B
i o
P
r o c e s s Technical
Implementation of Single-Use
Technology in Biopharmaceutical
Manufacturing
An Approach to Extractables and Leachables Studies,
Part Three — Single-Use Systems
Weibing Ding and Jerold Martin
T
he increasing application of
single-use components and
systems in bioprocessing
represents one of the most
significant changes in biopharmaceutical
manufacturing in recent times. Driven
by various factors such as improved
efficiency, flexibility, and economics,
this trend also presents specific
challenges to end users. In one industry
review by Langer, extractables and
leachable compounds from disposable
components were considered by end
users to be a major area of potential
concern regarding safety, efficacy, and
stability of the pharmaceutical product
(1). In a more recent survey by the BioProcess Systems Alliance (BPSA),
however, extractables and leachable
compounds were considered by only
13% of respondents to be a barrier in
integrating single-use technologies into
existing or new processes (2). The
Product Focus: All biologics
Process Focus: Upstream and
downstream processing
Who Should Read: Product/process
development, QA/QC, and analytical
Keywords: analytical methods,
process validation, disposables
Level: Intermediate
52 BioProcess International
N ovember 2010
apparent change in thinking may be
due, in part, to an increased amount of
information available in published case
studies, reviews, and industry guides
such as those issued by BPSA (3, 4)
covering regulatory issues, risk
assessment, and test programs.
Extractables are typically determined
with laboratory tests using standard
extraction fluids termed model solvents.
Such tests are designed to exceed worstcase product and process conditions and
reveal chemical entities that may migrate
from process components into the final
product as potential leachables.
The quality and quantity of materials
extracted from an organic polymer
component depends not only on the
materials of construction, but also on the
contact fluid composition, temperature,
contact area, and contact time. In a
multicomponent single-use system, the
importance of each parameter in the
migration of potential leachable
compounds will vary according to
component type and process conditions.
For example, a sterile connector may
have only transient exposure to a fluid
during transfer, whereas a flexible
biocontainer may be in contact with
product or process fluid for many hours,
days, or even months. Similarly, a typical
small capsule filter with an effective
filtration area of 0.15 m2 may have an
internal surface area of ~150 m2
compared with a 0.5-L flexible
Pall life sciences (www.pall.com)
biocontainer with a surface area of only
0.05 m2 ­— 3,000× smaller. For these
reasons, extractables studies should be
designed and performed under
conditions appropriate for each specific
component and process application. That
can provide an accurate and suitable
assessment of the potential for the
component or system, to release
leachables into the drug product.
We applied this customized approach
to two previous studies on extractables.
In the first study, we reported
extractables results from tests on sterile
connectors and membrane filter capsules
(5). The test articles were preconditioned
by gamma irradiation at 50 kGy to
represent worst-case sterilization
conditions and extracted under
exaggerated simulated conditions using a
recirculating system with extraction
times of four hours for ethanol and 24 h
for deionized (DI)water. Qualitative and
Table 1: Single-use components in a test
system
Component
Sterilizinggrade
capsule
filter
Figure 1: Fluid supply system and filter manifold
Materials of Construction
Membrane: PVDF
surface-modified with
hydrophilic acrylate
copolymer; support
and drainage =
nonwoven
polypropylene (PP)
capsule shell/filter;
hardware = PP
Sterile
connector
Quick
connector
Flexible
tubing
Polycarbonate
1-L biocontainer
Low-density
polyethylene
Fluid Supply
PTFE Tubing PTFE Diaphragm
Pump
System Sampling Point for
Negative Controls
Sterile
Connector
Filter Manifold
Sterilizing-Grade
Capsule Filter
Sterile
Connector
Acetal
SEBS* thermoplastic
elastomer; platinumcured silicone
Line 1
Flush Sample
Line 2
Time = 0
54 BioProcess International
N ovember 2010
Line 3
Time =
7 days
* styrene-ethylene-butylene-styrene/PP
copolymer
quantitative extractables results on sterile
connectors using 13 analytical methods
showed very low levels of extractables,
mostly below the limit of detection
(LOD) or no different from the controls.
