PEER-R EV IEW ED
Analysis of Degradation
Properties of Biopharmaceutical
Active Ingredients as Caused
by Various Process Cleaning
Agents and Temperature
Kathleen Kendrick, Alfredo Canhoto, and Michael Kreuze
ABSTRACT
INTRODUCTION
The biotechnology industry assumption in the cleaning of
product contact equipment is that both acidic and caustic
solutions and high temperatures effectively degrade residual
active pharmaceutical ingredients. The necessity of conducting research to qualify such assumptions is relevant when
considering the cleaning of equipment between batches
of the same product or cleaning for product changeover
within a manufacturing suite. The focus of this study was to
examine the claim that “typical” cleaning regimens found in
the biopharmaceutical industry effectively degrade residual
proteins from an equipment surface. Degradation studies seek to understand the breakdown effect a cleaning
agent and/or heat has on particular protein therapeutics.
By utilizing sodium dodecyl sulfate polyacrylamide gel
electrophoresis technology to quantify the degradation
effects on protein therapeutics, it was discovered that all
caustic cleaning agents did not completely degrade the
protein. While most basic and neutral pH cleaning solutions
did have a degradation effect on protein therapeutics, most
acidic cleaners did not. The small percentage of acidic cleaners that successfully degraded protein therapeutics required
the presence of elevated temperature. Through analysis
of degradation data, it was found that the combination of
heat and a caustic cleaning agent together yielded the most
degradation of a protein and was most effective.
The effects of cleaning agents used during cleaning operations on equipment utilized in the manufacture of protein
therapeutic products, including active pharmaceutical
ingredients (APIs), are not completely understood. A common perception in the biopharmaceutical industry is that
caustic cleaning agents degrade (or break down) protein
therapeutics, and that acidic, neutral, or enzymatic cleaners
may or may not have similar effects. Moreover, a similar
degradation effect may be observed with a high temperature and may be time dependent. While this mechanism
is not completely characterized, the assumptions and
potential implications may affect a manufacturer’s cleaning program philosophy. For example, should a manufacturer test for API residuals using a product-specific assay
when the cleaning agent breaks it down? Furthermore, a
strong science and risk-based approach to cleaning should
define cleaning conditions, such as concentration, time,
and temperature based upon a fully characterized cleaning process. The purpose of this report is to answer these
questions by presenting the results of a study that examined
and ultimately defined the degradation effects of cleaning solutions, temperature, and exposure duration on
biopharmaceutical active ingredients.
In addition to degradation, the mechanism investigated
in this report, a cleaning agent may employ sequestering,
For more Author
information,
go to
gxpandjvt.com/bios
[
gxpandjv t.com
ABOUT THE AUTHORS
Kathleen Kendrick is Validation Technology Manager at Wyeth Biotech in Andover, MA. Contact Ms.
Kendrick regarding this paper at kkendrick@wyeth.com or 978.247.1474. Alfredo Canhoto, Ph.D., is
Validation Manager, Cell Culture and Utilities at Genzyme Corporation in Allston, MA. Michael Kreuze
is Principal Engineer, Wyeth Biotech in Andover, MA.
Journal
of
Validation T echnology [Summer 2009]
69
PEER-R EV IEW ED
emulsification, dispersion, wetting, chelating, or saponification for removal of soils from surfaces. Some of these
mechanisms (i.e., sequestering, emulsification, chelating,
and saponification) are not immediately relevant to cleaning API. Moreover, mechanisms such as dispersion and
wetting are expected to be a precursor to the degradation
effect studied in this report. Degradation is, therefore, an
appropriate tool to study and characterize common cleaning regimes in an API manufacturing environment.
In order to characterize the degradation effects, a novel
application of existing technologies was developed. The
technique employed the use of sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) to determine whether the API degraded into smaller fragments
of the original protein therapeutic. SDS-PAGE separates
proteins solely by their size. Sodium dodecyl sulfate (SDS)
is an anionic detergent, which micellizes proteins by wrapping around or binding the polypeptide backbone. In so
doing, SDS confers a net negative charge to the polypeptide
in proportion to its length; the denatured polypeptides
become rods of negative charge with equal charge or charge
densities per unit length. In addition, SDS-PAGE also
separates proteins independently of pH and subsequent
amphoteric changes. These characteristics render SDSPAGE an appropriate analytical technique for quantifying
the degree of degradation of the studied proteins.
