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European Biopharmaceutical Review
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The usual method of controlling process-related impurities in
biopharmaceuticals leads manufacturers to consider new product-specific
assay development at early stages of drug development Host cell proteins
(HCP) are an inevitable impurity of biopharmaceuticals, regardless of
whether they are produced by recombinant fermentation or extracted from
natural sources. Even after multiple sophisticated purification steps,
HCPs remain or copurify. They represent a heterogeneous variety of
different proteins that need to be quantified in the drug substance and
in intermediates from the downstream purification process. The risk for
adverse effects, such as immunogenic reaction, does not necessarily
correlate with the amount of certain host cell proteins, and even small
traces of certain HCPs can be highly immunogenic (1-2).
Traditional protein detection methods such as HPLC and total proteins
stains are not suitable due to their insufficient sensitivity and
specificity. Consequently, optimised immunoassays (ELISA) have
established themselves as the method of choice for the measurement of
HCPs. In contrast to single contaminants or leachables such as trypsin
or protein A, which can be approached by a monoclonal antibody, the
heterogeneity of the HCPs always requires a polyclonal antiserum for the
assay development.
Generic Versus Product-Specific Assays
Information on HCP removal is necessary in early process development.
The development of a specific assay is time-consuming and consequently
generic assays or commercial kits are used.
Choice of Antigen
The selection of an appropriate antigen is crucial for the development
of any HCP assay. However, there are limitations that typically occur
for each type of expression system. For example, a prokaryotic
expression system, based on inclusion bodies, typically needs to start
with a whole cell lysate as an antigen for immunisation. In the case
that the protein of interest is secreted from the expression systems,
the cell-free supernatant can be used as an early stage antigen. In this
context, it is important to prevent the inclusion of potentially
antigenic material from non-HCP origin, such as components from the
growth media. Many media additives such as hormones or serum proteins
will result in non-HCP specific antibodies and significant background in
the immunoassay. Growing cells in serum-free media or thorough washing
of cells/ bacteria can help to eliminate such unwanted antigens. In some
cases it can make sense to start with material immediately following
the first capture step, or even material collected further downstream.
The later-stage antigen is more representative of the HCP in the final
product, where an antigen from the early downstream purification process
can help to demonstrate much better reduction factors of the HCP
content. However, there are some practical considerations, such as the
generation of sufficient antigen material for the immunisation and assay
development, which is usually in the range of 200 to 300mg of total
protein at a concentration of approximately 1mg/ml.
Immunisation Procedure
The immunisation procedure is the most critical and time consuming, but simultaneously the most unpredictable part of
assay development. Consequentially, all possible actions must be taken
to limit the disturbing parameters, which include the animals’ species,
health conditions and nutrition, and administration of the antigen and
collection and testing of antisera. The use of specific pathogen free
(SPF) animals, which are kept under controlled conditions and nourished
with a defined nutrition plan, helps to reduce the background, unwanted
immunogenic reactions against food-borne antigens. This applies to
bacterial and yeast-derived products, since many animal foods are based
on yeast extracts and peptones. A typical immunisation programme
utilises two species (four rabbits and two goats), which ensures
sufficient flexibility to set up a sandwich ELISA, as well as sufficient
quantity to supply several hundred, if not thousands, of ELISA
measurements.
Typically, an immunisation period of three months with three bleedings
results in the following quantities of antiserum, assuming that the
individual bleedings will be equivalent and suitable for the further
assay development:
- Goat: final blood volume per animal = approximately 700-1,000ml
- Rabbit: final blood volume per animal = approximately 120ml
From these crude antisera, 100ml are processed further by affinity
purification and labelling, resulting in 10ml of purified anti-HCP
antibody, which is sufficient for 500 ELISA plates (assuming a 1:500
dilution).
The literature describes several socalled ‘cascade’ immunisation
protocols, named as such because they involve repeated removal of
antigens, and production of further antisera directed against antigens
in the remainder (3). In theory, intermolecular immune competition can
be avoided by removing strong antigens from the extracts. The remaining
antigens, which give no immune response in the primary immunisation, are
used for further immunisation. However, this approach is quite
laborious, timeconsuming and costly and is therefore applied in
exceptional cases only.
Purification of the Polyclonal Antibodies from Crude Serum
The optimal performance of the HCP ELISA requires the purification of
the antibodies from crude serum. This is typically performed by affinity
chromatography against the HCP. The goal is to improve specificity and
sensitivity by the removal of non-specific antibodies and other
non-relevant components which can result in artificial signals during
the immunoassay. On average, when affinity-purified antibodies were
used, a 100-fold increase of the sensitivity is observed, compared to
the use of IgG antibody fractions only.