We concluded that the potential for
these connectors to release leachable
materials into compatible drug products
was very low. For the capsule filters,
higher levels of extractables were
obtained, as expected from the high
internal surface area of the microporous
membrane within the filter. Qualitative
analysis showed that all compounds
detected were consistent with the filter’s
materials of construction, which passed
biological safety tests such as USP <88>
“Biological Reactivity, in Vivo, for Class
VI Plastics.”
In the second study, we characterized
extractables from thermoplastic tubing
and flexible biocontainers (6). For the
tubing, a worst case was represented by
presterilizing with gamma irradiation at
50 kGy and agitating the test fluid of
ethanol or DI water within clamped
segments of tubing for 72 h. Very low
levels of extractables were detected,
many below the limit of quantification
(LOQ ). For the biocontainers, a worst
case was simulated by filling with test
fluid and storing at 40 °C for 30 days.
Results showed very few organic
compounds above the LOD for water
extracts and those identified were in the
low parts per million (ppm) or parts per
billion (ppb) range. Ethanol extracts
gave some additional identifiable
Extraction
Fluid Glass
Reservoir
Total Length of Tubing
from Point A to Final
A Connector Identical for
Flexible Tubing:
A All Six Lines
Platinum- Cured Silicone or SEBS
Thermoplastic Elastomer
Pinch Clamp 1
Pinch Clamp 2
Simultaneous Flow
Through Lines 2– 6 to
Biocontainers in
Line 6 Collection Manifolds
Time =
Line 5 12 months
Time =
Line 4
6 months
Time =
1 month
Table 3: TOC, conductivity, pH and ions in water extracts1
Sample
TOC (ppm) Conductivity
System flush
2.81 ppm
7.08 µS/cm
Storage time: 0
0.18 ppm
1.21 µS/cm
Storage time: 1 month
0.34 ppm
1.82 µS/cm
Storage time: 12 months
0.70 ppm
3.36 µS/cm
Negative control or
0.003 ppm
0.82 µS/cm
blank
1
pH
4.71
5.56
5.46
5.02
5.77
Acetate
0.025 ppm
0.006 ppm
0.006 ppm
0.310 ppm
<0.001 ppm2
Formate
0.245 ppm
0.020 ppm
0.039 ppm
0.043 ppm
<0.002 ppm2
Average values from two test systems; negative control values not subtracted; 2 limit of detection (LOD)
compounds, which were mostly
oligomers of the base polymers or
degradation products of the antioxidants
used in the polymer formulations.
Here we report on extractables studies
performed on a total system containing
all four components previously tested:
capsule filter, sterile connectors, plastic
tubing, and flexible biocontainers. The
test protocol was designed to simulate a
typical operating procedure for this type
of single-use system and under typical
worst-case conditions.
Objectives
We set out to meet four objectives
• To quantify and characterize
extractables from a model
multicomponent, single-use system.
• To study the effect of system
flushing
• To assess the effect of storage and
extraction times of up to one year in
biocontainers
• To develop a method of
determining extractable and potential
leachable compounds for process
validation of single-use processes in
biopharmaceutical manufacturing.
Table 2: Analytical methods used for
assessment of extractables
Extract
Analytical Method
Total organic carbon
(TOC)
Water
X
Ethanol
n/a
pH
X
n/a
Conductivity
X
n/a
Ion chromatography
(IC)
Nonvolatile residue
(NVR)
X
n/a
X
X
Fourier transform
infrared (FTIR)
Ultraviolet spectroscopy
(UV)
Direct-injection gas
chromatography/mass
spectrometry (GC/MS)
Headspace GC/MS
X
X
X
X
X
X
X
X
Derivatization GC/MS
X
X
High performance
liquid chromatography
with UV detection
(HPLC/UV)
Liquid chromatography
/MS (LC/MS; LC/MS/MS)
X
X
X
X
Inductively coupled
plasma/MS (ICP/MS)
X
X
Figure 2: Biocontainer manifold
Figure 3: Procedure for extraction on single-use system
Sterile Connector for
Attachment to Filter
Manifold
Pinch Clamp
Quick
Connector
Assemble
fluid supply
system.