Cleaning process efficacy is achieved through a combination of time, action, chemical/concentration, and temperature (TACT). This study did not evaluate the effect of action,
because it is directly related to how process equipment is
cleaned (i.e., static soak versus spray device). Because this
is a laboratory study not specific to any particular piece of
equipment, the “action” contribution was not examined.
However, the remaining factors were studied at length,
applying experimental values typically found in a biopharmaceutical manufacturing setting.
A number of cleaning agents and APIs were examined
in order to determine if the effects and results observed
were applicable categorically or simply specific to a certain
entity. Cleaning chemicals were divided into caustics,
acids, and neutrals including enzymatic cleaners in order
to determine if there were common effects related to a
particular pH range. The materials of construction chosen
represent the predominant equipment surface materials
typically found in bulk biopharmaceutical manufacturing processes. Refer to the “Experimental Materials and
Design” section for more detail on experimental variables
and study design.
This study investigated the effects of time, chemical, and
temperature on therapeutic protein degradation rate in
order to gain additional information on the independent
70
Journal
of
Validation T echnology [Summer 2009]
contribution of the variables to the efficacy of a cleaning
cycle. Analyzing the degradation characteristics of each
cleaning agent would be expected to aid in answering
questions such as: Which cleaning agent works best for
APIs? Do all basic and neutral cleaning agents degrade
APIs? Do all acidic cleaning agents degrade APIs? How
long does it take for the protein to degrade? Answering
questions such as these will result in more effective and
efficient cleaning procedures and could result in cost savings for a company (1).
EXPERIMENTAL DESIGN, MATERIALS/
EQUIPMENT, AND PROCEDURES
Design
The following components were utilized in the design
of the studies.
Cleaning Agents. An array of cleaning agents was
selected for these studies. The cleaning agents selected
were divided up into three categories based on their working pH: acidic, neutral, and basic. With a large number of
cleaning agents, it was probable that certain generalizations could be established representative of each of these
three categories. Tables I, II, and III list each cleaning
agent within its specific category as well as the name of
the manufacturer, pH of the solution (neat and actual
tested), and the concentration(s) tested.
The cleaning agent concentrations were determined
based on the manufacturer’s recommended concentrations for cleaning proteinaceous soils. For commodity
chemicals, such as NaOH, KOH, Cocktail, and phosphoric acid, the concentrations used for this experimentation are those typically used in cleaning cycles. The pH
values of the neat and diluted cleaning solutions were
measured using pH strips.
API Soils. Five representative API soils were selected
to use during these experiments (see Table IV). The soils
selected were chosen based on availability and as representatives of typical soils manufactured at Wyeth Biotech,
Andover, MA.
Degradation experiments were carried out for each
API in combination with each cleaning agent. The gels
were set up in a manner to test multiple variables at once.
Each gel tested for the effect of cleaning agent alone,
heat (temperature) alone, and the combination of heat
(temperature) and cleaning agent. Within each of these
testing criteria contact time was varied to determine if
there was time dependency. Time intervals of 6, 30,
and 60 minutes were tested to determine its influence.
The maximum of 60 minutes was chosen, as this was
anticipated to be an effective duration for the cleaniv thome.com
K AT H L E E N K E N DR IC K , A L F R E DO C A N HO T O, A N D M IC H A E L K R E UZ E
Table I: Basic (test solution pH greater than 9) cleaning chemicals and specifications.
