PHOTO COURTESY OF Q-ONE BIOTECH, GLASGOW, SCOTLAND.
Process Development
Kenneth Hoffman
B
iopharmaceutical products from
recombinant DNA can be
contaminated with components of the
host cell system used to manufacture
the drug. Even after multiple
purification steps, significant levels of those
impurities can be present. Although low levels
of most contaminants may be inconsequential,
patient safety demands that they be eliminated
or reduced to the lowest levels practical to
prevent problems such as adverse immune
reactions. Impurities can have significant cost
implications in drug development and
manufacture. Failure to identify and sufficiently
remove contaminates early in drug development
can result in reduced drug efficacy or adverse
patient reactions that could delay release or kill
a promising drug candidate. Reducing
contaminates to very low levels can necessitate
Strategies for Host Cell Protein Analysis
Developing methods to detect and
measure host cell protein
contamination in biopharmaceuticals
is technologically complex. A rational,
analytical quality control strategy
requires focusing on preparation of
the immunogen used to generate the
antibodies, purification of the
antibodies, calibration of the assay,
and combining both generic and
specific immunoassays to ensure
product safety.
Cygnus
Technologies
P.O. Box 1018
Wrentham, MA 02093
Tel: 508-769-8739
Fax: 508-883-9913
Email: cygnustec@aol.com
Web: www.cygnustechnologies.com
Kenneth Hoffman is the technical director of
Cygnus Technologies, 95 Comstock Drive,
Wrentham, MA 02093, 508.769.8739, fax
508.883.9913, cygnustec@aol.com,
www.cygnustechnologies.com.
multiple purification steps that reduce product
yield. Efforts to develop analytical methods for
host cell contaminants is an expensive
undertaking, which if not finished in time can
delay clinical studies.
The purpose of this article is to identify
the strengths and limitations of the various
analytical methods and then propose a
rational, cost-effective strategy to apply
those methods to process development and
routine quality control.
The host cells used for recombinant
expression are complex systems ranging
from bacteria and yeast to cell lines derived
from mammalian or insect species. The cells
contain hundreds to thousands of host cell
proteins (HCPs) and other biomolecules that
could contaminate the final product.
Although FDA requires analytical
methods demonstrating that contaminant
levels are minimized, the regulations and
guidelines covering the issue are somewhat
vague (1–3). Recognizing the complexity of
that task and the unique issues for each
product, FDA cannot state explicitly what
tests to use or even what levels of
contamination are acceptable. A few papers
have been published by scientists working in
the field, discussing product-specific
methodologies and theoretical rationales, but
no consensus exists on the best approach for
host cell protein analysis (4–6).
Analytical Methods for HCP
Table 1 shows the most commonly used
analytical methods for contaminant control,
with their inherent strengths and weaknesses.
A prudent analytical repertoire will include
most of those techniques. Methods with poor
sensitivity, such as HPLC, are of little value in
testing for clearance from final product. Still,
such methods are important as process
development tools and can find application as
control methods for lot-to-lot processes when
used to test intermediate product early
(upstream) in the purification. In combination
with a sensitive protein-staining method like
silver stain or colloidal gold, SDS-PAGE has
a sensitivity of 100 pg/band and thus is an
important method for both process
development and final product quality control
(QC). However, specific identification and
still lower sensitivities are required, and for
that an immunological analysis of HCPs has
proven indispensable. Immunoassays can
detect analytes in the subnanogram range,
which can translate to levels of parts per
billion in final products. In addition,
antibodies can yield the specific identity of
HCP contaminates. Such specificity is
necessary not only to identify individual
HCPs, but also to allow differentiation of
HCPs from other process contaminates. With
that high sensitivity and specificity, rational
and cost-effective decisions can be made
about the best approach to process
development and QC.
Two types of immunological methods are
usually applied to HCP analysis: Western
blotting (WB) and immunoassay (IA), which
includes techniques such as ELISA and
sandwich immunoassay or similar methods
using radioactive, luminescent, or
fluorescent reporting labels.
Limitations of IA and WB
The comparative advantages and limitations
of IA and WB are further expanded in
Table 2. Although WB and IA procedures
can use the same antibody and detect many
of the same HCP contaminants, they will
each detect some different HCPs. For
example, a WB procedure usually requires
some solubilizing or denaturing technique
before the electrophoresis step. That
typically consists of treatment with a strong
detergent, reducing agents, and heat, and it
can destroy some antigenic determinants.
