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Advances in Cleaning Validation

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Advances in Cleaning Validation
May – June 2022; Volume 6, Number 3
Simplify and Streamline Recovery Studies
Part II: Limitations and Drawbacks of TOC
Rizwan Sharnez, Ph.D.
Validation Solutions
Boulder Colorado USA
© 2022, Validation Solutions
In Part I of this series, we discussed
regulatory expectations for qualifying
analytical methods and the recovery of
contaminants from equipment surfaces.
We also discussed scientific principles and
methodologies for analytical method
development and bench-scale recovery
studies for cleaning validation. Evaluation of
swab and rinse recovery and the scalability
of bench-scale data were also discussed.
In this part, we discuss the limitations and
drawbacks of evaluating cleaning
validation samples based on Total
Organic Carbon (TOC). Viable alternatives
to TOC and tips to simplify and streamline
recovery studies will be discussed in Part III.
1. INTRODUCTION AND RECAP OF PART I
The quantitation of residual contaminants on
clean equipment surfaces is an essential and
challenging aspect of cleaning validation. A
critical element of this evaluation is to
develop a sampling method that can
accurately recover the target contaminant.
The target contaminant is the contaminant
for which we need to set an acceptance limit
for cleaning validation.
Advances in Cleaning Validation is a bimonthly
publication on new developments in Cleaning
Process Development, Validation and Monitoring.
ACV articles address scientific principles, strategies,
and approaches associated with cleaning that are
faced in everyday work situations. Reader
questions, comments, and suggestions are
requested for future discussion topics. These can be
sent to RSharnez@CleaningValidationSolutions.com
This can be tricky and problematic,
especially with non-specific analytical
methods such as Total Organic Carbon
(TOC).
1.1 Regulatory Expectations for the
Acceptable Carryover of Contaminants
An important regulatory expectation for
pharmaceutical cleaning validation is to
demonstrate that the carryover of potential
contaminants into the drug product is
acceptable from a patient safety standpoint.
The ability of the cleaning operation to
reduce contaminants to an acceptable level
is also an important regulatory expectation
in the preparation of newly manufactured
and reusable medical devices prior to
disinfection and sterilization. The criterion
for patient safety is generally specified in
terms of an acceptable exposure of the
contaminant [10-13]. Alternate approaches
could be used if adequately justified [12].
The acceptable exposure of a contaminant is
generally based on toxicity [10-13] and
immunogenicity (Sharnez 2012a, 2013).
The former is expressed on a per day basis
and is referred to as the Acceptable Daily
Exposure whereas the latter is expressed on
a per dose basis and is referred to as
Acceptable per Dose Exposure. These
terms are abbreviated ADE or PDE
(Permissible Daily Exposure or Permissible
per Dose Exposure).
Based on the above regulatory expectations,
an important criterion for reusable
equipment is that the carryover of the
previously processed active pharmaceutical
ingredient and its degradants (APIA*) into the
product that is subsequently processed in the
same equipment (Product B) should be
acceptable from a predictive safety stand-
point.
Figure 1: Schematic representation of
cleaning between batches of different
products.
This criterion for cross-contamination (A→B)
is often evaluated by collecting swab or rinse
samples and testing them for APIA*. The
results are used to determine whether the
carryover of APIA and its degradants into a
dose of Product B is less than the ADE of
APIA* (Sharnez 2011a, 2013).
1.2 Regulatory Expectations for
Analytical Methods and Sampling
Analytical methods that are widely used to
quantitate contaminants include total organic
carbon (TOC), chromatography, UV
absorbance and fluorescence spectrometry.
Additionally, electrophoretic and chromato-
graphic methods are used to assess
fragmentation and aggregation of proteins
during cleaning (Sharnez 2012b).
An important regulatory expectation for
analytical methods used for cleaning
validation is that sampling and analytical
methods should be qualified to demonstrate
that the contaminant – in the above example
APIA* – can be adequately recovered from
process residues on the equipment surfaces:
The firm should challenge the analytical
method in combination with the
sampling method(s) used to show that
contaminants can be recovered from
the equipment surface and at what
level; i.e., 50% recovery, 90%, etc.
This is necessary before any
conclusions can be made based upon
the sample results.
- FDA Guide to Inspections of
Validation of Cleaning Practices
1993.
Note that the stated numerical values for
recovery, viz., 50% and 90%, are included
as examples of possible outcomes of a
recovery study; they are not necessarily
acceptable recoveries. The acceptable
recovery of the contaminant is addressed
from first principles in Part III.