Chemical Name
Manufacturer
pH of Neat Solution
Concentration Tested
pH of Solution Tested
CIP100
Steris
~14
2.0%
>13
NaOH
Mallinckrodt
14
0.1N
>12
0.5N
1.0N
KOH
Mallinckrodt
14
0.1N
>13
CIP92
Ecolab
14
2.0%
10-11
CIP150
Steris
14
1.0%
11-12
CIP1000
Steris
~14
2.5%
>13
SoluJet
Alconox
~14
2.0%
~12
Cocktail
Proprietary Wyeth
14
N/A
>13
Foam 140
Steris
14
1.0%
~11
Chematic 82
Dober
14
2.0%
>14
Tergazyme
Alconox
N/A*
1.0%
10
CIP 95NA
Ecolab
14
2.0%
>13
0.5N
*The neat concentration of Tergazyme is a powder.
Table II: Neutral (test solution pH 7 ± 2) cleaning chemicals and specifications.
Chemical Name
Manufacturer
pH of Neat Solution
Chematic99
Dober
10
2.5%
~6
CIP90
Steris
11
2.0%
~6
CosaPur80
Ecolab
9
0.5%
~6
CosaPur84
Ecolab
~9
0.5%
6-7
DA-7645
Steris
10
2.0%
~6.5
ing agent exposure portion for a cleaning cycle. To
aid in the comparison of the results were two lanes of
controls. The controls were the same dilution as the
experimental lanes and added to the gel just prior to
execution. The final lane was filled with a molecular
marker. This marker aided in determining the amount
of effect (degradation or aggregation) that was occurring due to the varied conditions to which the protein
was introduced.
Materials and Equipment
The following materials and equipment were utilized
in the studies:
• Gels—ClearPAGE 4-20% gradient Cat# FK42012
• Gel box—C.B.S. Scientific Co model DCX-700
• Power supply—VWR model EPS 4000
• Orbital shaker—VWR model DS-500
• Coomassie Brilliant Blue
gxpandjv t.com
Concentration Tested
pH of Solution Tested
• Molecular weight markers—Lonza ProSieve Color
Protein Markers Cat #50550 (173,117,76,51,38,26,
8,12,and 9 kDa).
SDS-PAGE Solutions. The following SDS-Page solutions were used:
• Loading Buffer (4X Solution)
• Sodium Dodecyl Sulfate (SDS)
• Ethylenediamineteracetic Acid Disodium Salt
(EDTA)
• Tris pH 6.8
• Glycerol
• Purified Water
• Running Buffer (10X Solution)
• Tris Base
• Glycine
• SDS
• Purified Water
Journal
of
Validation T echnology [Summer 2009]
71
PEER-R EV IEW ED
Table III: Acidic (test solution pH less than or equal to 5) cleaning chemicals and
specifications.
Chemical Name
Manufacturer
pH of Neat Solution
Concentration Tested
CosaPur85
Ecolab
3
1.0%
4-5
Foam 240
Steris
1
1.0%
<2
CIP 72
Steris
1
2.0%
<2
CIP 200
Steris
1-2
2.0%
~1
CIP 220
Steris
1-2
1.5%
<2
Phosphoric Acid
EMD
1-2
0.33%
<2
Chematic91
Dober
1
2.0%
<2
Chematic9301
Dober
2
2.0%
~2
Table IV: API information.
Protein Name
Average
Concentration of
BDS (g/L)
Observed
Size (kDa)
Working
Concentration
Product A
4
55
1:20
Product B
17
150
1:50
Product C
15
20
1:100
Product D
55
150
1:200
Product E
45
200
1:100
Table V: Working concentration gel #1 loading
scheme.
Lane
Contents
1
38 μL of 1:50 BDS dilution 12μL of loading buffer
2
38 μL of 1:50 BDS dilution 12μL of loading buffer
3
Blank
4
38 μL of 1:100 BDS dilution 12μL of loading buffer
5
38 μL of 1:100 BDS dilution 12μL of loading buffer
6
Blank
7
38 μL of 1:200 BDS dilution 12μL of loading buffer
8
38 μL of 1:200 BDS dilution 12μL of loading buffer
9
Blank
10
38 μL of neat BDS dilution 12μL of loading buffer
11
38 μL of neat BDS dilution 12μL of loading buffer
12
12 μL molecular weight marker 4μL loading buffer
72
Journal
of
Validation T echnology [Summer 2009]
pH of Solution Tested
• Fixing Solution
• Methanol
• Acetic Acid
• Purified Water
• Coomassie Brilliant Blue Staining Solution
• Methanol
• Acetic Acid
• Purified Water
• Coomassie Brilliant Blue R-250 powder
• Destaining solution
• Methanol
• Acetic Acid
• Purified Water.
