the case of FLP (Fill-Label-Pack) operations, packaging head.
PROC-2.r
Product Matrix Approach - a chart that presents medical, toxicity, solubility, and other pertinent data so that a comparison can be made between products in the group in order that the
most risky product can be selected for cleaning validation. This 'worst case' approach obviates the need to perform cleaning validation studies on every combination of product and
equipment.
POL 1.2
Quality Authority - one or more persons who, collectively, have formal responsibilities for
specified quality-related operations, such as approval of manufacturing materials, release of
finished products, review and approval of documents, and adjudication of quality assurance
investigations. Titles of quality authority principals vary throughout the world; for example, in
the U.S., one term "the Quality Control (QC) unit," is all embracing; in the E.U. and Canada,
the head of quality control has some of the responsibilities, while a qualified person has others; terms as responsible head (or person) and quality assurance (and/or control) department are
also used in other areas.
PROC-2.r
Research and Development - The division of a company that is responsible for developing
the optimal manufacturing techniques and dosage form for a pharmaceutical product. It is also
responsible for the development of preliminary cleaning procedures for new products.
PROC-2.r
Revalidation - repeating the original validation or selected portions for the purpose of
demonstrating that the process is still in a state of control and delivers acceptable product and
processes. As applied to cleaning procedures, the purpose would be to demonstrate that the
cleaning procedures are still effective in removing residues. Revalidation is a natural consequence of making significant changes to equipment, manufacturing procedures, components,
cleaning procedures, and cleaning agents.
PROC-2.r
Rinse Sampling - a type of sampling of cleaned equipment used in cleaning validation and
cleaning verification studies to determine if product-contact manufacturing surfaces are clean.
Controlled amounts of solvent are subjected to the equipment either under pressure or allowed
to stand in the equipment to allow dissolution of the residues. Mixing, spraying, and recirculation may also be used to facilitate the detection of residues. Rinse solvents are usually
selected on the basis of residue solubility in that solvent. The rinses may be either heated or
at ambient temperature.
PROC-2.r
Scale-UplPilot Plant - Functionally, this area of responsibility is between development and
full-scale production. This group is charged with scaling a formulation up from small scale to
large production scale and troubleshooting problems that arise as a result of the scale-up process. They are also responsible for further refinements of the cleaning procedures handed over
by development.
POL 1.2
Site Validation Steering Committee (SVSC) - a standing committee with authority and
responsibilities for validation policies, practices, and adjudication of issues. Must include
quality authority and Production Authority representation, and often includes representatives
of other involved disciplines. The name of the SVSC may vary from firm-to-firm.
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Proposed Validation Standard VS-3
PROC-2.m
Spray Devices (Sprayballs) - a device that may be either permanently installed or inserted
into closed systems such as tanks specifically to provide a thorough, even coverage of the
equipment surfaces by the cleaning solutions. If fixed and spherical in shape, the device is
usually referred to as a "sprayball." Sprayballs may have fixed heads or they may rotate in
complex patterns.
PROC-2.1
Sprayball Pattern Analysis - a study that establishes that cleaning solution delivered by
sprayballs will reach all equipment surfaces, especially difficult to access or shadowed areas.
The study usually consists of coating equipment with easily detectable residue (dyes or fluorescent material), activating the sprayball mechanism for a normal cycle cleaning time, and
then examining the equipment to see if any material remains on the equipment surfaces.
PROC-2.r
Standard Operating Procedure (SOP) - A written document describing, in detail, a specific process or procedure. These written procedures are required by the current Good Manufacturing Practice regulations for all critical processes. These procedures must be current,
detailed, controlled, and revised when necessary. All personnel must be trained in a new or
revised SOP prior to its implementation. Some companies have function specific procedures,
e.g., cleaning procedures, that take the place of SOPs.
PROC-2.r
Swab Sampling - a type of sampling of cleaned equipment used in cleaning validation and
cleaning verification studies to determine if product-contact manufacturing surfaces are clean.
This type of sampling makes use of small pieces of fabric (usually polyester or other synthetic material) fused to the end of a plastic strip. The swab is typically wetted with solvent
(although they can be used dry). Defined surface areas of equipment, including the most difficult-to-clean locations, are swabbed. The swab is then immersed in a vial of solvent. The
residue on the swab is dissolved in the solvent, which is subsequently analyzed for product
residues. Limits are calculated on the basis of the area swabbed.