The concentration of the anti-HCP antibodies during the purification
step helps to increase the linearity of the assay as well as its working
range. Careful consideration needs to be taken for the potential loss
of specific anti-HCP antibodies during this immunoaffinity
chromatography procedure. A comparison of Western blots using crude
serum for detection with those obtained from purified antibodies can
indicate if relevant antibody fractions were lost.
Characterisation of the Antibody Population
The goal of the entire exercise is the detection of a maximum number of
antigen species in process intermediates and the final product (2,4).
Consequentially, the coverage of the antibody population for a relevant
HCP population needs to be checked and demonstrated. The gold standard
to address this coverage question is still the use of high resolution 2D
gels and Western blots. The antigens are put through a first run,
separated based on their isoelectric point, and a second run, separated
depending on their molecular weight in a high resolution SDS-PAGE. One
gel will undergo a total protein stain, whereas the second gel will be
developed in a Western blot using anti-HCP antibodies. The coverage is
calculated by an image analysis tool. Coverage of approximately 80 per
cent in the pI range of four to seven and a molecular weight between 10
and 120kDa is achievable and meets regulatory approval. The 2D gels and
immunoblots in Figure 5, 6 and 7 illustrate this characterisation
procedure and show the differences between a generic and specific
approach.
Assay Set-Up and Calibration
Once the antibodies are checked and qualified, the quantitative assay
development can be initiated using the following guidelines.
Ninety-six-well ELISA plates are coated using the unlabelled purifi ed
anti-HCP antibodies. After a blocking step using synthetically blocking
reagents, the reference standards, samples and control samples are
added. Depending on the detection system, the labelled anti-HCP
antibodies are added, followed by the addition of the relevant substrate
(dye, fl uorescence or chemiluminescence). After the incubation time,
signals are determined by using a 96-well-plate reader. A calibration
curve is prepared by using standard concentrations ranging from fi ve to
5,000ng/ml, and the statistical evaluation usually employs a four
parameter fi t. The overall performance of each individual experiment is
controlled by the following system suitability criteria:
- Recovery of the HCP spike – adding a known amount of HCP antigen
to the test item in order to check for any matrix infl uence of the test
item/matrix
- Detection limit – measuring of six background values and calculation of the detection limit where x = 3+s
- x = mean OD value of six single background signals
- s = standard deviation of the six determinations
Validation and Routine Testing
As HCP assays are used for process validation and the testing of drug
substances, a full ICHQ2R1 assay validation is mandatory before the
release of the product (5). The scope of the validation and typical
results from more than 20 different HCP validation projects (specifi c
and generic) are shown in Table 2 (see page 80).
The validation data, especially the working range of 1 to 1,000ng/ml,
clearly demonstrated that these assays are suitable for lot release
testing on drug substances, as well as the evaluation of HCP content in
samples from the downstream purification process. The results for
accuracy and precision are comparable for generic assays and those
specifically developed for a certain product (6,7).
Conclusion
Immunological methods are the only tool currently available for a
quantitative analysis of residual HCPs with a sufficient sensitivity
(more than 10-100ppm). The quantitative results are derived from
comparisons to a reference standard. Therefore, accurate results can
only be expected when the test item and reference standard are
comparable. In addition to an optimised sensitivity, the coverage of the
HCP population is of equal importance since, as recently described,
undetected HCP species can cause severe adverse effects in patients,
resulting in a setback to the entire drug development process. This does
require a thorough characterisation exercise, starting from the antigen
up to the final serum and antibody preparations. An optimised 2D
gel-electrophoresis is the method of choice to achieve evidence on the
coverage and finally the suitability of the antibodies used for the
quantitation. This applies for specific individual assay development as
well as for a generic approach based on commercially available sources
(kits). The evidence of the final HCP results strongly depends on the
quality of the immunological reagents, and consequentially unique
strategies need to be considered for individual projects, balancing
pressure for fast process development with the need for a precise and
specific assay.
References
1. Chamberlain P, Immunogenicity of therapeutic proteins, The Regulatory Review 5(5-6): 2002
2. Eaton LC, Host Cell Contaminant Protein Assay Development for Recombinant Biopharmaceuticals, J Chromatograph A 705: pp105-114, 1995
3. Thalhammer J and Freund J, Cascade immunisations: a method of
obtaining polyspecific antisera against crud fractions of antigens, J Immunol Methods 66(2): pp245-251, 1984
4. Hoffman K, Strategies for host cell protein analysis, BioPharm 13(6): pp38-45, 2000
5. ICH Q2A Text 011 Validation of Analytical Procedures (Guidance for
Industry), International Conference on Harmonisation of Technical
Requirements for the Registration of Pharmaceuticals for Human Use, 1995
6. Flatman S, Impurities in Biotechnology Products – Experience of
Setting Specifications, Jury Great Russell Street, London, Presentation,
1 July 2003
7. Wolter T and Richter A, Assays for Controlling Host-Cell Impurities in Biopharmaceuticals, BioProcess International 3(2): pp40-46, 2005
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