Experimental Procedures
The following procedures were used in the studies.
Working Concentration Determination. Dilutions of bulk drug substance (BDS) using USP water
for injection (WFI) to be studied were prepared in the
following concentrations: 1:50, 1:100, 1:200, 1:400,
and 1:800. Gels were loaded as shown in Tables V
and VI. All tubes of solutions were vortexed directly
before loading.
Running Gels. Gels were placed in the gel box
and run at 100 mA. A gel was considered complete
when the dye front progressed down to just above the
bottom of the gel casing.
Staining and Destaining Gels. Once the gel was
complete, it was rinsed with WFI. The gel was then
immersed in fixing solution and agitated at 50 RPM.
The gel was again rinsed with WFI and then immersed
in staining solution and agitated at 50 RPM. Finally,
the gel was rinsed once again with WFI and immersed in
destaining solution and agitated at 50 RPM twice.
Gel Analysis Using a Densitometer. Gels were
scanned using “Quantity One” software (version #4.2.2).
Data was analyzed by comparing the average optical
iv thome.com
K AT H L E E N K E N DR IC K , A L F R E DO C A N HO T O, A N D M IC H A E L K R E UZ E
densities (OD) of the bands in lanes 1-9 to OD for lanes
10 and 11 (control lanes). The difference indicated the
percent degradation of protein in a given lane.
Degradation Experiments. The procedure for
degradation experiments was executed as described
previously except gels for these experiments were
loaded as described in Table VII. All other steps for
running, staining, and analyzing gels were the same
as described previously.
RESULTS
The goal of the degradation experiment was to use SDSPAGE technology to quantify and qualify the degradative
effects of temperature and/or cleaning agents on various
APIs. This was accomplished by treating proteins with
cleaning agents at concentrations as recommended by
the vendor. The treatments that were quantified on an
SDS-PAGE gel included the effect heat (temperature)
had on the protein, cleaning agent effects on the protein,
and the combined effect of the cleaning agent and heat
(temperature) on the protein. Each gel also contained
samples of the untreated protein as a control as well as
protein molecular weight markers. Analysis of the gels
resulted in a total of seven different effect categories
(see Table VIII).
Figures 1-3 are some examples of the different effects
observed on the gels. Lanes 1, 2, and 3 were specifically designed to address the cleaning agent and protein
interaction vs. time. Lanes 4, 5, and 6 were specifically
designed to explain the interaction between cleaning
agent, elevated temperature, and the protein vs. time.
Lanes 7, 8, and 9 were specific to the interaction between
the protein and heat alone without the influence of a
cleaning agent. Lanes 10 and 11 (protein only) are the
control lanes. Lane 12 is a molecular weight marker
used to determine the approximate molecular weight
of the protein.
Degradation Effect Pattern 1—No Effect
Figure 1 illustrates the “No Effect” observed by use of 2%
Chematic 9301 with Product A BDS 1:20. The overall
effect observed with this gel is Pattern 1-No Effect.
From the gel and the corresponding densitometer readings, it can be seen that there was no apparent aggregative or degradative effect on the API. Lanes 1-9 remain
consistent with the controls in lanes 10 and 11.
Degradation Effect Pattern 4—Heat Alone,
Chemical Enhanced
Figure 2 illustrates the “Heat Alone, Chemical Enhanced”
observed by use of 1% Foam 140 with Product B BDS 1:50.
gxpandjv t.com
Table VI: Working concentration gel #2
loading scheme.