PROC-2.r
Validation Master Plan (VMP) - a comprehensive, project-oriented action plan that includes
or references all protocols, key SOPs and policies, existing Validation Task Reports *, and
other relevant materials on which the specific system or process validation effort will be
based. The plan also identifies resources to be allocated, specific personnel training, and qualification requirements of relevant, organizational structure, and responsibilities of the validation team, and planned schedules. The VMP is subject to periodic revisions as defined in
change control procedures.
Validation Task Report - a written report that summarizes results and conclusions following
execution of all or any portion of a Validation Master Plan (VMP) (often referred to as a final
report if summarizing all activities of the VMP).
PROC-2.r
Worst Case Product - the product selected from a group of similar products that presents the
greatest risk of carryover contamination to other products made in the same equipment by
virtue of its poor solubility, unstable chemical properties, potency, toxicity, or a combination
of these factors.
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Proposed Validation Standard VS-3
v.
SELECTED REGULATORY EXCERPTS
Regulatory Reference
FDA Proposed Amendments to current Good Manufacturing
Practice Regulations Section Ill. C (May 3, 1996)
Under CGMP, a manufacturer will set contamination limits on a substance-by-substance basis, according to both
the potency of the substance and the overall level of sensitivity to that substance.
Because other substances, such as cytotoxic agents, or other antibiotics, pose at least as great a risk of toxicity
due to cross-contamination, FDA is proposing to expand the contamination control requirements to encompass
other sources of contamination.
The Agency has refrained from establishing a list of drugs or drug products that present such an unacceptable
risk, because such a list would quickly become obsolete.
FDA Guidance for Industry: Manufacturing,Processing, or Holding
Active Pharmaceutical Ingredients Section IV.D (March, 1998)
Nondedicated equipment should be thoroughly cleaned between different products and, if necessary, after each
use to prevent contamination and cross-contamination. If cleaning a specific type of equipment is difficult, the
equipment may need to be dedicated to a particular API or intermediate.
The choice of cleaning methods, cleaning agents, and levels of cleaning should be established and justified.
FDA Guidance for Industry: Manufacturing, Processing, or Holding Active
Pharmaceutical Ingredients Section IV.E (March, 1998)
Equipment cleaning methods should be validated, where appropriate. In early synthesis steps, it may be unnecessary to validate cleaning methods where residues are removed by subsequent purification steps.
If various API's or intermediates are manufactured in the same equipment and the equipment is cleaned by the same
process, a worst-case API or intermediate can be selected for purposes of cleaning validation. The worst-case selection should be based on a combination of potency, toxicity, solubility, stability, and difficulty of cleaning.
The cleaning validation protocol should describe the equipment to be cleaned, methods, materials, extent of
cleaning, parameters to be monitored and controlled, and analytical methods.
Sampling should include swabbing, rinsing, or alternative methods (e.g., direct extraction), as appropriate, to
detect both insoluble and soluble residues. Swab sampling may be impractical when product contact surfaces are
not easily accessible due to equipment design and/or process limitations (e.g., inner surfaces of hoses, transfer
pipes, reactor tanks with small ports or handling toxic materials, and small intricate equipment such as micronizers and microfluidizers).
Validated analytical methods sensitive enough to detect residuals or contaminants should be in place.
Residue limits should be practical, achievable, verifiable, and based on the most deleterious residue.
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Proposed Validation Standard VS-3
FDA Guidance for Industry: Manufacturing, Processing, or Holding Active
Pharmaceutical Ingredients Section IV.F (March, 1998)
When practical, equipment in CIP systems should be disassembled during cleaning validation to facilitate inspection and sampling of inner product surfaces for residues or contamination, even though the equipment is not
normally disassembled during routine use.
FDA Part 211-Current Good Manufacturing Practice for Finished
Pharmaceuticals Subpart D, Section 211.67 (1990)
Equipment and utensils shall be cleaned, maintained, and sanitized at appropriate intervals to prevent contamination that would alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other
established requirements.
Written procedures shall be established and following for cleaning and maintenance of equipment, including
utensils, used in the manufacturing, processing, packing, or holding of a drug product.
FDA Guide to Inspections of Validation of Cleaning Processes, (July, 1993)
FDA expects firms to prepare specific written validation protocols in advance for the studies to be performed on
each manufacturing system or piece of equipment, which should address such issues as sampling procedures,
and analytical methods to be used, including the sensitivity of those methods.
FDA expects firms to conduct the validation studies in accordance with the protocols and to document the results
of studies.