Recirculate
ethanol to
clean system.
Validate cleanliness
using GC/MS UV,
and TOC.
Rinse with
DI water.
SEBS Thermoplastic
ElastomerBased Tubing
Quick
Connector
Pump 2 L of DI
water into Line
1 biocontainers.
Quick
Connector
1-L Biocontainer
Bag
1-L Biocontainer
Bag
Label as
“system
flush” and
analyze.
Flush
System
Figure 4: FTIR spectra of NVRs
Open pinch
clamps on all
Line 1 manifolds.
Close Line 1
and open
clamps to
Lines 2–6.
Connect assembled
manifolds to fluid
supply system.
Pump 5 × 2 L of DI
water simultaneously
to Lines 2–6 and
into biocontainers.
Analyze
Line 2
extract.
Pump 6 × 2-L
samples into PTFE
bottles for
negative controls.
Connect gammairradiated filter and
biocontainer manifolds
by sterile connectors.
Store remaining filled
biocontainers for
specified period and
analyze extracts.
System Flush
Table 4: Nonvolatile residues in extracts1
Flush Sample
4,000
4000
3,500
3500
3,000
3000
2,500
2,000
1,500
1,000
500
System one Year
Wave
2500 number
2000
1500
1000
500
System One Year
4000
4,000
12-Month
Sample
2500
2000
1500
1000
500
2,500
1,000
500
3500
3000
Wavenumber (cm-1)
3,500
3,000
2,000
Wavenumber (cm- )
1,500
1
2
Sample
System flush
Storage time: 0
Storage time:
12 months
Water
1.9 mg
0.4 mg
1.1 mg
Ethanol
119 mg
142 mg
216 mg
Negative control
<LOQ2
<LOQ2
Negative control values not subtracted.
LOQ (Limit of quantification) was 0.1 mg
Wave number
Experiment
Our test system included three
separate segments: f luid supply
system, filter manifold, and
biocontainer manifold (Figure 1).
The f luid supply system provided
clean extraction f luid for the singleuse system manifolds and a sampling
point for negative controls. To
maintain the extraction f luides at
highest purity, we used a
polytetraf luoroethylene (PTFE)
diaphragm pump and tubing with a
glass reservoir. The filter manifold
contained identical lengths of f lexible
tubing between the filter and the
final sterile connector to ensure the
same contact area and extraction
conditions.
Each line was connected to a
biocontainer manifold (Figure 2).
Line 1 was used for the system f lush
sample, and the other five lines were
used for extractables time tests from
zero to 12 months. The biocontainer
manifold was designed to collect 2 L
of extraction f luid using 2-L × 1-L
biocontainer bags. The test manifolds
were gamma irradiated at 50 kGy and
all extractables tests (including
biocontainer storage) were performed
56 BioProcess International
N ovember 2010
at 21 °C. Test solvents were 18
MΩ-cm DI water and 100% ethanol
HPLC grade. The complete study
was performed in duplicate.
The main components in this
model single-use test system were the
same as those used in our previous
extractables studies on the individual
components (5, 6). Table 1 shows
further details.
Test Procedure: Figure 3
summarizes the major stages of the
test procedure.
Analytical Methods: We performed
qualitative and quantitative analyses
on the extracts using 13 methods and
reported details about these methods
in previous studies (5, 6). Table 2 lists
our analyses on water and ethanol
extracts.
Results and Discussion
Because of space restrictions, we can’t
report here our detailed results
(information available from Pall Life
Sciences on request). However, the
main and the most significant results of
the extractables analyses are presented
here, which can be conveniently
divided into three groups.
Group 1 — TOC, Conductivity, pH,
and Ion Chromatography: We
performed these analyses on water
extracts only (Table 3).