Other antibody epitopes that might be
sterically hindered from binding will be
exposed by denaturation. Samples tested by
IA usually are not subjected to such harsh
treatment conditions, so HCPs are found in
more native configurations. The efficiency
with which electrophoretically separated
proteins can be transferred out of a gel onto
the blotting medium is also variable and
Process Development
technique dependent. Very high molecular
weight proteins cannot transfer sufficiently
and may remain undetected by WB.
Depending on its electrical charge, a protein
may not be released fully from the gel or
bound efficiently to the membrane during the
transfer step, resulting in underdetection.
Very low molecular weight contaminants can
pass through membrane pores without being
sufficiently adsorbed. Sandwich IA requires
that all contaminants have at least two
antigenic epitopes for detection to take place,
so the assay may fail to detect some lower
molecular weight species. In theory, WB
requires only a single epitope for detection.
Sensitivity. The advantage of WB is that it can
separate and help identify individual HCPs. A
multiple contaminate IA, on the other hand,
cannot identify or quantitate one component
from the next. However, IA is more sensitive
than WB. A typical colorimetric WB can detect
bands only at around 1 ng of protein. More
sensitive methods employing photon detection
techniques such as radioactivity or
chemiluminescence can provide detection at
around 100 pg per band, but the sensitivity of
WB is fundamentally limited by the sample size
that can be analyzed. For example, if a typical
10-mL sample is applied to the gel, and the
band detection sensitivity is 1 ng, the
concentration of analyte present in the sample
must be on the order of 100 ng/mL for detection
to occur. Detection limits for IA typically are
less than 1 ng/mL or about 100 fold more
sensitive than WB. That sensitivity is
demonstrated in Table 3 for the HCPs of an
antibody to Chinese hamster ovaries (CHO).
The same affinity-purified antibody directly
labeled with alkaline phosphatase was used in
both a microplate sandwich IA and the WB.
The IA has a 0.5 ng/mL limit of detection
(LOD). The substrate para-nitrophenyl
phosphate (PNNP) used in the IA whereas the
substrate 5-bromo-4-chloro-3-indolyl
phosphate/nitro blue tetrazolium (BCIP/NBT)
was used in the WB. A preparation of CHO
HCPs, solubilized from whole cells, was
subjected to limiting dilution until neither of the
assays could detect HCP. The IA detected CHO
HCPs to a dilution of 1:156,250 compared with
a WB final dilution of 1:1,250. The sensitivity
of the IA was approximately 125-fold greater
than that of the WB.
Measuring multiples. Although IA is
fundamentally a quantitative and objective
procedure, when the assay is trying to
measure multiple components simultaneously,
IA is not quantitative in the strictest sense.
Table 1. Common analytical methods for HCP analysis.
Method
Strengths
Weaknesses
SDS-PAGE/
Silver stain
Good sensitivity 100 pg/band
resolves multiple components
Subjective interpretation,
qualitative, complex, and
technique-dependent
HPLC
High resolution, quantitative
Low sensitivity, nonspecific,
subjective interpretation
Western blot
Immunological identity, resolves
multiple components,
sensitivity 0.1–1 ng/band
Qualitative, very complex,
antibody may fail to detect
some contaminants
Immunoassay
High sensitivity 1 ng/mL,
semiquantitative, objective
endpoint
No resolution of individual
components, antibody may
fail to detect some
contaminants
Table 2. Comparison of Western blot and immunoassay.
Western Blot
Immunoassay
Quantitative
Semiquantitative
Sensitivity 10 ppm
Sensitivity 1 ppm
Resolves multiple components and gives
relative molecular weight
Measures only “total” HCPs
Denaturation and solubilization steps can
destroy some native epitopes
No denaturation required, but can fail to
detect sterically hindered epitopes
Subjective interpretation and very
technique dependent
Objective results, instrument read
endpoints
Table 3. CHO HCP detection sensitivity. Comparison of immunoassay and Western blot.
Dilution of
CHO HCPs
Immunoassay Results
(ng/mL)
WB Results
(# of bands)
1:10
1:50
1:250
1:1,250
1:6,250
1:31,250
1:156,250
1:781,250
250
250
250
176
34
7.1
1.5
assay LOD
40
28
12
5
0
0
0
0
That is a common misconception of HCP
assay users, who expect the assay to conform
to the same linear accuracy validation criteria
applied to single-analyte IA. Although
linearity need not be an absolute HCP assay
validation criteria, the probable lack of
linearity should be appreciated as a potentially
important limitation of the technique. The lack
of linearity is caused by a number of factors.