Lane
Contents
1
38 μL of 1:400 BDS dilution 12μL of loading buffer
2
38 μL of 1:400 BDS dilution 12μL of loading buffer
3
Blank
4
38 μL of 1:800 BDS dilution 12μL of loading buffer
5
38 μL of 1:800 BDS dilution 12μL of loading buffer
6
Blank
7
38 μL of neat BDS dilution 12μL of loading buffer
8
38 μL of neat BDS dilution 12μL of loading buffer
9
Blank
10
12 μL molecular weight marker 4μL loading buffer
11
Blank
12
Blank
The overall effect observed with this gel is Pattern 4-Heat
Alone, Chemical Enhanced.
The gel and the densitometry data show that lanes 13 didn’t affect the API in comparison to the controls in
lanes 10 and 11. Heat only (lanes 7-9) aids in degradation
of the protein; however, the degradation is enhanced
with the addition of chemical (lanes 4-6). Increased
temperature alone will work but adding chemical will
enhance the effect.
Degradation Effect Pattern 5—Heat Effect,
Chemical Stabilized
Figure 3 illustrates the “Heat Effect, Chemical Stabilized” observed by use of 2% CIP 200 with Product
B BDS 1:50. The overall effect observed with this gel
is Pattern 5-Heat Effect, Chemical Stabilized.
In this gel and in the densitometry reading, it was
observed that when the solution is at ambient temperatures (lanes 1-3) there is no apparent degradation.
When the temperature is increased to 60°C (lanes 7-9)
a significant amount of degradation is seen. Lanes
4-6 are also exhibiting an effect but at a slower rate
than lanes 7-9 and the effect appears to have been
stabilized.
Summary of Effects
Table IX summarizes effects for the five biopharmaceutical proteins tested in this study.
Of the 139 total gel effects that were observed, Chemical Alone, Heat Enhanced (24%) and Only Chemical and
Heat (27%) were the two categories observed most often.
When only focusing on the basic and neutral cleaning
Journal
of
Validation T echnology [Summer 2009]
73
PEER-R EV IEW ED
Table VII: Loading scheme and experimental
sample preparation for degradation studies.
Lane
Contents
Protein
(working
concentration)
Chemical
(working
concentration)
Time
(minutes)
Temperature
Loading
Buffer
1
19ml
19ml
5
Ambient
12ml
2
19ml
19ml
30
Ambient
12ml
3
19ml
19ml
60
Ambient
12ml
4
19ml
19ml
5
60oC
12ml
5
19ml
19ml
30
60 C
12ml
6
19ml
19ml
60
60 C
12ml
7
38ml
N/A
5
60oC
12ml
8
38ml
N/A
30
60 C
12ml
9
38ml
N/A
60
60oC
12ml
10
38ml
N/A
N/A
Ambient
12ml
11
38ml
N/A
N/A
Ambient
12ml
12
Molecular weight standard (12ml) and 4ml loading buffer
o
o
o
agents, the same two categories were observed most often.
However, when acidic cleaning agents were analyzed the
top two effects that were observed were Heat Effect, Chemical Stabilizing (35%) and No Effect (30%).
DISCUSSION
The preceding experiments explored the degradation
characteristics of representative APIs in the presence
of various cleaning agents and temperatures. The degradation effect in most of the cases increased with an
increase in temperature and time of exposure. Some
cleaning agents had no degradation effect on protein
APIs alone. However, when combined with increased
temperature, they had an effect on either protein stability or aggregation. These findings and the novel use of
standard biochemical tools and techniques have several
implications for the biopharmaceutical manufacturing
industry specific to cleaning regimes and associated
cleaning validation efforts.
The degradation experiments were initially expected
to fall into one of four categories: no effect; cleaning agent (chemical) alone induced effect; heat alone
induced effect; or combination of cleaning agent
(chemical) and heat induced effect. Examination of
the empirical results revealed that there were more
subtleties in the resulting effects. The original effects
needed to be further subdivided to acknowledge subtleties such as: chemical alone heat enhanced; heat alone
chemical enhanced; chemical and heat synergy; only
74
Journal
of
Validation T echnology [Summer 2009]
chemical and heat effect; heat effect chemical stabilizing; and chemical effect heat stabilizing.