TOC: TOC levels, after a system
flush, were very low at <1 ppm, even
after 12 months storage and
extraction. The initial system-flush
sample showed, as expected, a higher
value (2.81 ppm) due to the removal of
readily soluble compounds by the 2-L
flush volume.
In our previous extractables
studies, we established that the bulk
of the extractables were derived from
the high-area filter membrane (5).
Those results suggest that best
practice for lowest leachable
compounds is to perform an initial
pref lush, whenever possible, of either
the full system or the filter, and to
discard the f lushed liquid.
Conductivity: The conductivity
values showed, after system flush, only
a small increase over negative control
and a slow rise in values over the 12
month storage and extraction period.
pH: After the system flush, the pH
values showed a shift of 0.21 units at
zero time rising to 0.75 units after 12
months. These shifts are within one
pH unit and in compliance with US
Pharmacopoeia requirements.
Ion Chromatography: We used ion
chromatography to measure acetate
and formate levels. Both compounds
can be found in small quantities in
most plastics and may come from the
raw materials, but they are also the
smallest breakdown products of
larger molecules and can be a strong
indicator of chemical instability of a
component, especially under extreme
conditions. All extracts, including
the system f lush sample and the 12
Table 5: Identity of compounds in deionized water (DI) and ethanol extracts by GC/MS analysis
Method
Headspace
GC/MS for
volatiles
Direct injection
GC/MS for
semivolatiles
DI Water
12- Month
Flush Sample
Sample
No peaks1
No peaks1
No peaks2
Derivatisation3
Oxalic acid
GC/MS for organic (0.056 ppm)
fatty acids
Succinic acid
(0.042 ppm)
Ethanol
Flush Sample
3-Methylpentane, hexane,
methylcyclopentane, acetal,
3-methylheptane and octane;
all <3 ppm
Decamethylcyclo-penta-siloxane,
dodecamethyl-cyclohexasiloxane, a
series of siloxanes, 1,3-DTBB4, 2,4DTBP5, 1-dodecanol, lauryl acrylate
and hydrocarbon isomer; all <1 ppm
No peaks2
Benzoic
acid (0.042
ppm)
Oxalic, malonic, lauric, succinic,
palmitic and stearic acid
all <0.3 ppm
12-Month Sample
3-Methylpentane, hexane, methylcyclopentane,
acetal, 3-methylheptane, octane, 2-methylpentane,
cyclohexane, and 1-octene; all <5 ppm
Decamethylcyclopentasiloxane,
dodecamethylcyclohexa-siloxane, a series of
siloxanes, 1,3-DTBB4, 2,4-DTBP5, 1-dodecanol,
lauryl acrylate, hydrocarbon isomers, 1-tridecanol,
1-pentadecanol, 1-octadecanol, and a series of
aliphatic hydrocarbons (C12 to C26); all <3 ppm
Succinic, palmitic and stearic acid; all <0.3 ppm
1
Limit of detection: 0.001 ppm using 2-methyl-2-propanol; 2 Limit of detection (LOD): 0.010 ppm using 2,4-ditertbutylphenol; 3 BF3/1-butanol used as derivatization
agent; 4 1,3-ditertbutylbenzene; 5 2,4-ditertbutylphenol
months storage sample, showed
levels <1 ppm.
Group 2 — Nonvolatile Residues
(NVR) and Fourier Transform
Infrared (FTIR) Spectroscopy: NVR
analyses provide a quantitative
measure of low volatility
extractables. Dried residues
(obtained after evaporation of test
solvent) from the extract can then be
analysed qualitatively by FTIR to
identify specific functional groups
and differences between test
extracts. Using the results of NVR
in water and ethanol extracts (Table
4) we can make several conclusions.
First, the NVR level in the flush
sample for DI water of 1.9 mg is
relatively low compared with the
ethanol extract of 119 mg. That is
typical of extractables data on singleuse components because ethyl alcohol
is a more aggressive solvent, especially
for additives and residual oligomers in
organic polymers.
Second, results show a timedependent, gradual increase in
extractables for both water and
ethanol. That indicates a continuous
but slow leaching of extractables from
the polyethylene biocontainer, the
only component in contact with the
f luid during storage.