The selection of an HCP preparation for use
as standards is arbitrary and will seldom
exactly match the numbers and relative
proportions of HCPs found in the various
samples. The relative affinities of the
antibodies for contaminant epitopes can vary
significantly. Should one contaminant be
present in high concentration, there may be
insufficient excess of both antibodies to
ensure a linear quantitation for that particular
contaminant. Therefore, multiple antigen
detection IAs should be realistically
considered as a semiquantitative indication of
the amount of HCPs present. Despite those
limitations, IA remains a sensitive technique
to detect HCP contaminants providing
important feedback for both process
development and lot release decisions.
Best effort. Any immunological procedure,
whether WB or IA, is only as good as the
antibodies used. It is unlikely that an HCP
assay can detect all HCPs found in a cell.
Some proteins are not sufficiently
immunogenic or present in high enough
concentrations to be detected by antibodies,
and methodological limitations mean that
some HCPs may not be detected. An HCP
assay is a best effort at measuring a large
number of HCPs. WB and IA, used with other
analytical procedures such as SDS-PAGE and
sensitive protein staining, can provide
cumulative evidence of process control with
reasonable assurance of product safety.
Generic Versus Specific Assays
HCP IAs are either generic or process specific.
Generic assays try to measure all the HCP
contaminants that could be present. Such
assays will use antibodies that are very broadly
reactive to as many HCPs as possible. A
process specific assay measures those HCPs
that typically copurify with the product. Such
assays often use antibodies developed from
antigens obtained by sham production and
purification runs. In a sham run the same cell
line is used, but it usually is not transfected
with the product gene. That cell line is then
subjected to the normal processing steps with
the HCPs concentrated and used as
immunogens to produce the antibody used in
the HCP assay. Sham runs make the critical
assumption that absence of the product from
the growth and purification procedure will not
alter the number or recovery of HCPs.
Both assay types have advantages and
limitations. Although it is generally agreed that
generic assays are indispensable in process
development, they also have important but
frequently overlooked value in lot release
testing. Process specific assays by definition are
not available until the process has been
determined. That is a significant limitation to
drug development because issues like yield and
cost will drive process developers to continually
optimize the process. Until the development
process is complete, development of the process
specific assay has to wait. Should a process
specific assay be developed before the process
is complete, the assay may become irrelevant.
Even subtle changes to a production or
purification process can have a significant effect
on the type, number, and quantity of HCP
contaminants present. We have seen that
problem manifest itself with clients who relied
on a premature process specific assay. In one
example, a company changed the growth
medium from a defined one to a protein-free
medium. The rationale for that decision was that
two of the protein additives in the defined
medium (bovine albumin and transferrin) were
problematic contaminants during purification. It
was reasoned that if those components were
eliminated from the growth medium, the final
product would be purer. When the final product
from the protein-free growth medium was
tested by the company’s process specific assay,
it failed to detect HCPs above its detection limit
of 1 ng/mL. However, SDS-PAGE silver stain
indicated the presence of some atypical bands,
and for that reason it was decided to test the
new product with a commercially available
generic assay. The generic assay, in
contradiction to the process specific assay,
yielded a total HCP value of over 1 mg/mL.
The true presence of new or atypical HCPs not
reactive in the process specific assay, but
reactive in the generic assay, was subsequently
confirmed by WB. A process specific assay can
become too specific not only from intentional
changes to a process but can also result from
spontaneous problems.
In another example, process parameter
irregularities were detected in a batch of
mammalian cells grown by standard
procedures. Cell viability counts were just
within specification but lower than normal.
Product yield was down. Tests for microbial
contamination were negative or
inconclusive. The client’s process specific
assay gave no detectable HCPs (3 ng/mL
limit of detection). However, testing by a
generic assay showed HCPs greater than
100 ng/mL. The examples demonstrate the
difficulty in relying solely on a process
specific assay to demonstrate HCP
clearance. Changes in a process (such as
new growth media, process scale-up,
stresses during culture, contamination with
mycoplasma or virus, or spontaneous
genomic transformation of the cell line) can
give rise to atypical HCP contaminants that
a process specific assay will fail to detect.