The results indicate that for basic and neutral chemicals,
the majority of the effects were primarily chemical with
a time dependent heat enhancement or synergy. Conversely, for the acidic chemicals, the primary effect was
heat induced with a secondary chemical effect of either
causing some degradation or aggregation. This portion
of the data matches with the common perception that
basic chemicals such as NaOH and KOH are the primary
cleaning agents for proteinaceous soils. This generalization cannot be extended to all formulated caustic or basic
cleaning agents as not all combinations of cleaning chemistry and protein displayed degradation of the proteins
tested. There should be an attempt made by organizations involved in cleaning development and validation
to understand the effect of their chosen cleaning agent
and regimen on their API or residual process soils. This
knowledge is fundamental in justifying cleaning agent
chemistries, concentrations, times, and temperatures
chosen in validating a robust cleaning cycle.
Furthermore, by understanding the strength and effects
of cleaning cycle parameters, the risks associated with the
changeover of equipment from one manufacturing process
or API to the next can be mitigated. Likewise, residual
drug substance carryover concerns and testing should
be viewed in light of the effect of the cleaning agent on
the process soils and API. If a protein is clearly degraded
with a chosen cleaning chemistry, concentration, time
and temperature, then the use of product specific assays to
demonstrate residual removal are not appropriate markers
of cleanliness. A non-specific assay such as total organic
carbon (TOC) is more applicable and appropriate to ensure
the removal of organic APIs or proteins that, with the use of
the above-described techniques, have been demonstrated
to be degraded.
The benefits of this work can be illustrated in a multiproduct production services area. By having these degradation studies complete for multiple soils, an argument can be
made that small equipment parts may be cleaned together
in a single load of a washer, assuming that the washer has
been preprogrammed to deliver cleaning parameters that
meet the threshold necessary for all individual soils contained within the load. This understanding could justify
the necessary wash and rinse times and temperatures of
a cleaning cycle.
Fundamentally this set of experiments sought to address
the axiom of “Know thy Process.” Because the cleaning process is so integral to the biopharmaceutical manufacturing
industry, many resources and time are utilized to qualify a
cleaning procedure. By performing studies to understand
iv thome.com
K AT H L E E N K E N DR IC K , A L F R E DO C A N HO T O, A N D M IC H A E L K R E UZ E
Table VIII: Gel effect categories.
Classification #
Name of Effect
1
No Effect
Description
None
No effect from any independent or combined temperature or chemical interaction
Primarily Chemical
2
Chemical Alone, Heat Enhanced
An increased effect when chemical and temperature
were combined, but no effect with temperature alone
3
Chemical Effect and Heat Stabilizing
Chemical had an effect, but with the addition of heat
the strength of the effect decreased (aggregation
and/or polymerization fall into this category)
4
Heat Alone, Chemical Enhanced
An increased effect when heat and chemical were
combined, but no effect with chemical alone
5
Heat Effect and Chemical Stabilizing
Heat had an effect, but with the addition of chemical the strength of the effect decreased (aggregation
and/or polymerization fall into this category)
Primarily Heat
Combined Chemical and Heat
6
Only Chemical and Heat Effect
No effects for heat alone or chemical alone, only an
effect when chemical and heat were combined
7
Chemical and Heat Synergy
Chemical had an effect and heat had an effect, but
combined there was a stronger effect (aggregation
and/or polymerization fall into this category)
the effects of cleaning chemicals on process soils, companies can design a cleaning cycle that is more efficient and
increase the level of compliance with regulatory expectations. As required by risk assessment tools such as hazard
analysis and critical control points (HACCP) and failure
mode and effects analysis (FMEA), there is an increased
emphasis on scientific fundamentals and reducing risk
by characterizing process soils and their interactions with
cleaning agents and parameters that may be necessary to
address the same. In addition, acute process understanding, done in a proactive manner outside the manufacturing
facility, may lead to cost savings.