The recovery of a contaminant involves two
steps:
1. Surface recovery which is the
recovery of the contaminant from
the equipment surface into the
sample; and
2. Sample recovery which is the
recovery of the contaminant from
the sample by the analytical
instrument, in this case the TOC
analyzer.

Figure 2: The recovery of a contaminant
consists of two steps: 1. surface recovery
and 2. sample recovery.
For swab samples, the first step, surface
recovery, has two components:
(1a) Recovery from the surface to
the swab; and,
(1b) Recovery from the swab into
the solvent that is used to
extract the contaminant from
the swab, i.e., the swab
extractant.
The recovery of the contaminant is used to
determine its “recovery factor.” The
recovery factor is used to “correct” the
measured value (mass or concentration) of
the contaminant to account for any loss of
the contaminant associated with the
sampling and analysis of the process
residue. For example, if the measured
value (MV) of the contaminant for a swab
sample is 0.5 µg/cm2 and the swab
recovery factor (SRF) for the contaminant
is 50% (or 0.5), the actual or corrected
value CV (i.e., the MV that would be
obtained if SRF was 100%, and as a result,
there was no loss of the contaminant during
sampling) would be 1 µg/cm2 (0.5 µg/cm2
÷ 0.5). Alternatively, the recovery factor
can be used to modify the acceptance limit
(AL) for the contaminant. For example, if
AL is 1 µg/cm2 and the SRF is 50%, the
modified value of AL would be 0.5 µg/cm2
(1 µg/cm2 x 0.5). An advantage of the
latter approach of modifying the
acceptance limit over the former approach
of correcting the measured value is that it
obviates the need to correct every
measured value, thereby minimizing
human errors and the resulting
misinterpretation of data.
1.3 Non-specific Analytical Methods
Non-specific analytical methods used for
cleaning validation and process
development include TOC, conductivity and
pH. Additionally, SDS PAGE, Capillary
Electrophoresis (CE) and size-exclusion
chromatography (SEC) are used to
characterize fragmentation and
aggregation of proteins during cleaning;
and microgravimetry is used to quantitate
process residues in bench-scale cleaning
studies.
TOC is the most commonly used non-
specific test methods for cleaning
validation. TOC analysis involves the
oxidation of organic carbon in the sample
and subsequent detection of the resulting
carbon dioxide.
TOC was originally developed to monitor
water quality and is still widely used for this
purpose. It has some distinct advantages
for evaluating water quality, especially
when used in conjunction with conductivity.
Together, the two tests provide an effective
way to monitor changes in the total
concentration of chemical contaminants,
and therefore water quality in general. The
limit of quantitation (LOQ) of TOC for swab
samples is typically on the order of 0.1
ppm, which equates to about 1 µg/cm2 for
swab samples. This conversion is based on
a swab extractant volume of 40 ml, a
carbon fraction and an SRF of 0.5 and 50%
for the target contaminant, and a swabbed
surface area of 16 cm2). In comparison,
the visible limit of detection for most
process residues is between 1 and 4 µg/cm2
(Forsyth 2009).
The limitations and drawbacks of TOC and
other non-specific test methods for cleaning
validation are discussed in Section 2.
1.4 Specific Analytical Methods
Specific analytical methods used for cleaning
validation include HPLC, gas chromatography,
mass spectrometry and infrared spectro-
metry. Of these methods, HPLC is the most
well-established and commonly used specific
test method. In HPLC, the target contaminant
is separated from other components of the
process residue by chromatography. The
sensitivity and accuracy of HPLC are generally
adequate for quantifying contaminants for
cleaning validation; a drawback of HPLC,
however, is that sample turnaround times can
be up to 45 minutes per injection, which is
relatively lengthy compared to other test
methods used for cleaning validation.
Product-specific assays such as ELISA and
other immunoassays are sometimes used for
biological contaminants such as active
proteins. They overcome many of the
limitations and drawbacks of TOC. They also
have greater specificity and sensitivity; for
example, the LOQ of most immunoassays is
on the order of 1 ppb.
A major drawback of product-specific assays
(PSA) for cleaning validation, however, is that
they can lead to false positives and negatives.
That is because most PSAs are designed to
detect an intact epitope in the protein
molecule, but not whether the protein is
functionally active. Thus, if the protein
degrades and becomes inactive during
cleaning, but the epitope remains intact, the
test would result in a false positive.