Multiple uses. A generic assay should
therefore be used during process
development and also as a lot release test. If
the generic assay is sensitive enough, it can
be a good indicator of problems in the
growth or purification process. Process
specific assays can provide some help in
determining process control, but they
frequently may be redundant to a good
generic assay. The value of using a process
specific assay in addition to a generic assay
should be considered product-by-product. In
cases with a defined, persistent, and
problematic HCP contaminant (such as a
particularly immunogenic or biologically
active HCP that persists even after the final
purification step), a downstream process
specific assay may be justified. Relying
solely on a process specific assay is ill
advised and can result in failure to detect
atypical process contaminants.
Developing HCP Assays
IA development is a complex task requiring
multiple technical disciplines. Many
companies find it cost effective to outsource
some of that task. Companies can waste
money and delay product development if
they fail to appreciate the difficulties and
limitations of HCP analysis. As a complex
R&D task, the development of an HCP
assay should begin with a plan. The logical
disciplines involved in project management
(as described in CGMP guidelines) are
useful in determining team goals, and are
paraphrased in the “The HCP Development
Team Priorities” box.
Producing the antibody. Before embarking on
the difficult task of producing antibodies to
complex antigenic mixtures, the possibility
of using commercial antibodies should be
considered. In many cases, commercially
available polyclonal antibodies exist for
many of the common host cell expression
systems. If those antibodies have been
competently produced and purified, they
may be adequate or better than those
generated against a company’s proprietary
strain of the same cell line. The concern that
commercial antibodies may have been raised
against a different strain of the cell line (and
thus cannot recognize another strain) is
largely unfounded because the majority of
proteins are highly conserved from strain to
strain. We have confirmed that by
performing SDS-PAGE silver staining and
Western blots of multiple strains against an
antibody generated against a particular
strain. We typically see protein and
antigenic homologies of greater than 95%
among different strains.
If an acceptable antibody is not
commercially available, it will be necessary
to obtain an appropriate source of antigen for
use in producing the antibodies. For generic
assays, whole cell lysates are typically used
as the source of immunogen. If the product
itself is secreted, easily soluble or secreted
HCPs may be more representative of the
fraction of HCPs likely to be obtained from
purification. Subfractions can eliminate
irrelevant immunogens from the immunogen
cocktail, but also run the risk of excluding
ones that could be a problem in the event of
process irregularities or if the process should
later be changed intentionally. Our
experience has been that it is best to use a
whole cell lysate because qualitatively nearly
all proteins found in lysates also can be
detected in the supernatant of carefully
grown, nonlysed cultures of both prokaryotic
and eukaryotic cells when using a sensitive
immunoblotting assay.
For process specific assays, the
immunogen is usually obtained from sham
HCP DEVELOPMENT TEAM PRIORITIES
Establish design goals for the assay.
Get suggestions from other departments
and approval for design goals.
Know the limitations of the assay and
consider how it will be used with other
analytical procedures.
Assess needed resources and costs.
Determine whether assay can or should
be developed in-house or by an outside
contractor or purchased (if commercially
available assays already exist).
Establish the timetable for developing
the assay, and make it consistent with
the product development timetable.
production runs that have been concentrated
after each purification. The question is, At
what point in the purification should HCPs
be collected for the immunogen? If done
early in the process (upstream) as is reported
in most publications, the resulting antibodies
will be against a wide array of HCPs, and
the assay may not be significantly different
from a generic assay (4–6). Furthermore, if
that early process specific assay is calibrated
with the same early HCP cocktail, it may not
accurately detect downstream and final
process HCPs, which can be more limited in
number and relative proportions.
An important point to consider in
preparing the immunogen is to prevent
inclusion of antigenic material of non-HCP
origin. The major source of such material is
the growth medium. Serum proteins or other
growth hormone additives can be
immunogenic and are typically found in
higher concentration than HCPs. If they are
present in the HCP immunogen, such
components will result in antibodies that are
not confined to detecting HCP but can
instead react predominately with growth
media components. Inclusion of growth
media antigens in the HCP immunogen
seems to be a common mistake in
developing HCP assays. We have found that
some process specific HCP assays actually
have greater than 90% of their activity
directed against materials such as serum
albumin or transferrin. Eliminating growth
media antigens from the HCP immunogen
can be done by extensively washing cells
before lysing, by purging the cell line in a
medium containing no antigenic
components, or by growing the cell line in
protein-free media. Regardless of the
method used to reduce media antigens, the
resulting HCP antisera should be tested
against the medium to demonstrate its lack
of reactivity. If antibody activity is found
due to trace contamination of the HCP
immunogen, those antibodies can be
removed by subtractive affinity
chromatography. An HCP assay should
detect only HCP. Growth media
contamination of the final product is best
measured by separate monospecific assays
for the individual components. Developing
an assay for a single antigen is an easier task
than developing an HCP assay.