CONCLUSIONS
The application of SDS-PAGE to investigate and characterize the effects of cleaning processes on APIs provides a
powerful tool in support of cleaning process development
and validation. Gel electrophoresis indicates degradation
(or aggregation) products due to contact with cleaning
solution and/or elevated temperature.
Regarding what these techniques demonstrated for
actual effects of cleaning agents on API degradation, conclusions are made categorically as follows:
• Most but not all caustic cleaning agents degrade protein
therapeutics. This study showed that when cleaning
gxpandjv t.com
Table IX: Overall effects observed for the five
biopharmaceutical protein therapeutics tested.
Effect
Total
%
Base/
Neutrals
%
Acids
%
No Effect
22
16
10
10
12
30
Chemical Alone,
Heat Enhanced
34
24
34
33
0
0
Chemical Effect,
Heat Stabilizing
4
3
4
5
0
0
Heat Alone, Chem- 8
ical Enhanced
6
6
6
2
5
Heat Effect,
Chemical Stabilizing
18
13
4
5
14
35
37
27
28
28
9
23
Chemical and Heat 16
Synergy
12
13
13
3
7
Chemical Effect
Heat Effect
Combined Effect
Only Chemical and
Heat
Totals
139
Journal
of
99
40
Validation T echnology [Summer 2009]
75
PEER-R EV IEW ED
Figure 1: 2% Chematic 9301 with PRODUCT A BDS (1:20).
The overall effect observed with this gel is Pattern 1- No Effect.
Lane Number
Lane Contents
Densitometer Reading
1
Protein and Solution, 5 min, Ambient
90.32
kDa
174
2
Protein and Solution, 30 min, Ambient
90.32
117
3
Protein and Solution, 60 min, Ambient
87.10
4
Protein and Solution, 5 min, 60°C
90.32
5
Protein and Solution, 30 min, 60°C
93.55
6
Protein and Solution, 60 min, 60°C
106.45
7
Protein, 5 min, 60°C
83.87
8
Protein, 30 min, 60°C
77.42
9
Protein, 60 min, 60°C
87.10
10
Protein Control
93.55
11
Protein Control
103.23
12
Protein Markers
N/A
1 2 3 4 5 6 7 8 9 10 11 12
76
51
39
26
19
13
Figure 2: 1% Foam 140 vs. PRODUCT B BDS (1:50).
The overall effect observed with this gel is Pattern 4- Heat Alone, Chemical Enhanced.
1 2 3 4 5 6 7
8 9 10 11 12
kDa
174
117
76
51
39
26
19
13
Lane Number
Lane Contents
Densitometer Reading
1
Protein and Solution, 5 min, Ambient
100.00
2
Protein and Solution, 30 min, Ambient
70.59
3
Protein and Solution, 60 min, Ambient
100.00
4
Protein and Solution, 5 min, 60°C
58.82
5
Protein and Solution, 30 min, 60°C
35.29
6
Protein and Solution, 60 min, 60°C
11.76
7
Protein, 5 min, 60°C
64.71
8
Protein, 30 min, 60°C
35.29
9
Protein, 60 min, 60°C
41.18
10
Protein Control
105.88
11
Protein Control
94.12
12
Protein Markers
N/A
agents were effective at degrading API, they were both
time and concentration dependent.
•T
he assumption that all high temperature-cleaning
cycles cause a break down of protein therapeutics has
also been refuted. Furthermore, elevated temperatures
combined with caustic cleaning agents were shown
to be the most effective combination; however, this
generalization was not true for all situations.
• With respect to the acidic cleaning agents, in general
they did not exhibit a high degree of degradation.
When degradation was observed, higher temperature
enhanced the cleaning effect.
• The industry-wide assumption that heat and caustic
76
Journal
of
Validation T echnology [Summer 2009]
cleaning together are the most effective combination
was confirmed.