FDA expects a final validation report which is approved by management and which states whether or not the cleaning process is valid. The data should support a conclusion that residues have been reduced to an "acceptable level."
Examine the design of equipment, particularly in those large systems that may employ semi-automatic or fully
automatic clean-in-place (CIP) systems since they represent significant concern. For example, sanitary type piping without ball valves should be used. When such nonsanitary ball valves are used, as is common in the bulk
drug industry, the cleaning process is more difficult.
Examine the detail and specificity of the procedure for the cleaning process being validated, and the amount of
documentation required.
When more complex cleaning procedures are required, it is important to document the critical cleaning steps (for
example certain bulk drug synthesis processes).
Determine the specificity and sensitivity of the analytical method used to detect residuals or contaminants.
The firm's rationale for the residue limits established should be logical based on the manufacturer's knowledge
of the materials involved and be practical, achievable, and verifiable.
Check the manner in which limits are established.
If a detergent or soap is used for cleaning, determine and consider the difficulty that may arise when attempting
to test for residues.
FDA Guide to Inspections of Bulk Pharmaceutical Chemicals (May, 1994)
Cross contamination is not permitted under any circumstances.
Institute of Validation Technology
Proposed Validation Standard VS-3
The cleaning program should take into consideration the need for different procedures depending on what product or intermediate was produced.
Where mUltipurpose equipment is in use, it is important to be able to determine previous usage as an aid in investigating cross-contamination or the possibility thereof.
Cleaning of multiple use equipment is an area where validation must be carried out.
Validation data should verify that the cleaning process will remove residues to an acceptable level.
There should be a written equipment cleaning procedure that provides details of what should be done and materials to be utilized.
We expect the manufacturer to establish an appropriate impurity profile for each BPC based on adequate consideration of the process and test results.
PIC Document PR 1/99-2 "Cleaning Validation" Section 1.0 (April, 2000)
Cleaning procedures must strictly follow carefully established and validated methods of execution. This applies
equally to the manufacture of pharmaceutical products and bulk active ingredients.
PIC Document PR 1/99-2 "Cleaning Validation" Section 2.1 (April, 2000)
Normally only cleaning procedures for product contact surfaces need to be validated.
PIC Document PR 1/99-2 "Cleaning Validation" Section 2.2 (April, 2000)
Cleaning procedures for product changeover should be fully validated.
PIC Document PR 1/99-2 "Cleaning Validation" Section 2.6 (April, 2000)
At least three consecutive applications of the cleaning procedure should be performed and shown to be successful in order to prove that the method is validated.
PIC Document PR 1/99-2 "Cleaning Validation" Section 2.8 (April, 2000)
Control of change to validated cleaning procedures is required. Revalidation should be considered under the following circumstances:
(a) Revalidation in cases of changes to equipment, products or processes.
(b) Periodic revalidation at defined intervals.
PIC Document PR 1/99-2 "Cleaning Validation" Section 3.1 (April, 2000)
A validation protocol is required laying down the general procedures on how cleaning processes will be validated. It should include the following:
• The objective of the validation process
• Responsibilities for performing and approving the validation study
• Description of the equipment to be used
• The interval between the end of production and the beginning of the cleaning procedures
• Cleaning procedures to be used for each product, each manufacturing system or each piece of equipment.
• Any routine monitoring requirement
• Sampling procedures, including the rationale for why a certain sampling method is used
• Data on recovery studies where appropriate
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Proposed Validation Standard VS-3
• Analytical methods including the limit of detection and the limit of quantitation of those methods
• The acceptance criteria, including the rationale for setting specific limits
• When revalidation will be required
PIC Document PR 1/99-2 "Cleaning Validation" Section 3.3 (April, 2000)
A final validation report should be prepared. The conclusions of this report should state if the cleaning process has
been validated successfully. Limitations that apply to the use of the validated method should be defined (for example, the analytical limit at which cleanliness can be determined). The report should be approved by management.
PIC Document PR 1/99-2 "Cleaning Validation" Section 3.4 (April, 2000)
The cleaning process should be documented in an SOP. Records should be kept of cleaning performed in such a
way that the following information is readily available:
• The area or piece of equipment cleaned
• The person who carried out the cleaning
• When the cleaning was carried out
• The SOP defining the cleaning process
• The product which was previously processed on the equipment being cleaned
PIC Document PR 1/99-2 "Cleaning Validation" Section 3.5 (April, 2000)
The cleaning record should be signed by the operator who performed the cleaning and by the person responsible for the Production and should be reviewed by Quality Assurance.