Alternatively, testing laboratories can
consider purchasing commercially available,
well validated assays for typical media
components such as albumin, transferrin,
insulin, and bovine gamma globulin.
An HCP preparation free of growth media
antigens can require still further
modification to be a satisfactory immunogen
because many potential HCP contaminants
are weak immunogens that do not elicit an
adequate antibody response in the host
animal. Comparing the protein stain of
electrophoretically separated HCPs to a WB
shows that not all the protein bands have a
corresponding WB band. Although it would
be naïve to expect that every protein band
should have a WB band, it is the goal of a
generic HCP assay to detect as many of the
HCPs as reasonably possible. The number of
immunoreactive HCPs can be increased by
modifying the immunogen itself or by
employing special immunization protocols.
The literature describes various cascade
immunization protocols that report
improvements to the spectrum of
immunoreactivity (7,8). We have found the
cascade processes to be somewhat tedious
and not totally effective and instead prefer
chemical modification of the immunogen to
improve the immunogenicity of the HCP
preparation. Our chemical modification
techniques are similar to those described in
the literature for generating clinically
diagnostic antibodies to haptens and
nonimmunogenic and species-conserved
peptides and proteins. The potential of a
chemical modification to generate irrelevant
antibodies to unique, chemistry-created
epitopes is not a problem, provided that the
resulting antisera is affinity purified against
HCP antigens that have not been chemically
modified. By using chemical antigenic
enhancements, we have been able to
demonstrate up to a twofold increase in the
number of WB bands relative to
nonmodified immunogen.
Purification of the antibody. Optimal
performance of a WB or IA requires affinity
Process Development
purification of the antiserum against the
HCPs. Affinity purification can improve
both specificity and sensitivity. Specificity is
enhanced by the removal of preexisting
irrelevant antibodies and other nonspecific
factors from the polyclonal serum that could
result in artifactual HCP bands in WB or
falsely elevated HCPs in the IA. We
typically see an increase in sensitivity of
greater than 100-fold for IA when affinity
purified antibodies are used in place of an
IgG antisera fraction. Selective enrichment
of HCP-reactive antibody species allows for
lower assay backgrounds, at the same time
increasing the concentration of HCPreactive antibodies. That affinity enrichment
also improves the condition of antibody
excess in the assay relative to the highconcentration HCPs that could be present in
the test sample. By maintaining a high
concentration of antibodies, assay linearity
and analytical range can be extended.
Affinity purification of a polyclonal
antibody preparation using multiple antigens
is not a trivial undertaking, and careful
thought should be given to the strategy.
Although the objective is to enrich the
concentration of HCP-reactive antibodies,
certain HCP antibody species can be lost
because the HCP affinity absorbent does not
contain those antigens. Comparison of WBs
from raw antiserum with those obtained
from affinity-purified antibody should
indicate whether significant antibody
populations have been lost. A perfect
correlation of bands between the antisera
and the purified antibody is unlikely. The
antisera may show non-HCP bands from
nonspecific binding or other artifacts.
Affinity-purified antibody can yield
additional bands over the raw antiserum
because of the selective enrichment and
sensitivity improvement. Multiple WB
bands can be somewhat subjective to count,
but as a general rule we are content with
affinity purification when fewer than 5% of
the raw antiserum HCP bands are missing
afterward. Alternatively, the use of twodimensional gels can be an even higher
resolving method in determining the
reactivity of an antibody preparation.
Calibration of the IA. Much of the difficulty
and misconceptions surrounding assay
calibration are due to a failure to appreciate
the limitations of multiple analyte HCP IA.