Figure 4 shows FTIR spectra of
residues in the ethanol extract from
the system f lush and the extracts
after 12 months storage. The major
difference between spectra is the
presence of some peaks (circled) in
the system-f lush sample, which are
58 BioProcess International
N ovember 2010
Table 6: Elements in water and ethanol extracts
Element
B
Na
Mg
Water Extract
Flush Sample
12-Month
ppb1
Sample ppb1
1.25
1.93
2.82
0.27
0.57
0.17
Ethanol Extract
12-Month Sample
Flush Sample ppb1
ppb1,3
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
Al
K
Ca
1.15
0.32
5.6
<0.14
<0.15
3.0
<LOD
<LOD
5.9
<LOD
<LOD
<LOD
Ti
Cu
Zn
0.11
0.18
0.34
<LOD2
0.2
0.57
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
1
Corrected with negative control or solvent blank; 2 LOD = 0.04 ppb; 3 LOD of 1­­–5 ppb due to dilution
not present in the extracts stored for
12 months. This result indicates that
these compounds can be easily
removed by system f lushing to
minimize extractables. The absence
of additional peaks after storage
indicates a relatively stable
environment during storage with no
significant extraction of additional
nonvolatile compounds or release of
nonvolatile breakdown products.
Group 3 — GC/MS, LC/UV/MS, and
ICP/MS: These methods are powerful
tools for identifying a range of organic
compounds and inorganic elements in
extraction fluids and can quantify
them down to ppm, ppb, and, in some
methods, ppt (parts per trillion) levels.
GC/MS Analyses: We applied the
following analytical strategies for GC/
MS when investigating trace levels of
extractables:
• Concentrate extracts to maximize
detection
• Carefully interpret MS data
• Use authentic standards
• Match the mass spectra and
retention times
• Ensure accurate calibration and
quantification
• Perform recovery studies.
Results show no detectable peaks
in water samples for volatile and
semivolatile compounds in both the
f lush sample and after 12 months
storage and extraction (Table 5).
Derivatization results for organic
fatty acids identified only three
acids, all <0.1 ppm.
Because most pharmaceutical
products are water-based, GC/MS
results indicated very low extractables
for these types of compounds can be
expected for this type of
multicomponent single-use system.
The ethanol extracts revealed several
identifiable volatile and semivolatile
compounds in the flush sample (Table
5) and some additional compounds
(shown in italics) after 12 months of
storage and extraction. The data
demonstrate a much stronger solvent
action of ethanol. However, all
compounds can be traced back to the
individual single-use components in
the test system, as reported previously
(5,6). Furthermore, all were present at
a low concentration of <5 ppm.
Similarly, some additional fatty acids
were identified in ethanol extracts but
all at concentrations <0.3 ppm, with
no additional compounds present after
12 months of storage.
LC/UV/MS analyses are ideal for
detecting trace levels of antioxidants
and their degradation products, as well
as heat-sensitive organics in extraction
fluids. An analytical strategy to achieve
precise identification and highest
possible resolution must include careful
evaluation and interpretation of MS
data, use of authentic standards,
matching mass spectra and retention
times, and precise calibration and
quantification.
Analysis of water samples by LC/
UV/MS showed no peaks at LOD
between 0.1 ppm and 1 ppm using a
mixture of antioxidants as standards.
For the ethanol extracts, the compounds
identified included
1,3-ditertbutylbenzene (1,3-DTBB) and
2,4-ditertbutyl phenol (2,4-DTBP),
which we identified in previous studies
as degradation products from polymer
additive (Irgafos 168 phosphate) in
specific single-use components of the
test system (5, 6).
ICP/MS Analyses: Inorganic elements,
especially heavy metals, can influence
the efficacy, safety, and stability of some
drug products. Analysis with ICP/MS
provides a convenient and highly
sensitive method for identifying and
quantifying a wide range of metallic
ions.
The ethanol extracts showed no
elements higher than the LOD or
LOQ , except for calcium at 5.9 ppb
(Table 6). For water extracts, nine
elements were detected in the flush
sample at low ppb concentrations.