PIC Document PR 1/99-2 "Cleaning Validation" Section 4.1 (April, 2000)
Operators who perform cleaning routinely should be trained in the application of validated cleaning procedures.
Training records should be available for all training carried out.
PIC Document PR 1/99-2 "Cleaning Validation" Section 5.1 (April, 2000)
The design of the equipment should be carefully examined. Critical areas (those hardest to clean) should be identified, particularly in large systems that employ semi-automatic or fully automatic clean-in-place (CIP) systems.
PIC Document PR 1/99-2 "Cleaning Validation" Section 5.2 (April, 2000)
Dedicated equipment should be used for products, which are difficult to remove (e.g., tarry or gummy residues
in the bulk manufacturing), for equipment, which is difficult to clean (e.g., bags for fluid bed dryers), or for products with a high safety risk (e.g., biologicals or products of high potency which may be difficult to detect below
an acceptable limit).
PIC Document PR 1/99-2 "Cleaning Validation" Section 6.1 (April, 2000)
The existence of conditions favorable to reproduction of microorganisms (e.g., moisture, sub-strength, crevices,
and rough surfaces) and the time of storage should be considered. The aim should be to prevent excessive microbial contamination.
PIC Document PR 1/99-2 "Cleaning Validation" Section 7.1 (April, 2000)
Samples should be drawn according to a written sampling plan.
Institute of Validation Technology
Proposed Validation Standard VS-3
PIC Document PR 1/99-2 "Cleaning Validation" Section 7.2 (April, 2000)
There are two methods of sampling that are considered to be acceptable: direct surface sampling (swab method)
and the use of rinse solutions. A combination of the two methods is generally the most desirable, particularly in
circumstances where accessibility of equipment parts can mitigate against direct surface sampling.
PIC Document PR 1/99-2 "Cleaning Validation" Section 8.1 (April, 2000)
The efficiency of cleaning procedures for the removal of detergent residues should be evaluated. Acceptable limits should be defined for levels of detergent after cleaning. Ideally, there should be no residues detected. The possibility of detergent breakdown should be considered when validating cleaning procedures.
PIC Document PR 1/99-2 "Cleaning Validation" Section 9.1 (April, 2000)
The analytical methods should be validated before the cleaning validation study is carried out.
PIC Document PR 1/99-2 "Cleaning Validation" Section 9.2 (April, 2000)
The analytical methods used to detect residuals or contaminants should be specific for the substance to be assayed
and provide a sensitivity that reflects the level of cleanliness determined to be acceptable by the company.
PIC Document PR 1/99-2 "Cleaning Validation" Section 10.1 (April, 2000)
The pharmaceutical company's rationale for selecting limits for product residues should be logically based on a consideration of the materials involved and their dosage regimes. The limits should be practical, achievable, and verifiable.
PIC Document PR 1/99-2 "Cleaning Validation" Section 10.2 (April, 2000)
The approach for setting limits can be:
• Product specific cleaning validation for all products
• Grouping into product families and choosing a "worst case" product
• Grouping into groups of risk (e.g., very soluble products, similar potency, highly toxic products, difficult to detect)
PIC Document PR 1/99-2 "Cleaning Validation" Section 10.3 (April, 2000)
Carry-over of product residues should meet defined criteria, for example the most stringent of the following three criteria:
(a) No more than 0.1 % of the normal therapeutic dose of any product will appear in the maximum daily dose of
the following product.
(b) No more than 10 ppm of any product will appear in another product.
(c) No quantity of residue will be visible on the equipment after cleaning procedures are performed. Spiking
studies should determine the concentration at which most active ingredients are visible
(d) For certain allergenic ingredients, penicillins, cephalosporins, or potent steroids and cytotoxics, the limit should
be below the limit of detection by best available analytical methods. In practice, this may mean that dedicated
plants are used for these products. 0
About the Author
William E. Hall, PhD., is the President of Hall & Associates, where he provides consulting on cleaning validation,
process validation, and compliance issues for the pharmaceutical industry. Dr. Hall is internationally recognized
as an authority on the subject of cleaning validation. Dr. Hall serves on the Editorial AdviSOry Board of the Journal
of Validation Technology, and is a member of the Institute of Validation Technology Hall of Fame. Dr. Hall received
his PhD. from the University of Wisconsin, and is a former professor at the University of North Carolina. Dr. Hall
can be reached by phone at 910-458-5068, by fax at 910-458-5068, or bye-mail at CleanDoct@aol.com.
Journal of Validation Technology