The most common mistake is to solubilize
cell proteins and then perform a protein
assay, such as biuret or bicinchoninic acid
Process Development
(BCA), to establish the concentration units
for the IA. Unfortunately, that approach will
rarely yield an accurate or even close
estimation of HCP levels in the product
because dye-binding protein assays and
HCP IAs measure different things. Not all
the proteins are immunogenic. Some, such
as highly repetitious membrane or structural
constituents, will make up most of the total
protein as measured by the protein assay.
However, the HCP assay contains an
insufficient excess of antibody to quantitate
those very high concentration components.
Another reason that solubilized cell proteins
make unreliable determinants of IA
concentration units is that nonprotein
components in the cell lysate, such as the
detergent used for solubilization and cellderived lipids and peptides, can interfere
with the dye-binding protein assay. The use
of IA units calibrated by a protein assay
from whole cell lysate will show falsely
that the IA is relatively insensitive and
will typically result in a significant
overestimation of HCP contamination in
test samples.
Arbitrary units. Analytical purists would like
to report absolute mass or concentration
units, but the practical limitations to
linearity coupled with the arbitrary selection
of antigens for use in both antibody
generation and assay standards make an
absolute measurement nearly impossible.
HCP results should be reported, instead, by
recognizing the limitations of the method
and delivering assay results in units that
acknowledge those limitations. We like to
use the concentration unit of “ng/mL of
immunoreactive host cell protein
equivalents.” Such an arbitrary unit implies
the uncertainty of calibration yet still
acknowledges that the methodology is
inherently sensitive, semiquantitative, and
capable of providing useful information on
process control. Arbitrary concentration
units have been accepted for years in clinical
diagnostics for many protein hormones and
tumor markers for which a homogenous,
highly purified, well-characterized standard
does not exist. For example, the human
chorionic gonadotropin (hCG) hormone
used to diagnose pregnancy and some other
conditions is assayed by immunological
methods, but the results are reported in
biological activity units of mIU/mL. Those
biological units are based on a historic
nonimmunological bioassay. Because the
immunoassay measures immunoreactive
hCG, whereas the bioassay measured only
biologically active hCG, the use of
biological units in reporting immunoassay
results is arbitrary. The point is that
definitive mass units are unimportant; it is
the correlation with clinical conditions that
is significant. Similarly, with HCP analysis,
it is not the unqualified value that matters
but rather the correlation of the assay result
to process control that is important.
HCP Assay Calibration
The three approaches described below give a
somewhat arbitrary but logically justifiable
and methodologically traceable method for
calibration of HCP assays.
Sham production and purification. Perform a
sham production run and concentrate HCPs
from the purification process. The resulting
antigen can be considered “process
representative” and assigned a value based
on gravimetric or protein assay. Non-HCPs,
such as growth media, will interfere in this
method if they become concentrated in the
HCP sample. If present, they should be
removed or corrected for. This method can
Figure 1. Bioprocess contaminant testing.
be costly, is often tedious to perform, and
can be based on critical assumptions about
how representative the resulting HCPs are.
Still it can provide a reasonably accurate
calibration for HCP activity.
Affinity isolation. We also have developed
HCP standards by use of immunoaffinity
purification. In this technique the antibody
generated from an HCP whole cell lysate
immunogen is immobilized onto an affinity
support. Solubilized HCPs are then passed
over the support. Nonimmunoreactive HCPs
and excess antigens from very high
concentration HCPs are not retained on the
column. The bound immunoreactive
material is eluted and that protein
quantitated by a protein assay for use as the
HCP assay calibrator.
Theoretical and mathematical calibration. A
calibration curve also can be theoretically
derived with acceptable accuracy using the
physical laws governing sandwich
immunoassays and determining certain
critical parameters.
Determine the amount of functional solidphase antibody used for capture and the
Process Development
amount of antibody used as the detection
reagent. Because the upper limit of detection
for the assay is determined fundamentally by
the quantity of antibodies, a reasonably good
estimate of the assay range can be obtained by
knowing the quantity of those two antibodies.
The least detectable concentration is more
difficult to estimate because it depends on
other factors such as antibody affinity
constants, nonspecific binding (NSB), and
attributes of the detection and signal
measurement system. With those variables
reasonably estimated, it is possible to estimate
the lower limit of detection based on the
physics of ligand binding.