It is also important to note that aggregation was
observed in many of the SDS-PAGE results. This
aggregation was observed with both acidic and
basic cleaning agents. It has been demonstrated that
aggregation is affected by external factors including
temperature, pH, and protein concentration, all of
which were factors in this study (2). For this reason,
more testing should be done to determine the cause of
aggregation in the select gels where it was observed.
iv thome.com
K AT H L E E N K E N DR IC K , A L F R E DO C A N HO T O, A N D M IC H A E L K R E UZ E
Figure 3: 2% CIP 200 with PRODUCT B BDS (1:50).
The overall effect observed with this gel is Pattern 5- Heat Effect, Chemical Stabilized.
1 2 3 4 5 6 7
8 9 10 11 12
kDa
174
117
76
51
39
26
19
13
Lane Number
Lane Contents
Densitometer Reading
1
Protein and Solution, 5 min, Ambient
285.71
2
Protein and Solution, 30 min, Ambient
228.57
3
Protein and Solution, 60 min, Ambient
207.14
4
Protein and Solution, 5 min, 60°C
207.14
5
Protein and Solution, 30 min, 60°C
121.43
6
Protein and Solution, 60 min, 60°C
114.29
7
Protein, 5 min, 60°C
78.57
8
Protein, 30 min, 60°C
35.71
9
Protein, 60 min, 60°C
21.43
10
Protein Control
114.29
11
Protein Control
78.57
12
Protein Markers
N/A
ACKNOWLEDGMENTS
The authors thank Richard Wright and Priscilla Jennings
for their technical assistance and numerous discussions
throughout the project. In addition, thanks to all involved
with long hours of degradation runs, data entry, review,
and technical assistance: Meghan Pearson, David Hindson,
Rod Azadan, Kristen Nobles, James Snow, Jennifer Strand,
Stanley Garib, Kate Crotty, Kelli Barrett, Linda Marshall,
Debra Pierzynski, and Brandon Sullivan
REFERENCES
1.FDA, Guide to Inspections, Validation of Cleaning Processes,
1993.
2.W. Wang, “Protein Aggregation and its Inhibition in Biopharmaceutics,” International Journal of Pharmaceutics, 289, 1-30,
2005. JVT
GENERAL REFERENCES
Bailey, Rochelle, “Effect of 0.1N NaOH on rAHF Activity and
Structural Integrity,” Genetics Institute, 1991.
Bowser, Tim, “CIP Systems Critical to Validatable, Cost-Effective
Cleaning,” Cleanroom, Volume 21, No.02, February 2007.
Dawn Chemical, Inc. Website: www.dawnchemical.com/shop/
scripts/page.asp?p=_chemistryofcleaning&s=dawnchemical,
viewed on August 3, 2006.
Tsai, A.M., van Zanten, J.H., Betenbaugh, M.J., “Study of Protein Aggregation Due to Heat Denaturation: A Structural
Approach Using Circular Dichroism Spectroscopy, Nuclear
Magnetic Resonance, and Static Light Scattering,” Biotechnology and Bioengineering, 59 no.3, 273-280 (1998).
gxpandjv t.com
Lemmer, K., Mielke, M., Pauli, G., Beekes, M., “Decontamination of surgical instruments from prion proteins: in vitro
studies on the detachment, destabilization and degradation
of PrPSc bound to steel surfaces,” Journal of General Virology,
85, 3805-3816 (2004).
Cabra, V., Arreguin, R., Vazquez-Duhalt, R., Farres, A., “Effect
of temperature and pH on the secondary structure and processes of oligomerization of 19kDa alpha-zein,” Biochimica
et Biophysica Acta, 1764, 1110-1118 (2006).
ARTICLE ACRONYM LISTING
APIsActive Pharmaceutical Ingredients
BDSBulk Drug Substance
EDTAEthylenediamineteracetic Acid Disodium Salt
FMEAFailure Mode and Effects Analysis
HACCPHazard Analysis and Critical Control Points
ODOptical Densities
SDSSodium Dodecyl Sulfate
SDSPAGESodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis
TACTTime, Action, Chemical/Concentration, and
Temperature
TOCTotal Organic Carbon
WFIWater for Injection
Journal
of
Validation T echnology [Summer 2009]
77