Conversely, if the protein remains functionally
active during cleaning, and the epitope is
rendered undetectable by the cleaning
chemicals, the test would result in a false
negative. This issue has been cited in some
guidance documents such as Health Canada’s
Guideline on Cleaning Validation. Another
drawback of most PSAs is that they require
relatively more time and expertise to develop.
2. LIMITATIONS AND DRAWBACKS OF
TOC FOR CLEANING VALIDATION
The limitations and drawbacks of TOC
analysis for biological samples stem mainly
from the following issues:
2.1 Overestimation of Target
Contaminant
Since TOC is a non-specific test method, the
organic carbon that is measured by the
instrument is from all sources of organic
carbon in the residue, not just the
contaminant. Thus, all of the measured
carbon in the residue has to be attributed to
the contaminant, a worst-case assumption
that can greatly overestimate the
concentration of the contaminant in the
sample.
2.2 Sample Contamination
A significant drawback of TOC is that samples
can become contaminated with organic
solvents and disinfecting agents, such as
alcohol. Effective environmental controls can
mitigate some of the risk associated with
sample contamination; however, since
organic matter is ubiquitous in testing
environments there is always a significant
risk of sample contamination.
The above issues can lead to apparent
failures (false positives) and complex and
avoidable investigations. Appropriate control
samples should be collected to facilitate the
investigation of failures caused by sample
contamination.
2.3 Range of the method is limited by
solubility
TOC analysis requires the target contaminant
to be soluble in the extractant. This can be
a significant disadvantage for biological
contaminants that are sparingly soluble
because it can limit the effective range of the
method. Further, the concentration and
solubility of the target contaminant in the
rinse water – typically at a pH of around 5.2
– are often considerably lower than that of
the other organic components. And since
recovery depends on concentration and
solubility, the recovery of the contaminant
would be commensurately lower than that of
the various other components in the process
residue. Significant reasons for this
discrepancy and the resulting error in
estimating the measured value and the
recovery of the target contaminant are
discussed in Part III in terms of the
underlying physicochemical phenomena.
2.4 Inadequate Recovery
Another drawback of TOC for biologicals is
that the recovery is sometimes too low to
accurately quantify the amount of
contaminant in a sample from a statistical
perspective. This issue stems from the fact
that there are multiple sources of organic
carbon in the residue, such as water, inactive
ingredients, detergents, leachables from
elastomers, and various impurities that are
carried over from the previously
manufactured batch. Thus, recovery based
on TOC analysis includes organic carbon from
a host of organic compounds, not just the
target contaminant or its degradants in the
residue, which is what is required to
determine the carryover of the target
contaminant for cleaning validation. Further,
the concentration of the target contaminant
in the soilant can be relatively low: 1 to 10%
for the protein and its degradants. It is
therefore possible for the overall recovery
based on TOC to be >90% and yet the
recovery of the target contaminant itself to
be virtually zero.
Thus, while non-specific methods such as
TOC are useful for determining the recovery
of the process residue as a whole – i.e., from
all organic components – they may not be
useful for determining the recovery of a
specific contaminant. This issue is especially
significant when the contribution of the
target contaminant to the TOC content of the
entire process residue is relatively low
(<10%), and as a consequence, the
contaminant cannot be quantified accurately
and reproducibly.
2.5 Limitations Associated with Culture
Media and other Complex Soils
Another limitation of TOC is that the
recovery of a specific contaminant based on
organic carbon is often not possible to
estimate for complex soils such as cell-
culture media and upstream residues. To
accurately determine the recovery of a
contaminant, the theoretical (i.e., actual,
not measured) carbon fraction of the
contaminant in the residue is required. This
information is seldom available for complex
soils, such as human, plant and animal-
based materials; proprietary culture media;
and degraded biological residues. These
drawbacks are especially significant when
the contaminant comprises of a relatively
small fraction of the overall amount of
carbon in the residue as discussed in Section
2.4.
2.6 Compounding Factors
The above issues are often compounded by
experimental factors such as degradation of
proteins during cleaning (Sharnez 2011b,
2013), low recovery of protein aggregates in
post-cleaning residues, poor scalability of
bench-scale recovery data and high
experimental variability. Of these factors,
degradation of proteins during cleaning is
especially significant because proteins can
form aggregates when exposed to
aggressive cleaning conditions such as high
pH (>13) and temperature (>75°C).
Figure 3: Antibodies and other therapeutic
proteins can degrade and form aggregates
during cleaning (Sharnez 2012a, 2012b and
2013). The aggregates are generally less
soluble and have greater affinity for
equipment surfaces than the monomeric
form of the protein. Thus, the recovery of
the aggregated protein can be substantially
lower than that of the monomeric protein.