By the time that the estimated NSB and
detection system background signal is
subtracted, the resulting sandwich assay
standard curve should give a slope of
approximately one over a concentration range
in which the antibodies remain in excess. In
mathematics, that means the slope has been
determined and the upper and lower
asymptotes of the assay ascertained. With the
previous parameters estimated, an antigen
preparation intended for use as a standard then
can be reasonably assigned a value based on its
actual assay dilutional performance compared
with the theoretical mathematic curve. In our
experience, the mathematical method yields a
concentration value for the standards antigen
that agrees closely with the value obtained by
the affinity isolation method.
Strategies for Contaminate Testing
The larger picture of bioprocess contaminate
analysis now can be outlined. Following is a
general outline or systematic strategy for
both process development and validation
and for routine quality control. Many
analytical methods used for process
development and validation also should be
used in routine QC and lot release decisions.
A guidance published by the International
Conference on Harmonisation (ICH) implies
that testing for some process impurities such
as HCP and DNA can be discontinued if the
process can be validated to show removal of
those impurities (3). The use of process
validation and/or clearance studies as
justification to minimize the amount of lotto-lot testing procedures puts the burden on
very tight process control.
Processes can change unintentionally, and
often the most sensitive methods to detect
those changes are the contaminant assays.
Valuable lot specific and process control
information can be obtained by making those
methods a part of routine QC. The bioprocess
contaminant testing flow diagram is suggested
as a general strategy for both process
validation and lot release testing (Figure 1).
Although samples from the intermediate
process steps will need to be tested as a part of
process validation, the necessity of routine lotto-lot testing for in-process samples can be
considered product by product.
Sometimes in-process testing will prove
cost effective by identifying problems and
allowing for timely remedial action before
significant value is added to the product. A
highly sensitive, well-validated, generic
HCP assay, together with the other sensitive
and complementary analytical methods,
should provide adequate process quality
control and product safety assurances.
To prevent redundancy with a good generic
assay, a process specific assay should be
directed and calibrated against downstream
HCPs that have previously caused adverse
reactions. We have yet to encounter such a
problematic case, but it is conceivable that a
downstream process specific assay could be
needed. In a recent European commission
publication about the safety of biological
products prepared from mammalian cell
culture, data review suggested that “clinical
experience with different biopharmaceuticals
shows that residual protein in these highly
purified products does not represent a danger
to the health of the patients” (9). Given that
determination and because process specific
assays will most probably fail to detect
atypical HCP contaminants, process specific
assays may only rarely be useful or necessary.
A Battery of QC Procedures
IA methods are very sensitive and specific tools
for determining the presence of HCPs. Although
IAs have limitations and are technically complex
to develop, they are valuable tools. Some people
suggest that HCP assays are necessary only for
process validation and that the routine use of
HCP assays for lot release analysis may not be
required based on validation studies using
somewhat downstream, process specific assays
that indicate acceptable clearance. Our
experience with numerous cell lines and
biopharmaceutical products indicates that the
exclusion of a generic IA from the lot release
©Reprinted from BIOPHARM, Volume 13, Number 6, pages 38-45, May 2000
AN
battery of tests is contrary to prudent QC and is a
risk not justified by the cost savings. Processes
can and do change in unforeseeable ways.
Failure to use the most sensitive method
available (because it is difficult to develop or has
limitations in absolute quantitation) can increase
the costs of manufacturing beyond those
associated with maintaining an efficient and
complementary battery of QC procedures.
Although downstream process specific assays
may not be required in most cases, a highly
optimized and well-validated generic IA is a
necessity. Such an assay will not only increase
the likelihood of detecting unintentional process
changes or irregularities, but can, along with
other analytical methods, yield a repertoire of
tests that cumulatively assure control of
processes and product purity. Additionally, such
a test repertoire can have the economic benefit of
allowing for some flexibility in the production
process to take advantage of opportunities to
improve yield and reduce costs without the
worry of compromising product safety.
References
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Drugs and Biologicals Produced by
Recombinant DNA-Technology (FDA,
Rockville, MD, 1995).
(3) International Conference on Harmonisation of
Technical Requirements for Registration of
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on Specifications: Test Procedures and
Acceptance Criteria for
Biotechnological/Biological Products”
(Geneva, Switzerland), Federal Register 64
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(4) V. Anicetti et al., “Immunoassay for the
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(5) A. Chen et al., “Quantitation of E. Coli Protein
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(9) J. Lupker, “Residual Host Cell Protein from
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Switzerland, 1998), pp. 61–65. BP
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