The 12-month sample showed
reduced levels, mostly <1 ppb or the
LOD, indicating that the system flush
had generally removed the majority of
the extractable elements.
Discussion and Conclusions
In this study, we generated additional
information about extractables from
single-use systems that could impact
the qualification and validation of
biomanufacturing processes. In our
previous studies, we took one type of
component (e.g., a capsule filter or a
biocontainer) and designed a specific
extractables procedure to represent a
worst-case condition for that
component (e.g., continuous
recirculation of extraction fluid
through a filter or storage of filled
biocontainers at 40 °C for 30 days). In
this way, a worst-case extractables
profile (qualitative and quantitative)
could be established for each
component. This data library
facilitates selection and qualification
components for single-use systems.
Furthermore, during process
validation, if such data from all
components of single-use system are
available and the test conditions
represent a worst case to process
conditions, then little or no further
extractables testing may be required.
Otherwise, a process- and productspecific extractables validation study
may need to be performed on the
whole single-use system for
determining potential leachables and
assess any toxicity or impact on
quality of a final drug product.
The study results for components
(5, 6) and model single-use system (this
article) can facilitate process- and
product-specific extraction tests
(potential leachables) using model
solvent as well as process fluid because
the potential leachable compounds (at
least the majority of them) are already
identified. Furthermore, the effect of
preflushing the system could provide
supporting data for preparing suitable
standard operating procedures (SOPs)
to minimize leachable compounds.
From the results, we showed:
• It is feasible to perform
extractable studies on complete singleuse systems.
• A short system flush can remove
a substantial proportion of extractable
material.
• Water extractables were
substantially lower than ethanol
extractables for organic compounds.
• In most cases, no significant
increase in extractables was seen after
storage for up to 12 months.
• Identified compounds were also
found in previous studies on the
individual components.
• Those compounds could be traced
to the materials of construction of
single-use components.
• With advanced analytics, a wide
range of compounds can be identified
and quantified. Such characterization
is ideal and suitable for analyzing
extractables from single-use systems.
We conclude that a suitable
method for extractables testing of
multicomponent single-use systems
has been developed and qualified. The
data generated can also be available
and of benefit to end users in their
qualification and validation of similar
bioprocesses.
References
1 Langer E. Advances in Large-Scale
Pharmaceutical Manufacturing; 2nd Edition,
BioPlan Associates Inc., Rockville USA.
November 2007.
2 Caine B. BPSA Survey: The Impact of
Single-Use Technologies. Bio-Process Systems
Alliance, 2009; www.bpsalliance.org.
3 Colton R, et al. Extractables and
Leachables Subcommittee of the Bio-Process
Systems Alliance. Recommendations for
Extractables and Leachables Testing. BioProcess
Int. 6(3) 2008: S28–S39.
4 Martin J, et al. Recommendation for
Testing and Evaluation of Extractables from
Single-Use Process Equipment. Bio-Process
Systems Alliance, April 2010; www.bpsalliance.
org.
5 Ding W, Martin J. Implementing SingleUse Technology in Biopharmaceutical
Manufacturing: An Approach to Extractables/
Leachables Studies, Part One — Connectors and
Filters. BioProcess Int. 6(9) 2008: 34–42.
6 Ding W, Martin J. Implementing SingleUse Technology in Biopharmaceutical
Manufacturing: An Approach to Extractables/
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and Biocontainers. BioProcess Int. 7(5) 2009:
46–51. •
Weibing Ding is technical manager at Pall
Life Sciences Scientific and Laboratory
Services, David B. Pall Technical Center, 25
Harbor Park Drive, Port Washington, NY 11050;
1-516-801-9254, weibing_ding@pall.com.
Jerold Martin is senior vice president of
scientific affairs at Pall Life Sciences; 25 Harbor
Park Drive, Port Washington, NY 11050; 251516-801-9086, fax 1-516-484-5228; jerold_
martin@pall.com; www.pall.com.