Aggregates are generally less soluble and
have greater affinity for equipment surfaces
than the monomeric protein. Thus, the
recovery of the aggregated protein in the
post-cleaning residue can be substantially
lower than that of the monomeric protein in
the process soilant before cleaning.
Aggregates are also more immunogenic
than the native protein and this can have
implications or setting acceptance limits for
cleaning validation (Sharnez 2012a, 2013).
The above factors and their impact on the
recovery of the target contaminant will be
discussed in Part III in terms of the
underlying physicochemical phenomena.
Viable alternatives to TOC and tips to
simplify and streamline recovery studies will
also be discussed.
3. CONCLUSION
Regulatory expectations for the acceptable
carryover and quantitation of process
contaminants, and the limitations and
drawbacks of TOC in this context were
discussed. The underlying principles and
concepts, however, are also applicable to
other non-specific test methods used to
quantitate contaminants in process
residues. Common examples of such non-
specific methods include SDS PAGE,
Capillary Electrophoresis (CE) and SEC for
proteins and their degradants, electrical
conductivity and pH for detecting ionic
contaminants in rinse water samples, and
microgravimetry for bench-scale cleaning
process development studies.
The limitations and drawbacks of TOC stem
mainly from the following issues:
▪ Overestimation of the target
contaminant due to the non-specific
nature of the method;
▪ Contamination of samples with
organic matter that may be present
in the environment;
▪ Relatively low solubility and stability
of proteins in aqueous extractants.
▪ Aggregation of proteins during
cleaning.
▪ Low concentration of the target
contaminant in the process residue; and,
▪ The precise composition and organic
carbon content of each component in
 commercially available culture media
and complex or degraded biological
residues are generally unknown.
The above shortcomings of TOC and
other non-specific methods and their
impact on the recovery of the target
contaminant will be discussed in Part
III. Scientifically sound and readily
implementable solutions will be
discussed in a future article.
4. REFERENCES
1. Forsyth, R., (2009), Ruggedness of
Visible Reside Limits for Cleaning
Validation, Pharmaceutical Technology,
p. 102 – 111.
2. Fourman, G. L., and M. V. Mullen (1993).
Determining Cleaning Validation
Acceptance Limits for Pharmaceutical
Manufacturing Operations. Pharma-
ceutical Technology 14 (4): 54-60.
3. Sharnez, R. (2010), Strategies for
Setting Rational MAC-based Acceptance
Limits for Cleaning Validation, Part I:
Reassessing the Carryover Criterion;
Journal of Validation Technology, Vol 16,
No. 1, p.71-74.
4. Sharnez, R., et al. (2011a), Strategies for
Setting Rational MAC-based Acceptance
Limits for Cleaning Validation, Part II:
Application to Rinse Samples; Journal of
Validation Technology, Vol 17, No. 2, p.
43-46, 2011.
5. Sharnez, R., et al. (2011b), Strategies for
Setting Rational MAC-based Acceptance
Limits for Cleaning Validation, Part III:
Leveraging Toxicology and Cleanability
Data; J. of Validation Technology, Vol 17,
No. 3, p. 24-28.
6. Sharnez, R., (2012a), Leveraging
Acceptable Exposure of Host Cell Protein
to Set Acceptance Limits for Inactivated
Product, J. of Validation Technology, Vol.
18, No. 3, p. 38-44.
7. Sharnez, R., et al. (2012b), Methodology
for Assessing Product Inactivation during
Cleaning – Part I: Experimental Approach
and Analytical Methods, J. of Validation
Technology, Vol 18, No.4, p 42-45.
8. Sharnez, R., et al. (2013), Acceptance
Limits for Inactivated Product based on
Gelatin as a Reference Impurity; J. of
Validation Technology, Vol 19, No.1.
9. Guide to Inspections of Validation of
Cleaning Practices; U.S. Food and Drug
Administration, 1993.
10. ICH Q7 Good Manufacturing Practice for
Active Pharmaceutical Ingredients, 2000.
11. ISPE Baseline Guide; Risk-Based
Manufacture of Pharmaceutical Products
(Risk-MaPP), 2017.
12. EMA Guideline on setting health-based
exposure limits for use in risk
identification in the manufacture of
different medicinal products in shared
facilities, 2014.
13. EU GMP Annex 15; Qualification and
Validation, Section 10 Cleaning
Validation, 2015.
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