2.6.34. HOST-CELL PROTEIN ASSAYS 3

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Reference: PA/PH/Exp. HCP/T (15) 1 1
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2.6.34. HOST-CELL PROTEIN ASSAYS 3
This general chapter provides guidance for the development and validation of host-cell protein (HCP) assays
used to test products obtained by recombinant DNA technology. It does not exclude the use of alternative
approaches that are acceptable to the competent authority. 4
INTRODUCTION 5
Host-cell proteins (HCPs) are process-related impurities derived from the host organism used for the production
of a medicinal product by recombinant DNA technology. In order to mitigate their potential adverse effects (e.g.
immunogenicity), HCP content is reduced to the lowest possible level. 6
HCP clearance during the purification process must be assessed and the HCP content determined using an HCP
assay that has been evaluated and validated for a given product. 7
The HCP acceptance limit, typically expressed in nanograms of HCP per milligram of active substance (ppm),
must be justified with regard to the HCP clearance capacity of the purification process and with regard to the
potential impact of residual HCP on patients, taking into account the worst-case amount of HCP that could be
administered with the product. 8
HCPs are generally measured using an immuno-based assay containing, as reagents, the HCP antigen (HCP
reference standard) and the corresponding polyclonal antibodies (antisera). Antisera must cover a broad
spectrum of HCPs representative of the product concerned. 9
Sandwich-type enzyme-linked immunosorbent assays (ELISA) are the most commonly employed assays to
quantitatively assess the level of HCPs. The sensitivity is the result of the observed cumulative responses of
many individual HCPs in comparison to the response of an HCP reference standard. The use of orthogonal
analytical methods (e.g. electrophoresis, HPLC, western blot, mass spectrometry) is recommended to support
the development and selection of the assay, as well as the characterisation of process HCPs. 10
ASSAY SELECTION 11
Several types of assays are available, with selection taking into account several factors, including the stage of
development of the product, the nature of the host cell and the protein immunogenicity, the expression mode, the
manufacturing process, and prior knowledge. When selecting and developing the assay, its life cycle (e.g.
reagent supply, consistency, assay validation, process change) must also be considered. 12
TYPES OF ASSAY 13
Process-specific assays 14
Process-specific HCP assays (also called product-specific HCP assays) are developed and validated taking into
account the specificity of the production process, and using the host organism expressing the recombinant
product. 15
The antigen is derived from a mock run of the active substance manufacturing process (or a process
representative of it) up to a step capable of generating a broad spectrum of HCPs in sufficient quantities. 16
The antisera raised must cover a broad range of HCPs, in order to detect as many different HCPs as possible
and to also accommodate process variations. 17
Platform assays 18
Platform assays are developed by individual manufacturers and customised for their expression host and
processes. The same sets of reference standards and reagents may be used to monitor HCPs in several
products manufactured in the same expression host, provided that upstream processes (and downstream, if
relevant) are sufficiently similar for these products. 19
Generic assays 20
Commercially available HCP test kits are commonly referred to as generic HCP assays. They are intended to
work broadly across similar expression hosts. Detailed information on the preparation of the assay may not be
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scenarios (e.g. to deliver an antigen covering a broad spectrum of HCP species). For example, the antigencontaining cell culture supernatant may be harvested beyond the minimum level of cell viability in order to include
more cytosolic proteins, which are released by additional cell lysis. 38
Downstream 39
The immunogens derived from the upstream process are usually only minimally processed (filtration,
concentration), in order to obtain a broad spectrum of HCPs. Further purification is generally not recommended.
40
However, in cases where the antigens are not representative or do not yield a suitably broad HCP coverage,
mixing of mock materials from different processing steps can be considered. Enrichment may also be achieved
by pooling materials from mock fermentation or purification runs using different operating conditions, or from
selective purification steps (e.g. to reduce large amounts of the few immunodominant HCPs). 41
Cross-contamination with the protein of interest 42
The antigen must be produced in a manner that avoids contamination with even minute traces of the product in
order to avoid cross-reactivity with the polyclonal antibodies. 43
To achieve this goal, dedicated or single-use equipment is used as much as possible. Where multi-purpose
equipment is used, it must be cleaned appropriately. In addition, the risk of contamination when filling or handling
the antigen in the laboratory environment must also be considered. 44
Characterisation and testing 45
Before using the antigen for immunisation, the protein content is assessed (total protein assay) and the absence
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Immunisation 64
One of the challenges of the immunisation step is to generate a polyclonal antibody that is highly specific and
sensitive for each of the antigenic proteins in the complex mixture of HCPs used as an immunogen. An animal’s
immune response must be stimulated against both the stronger and weaker antigens. 65
An animal host that yields sufficient amounts of immune-specific immunoglobulin G (IgG) is selected. Rabbits,
goats or sheep are the most common animal species for this purpose. 66
Where both the polyclonal capture and detection antibodies are from the same source, it can be assumed that
they recognise different epitopes on the same HCP. Alternatively, polyclonal anti-HCP antibodies from different
animal species may be used. Using several animals for a given species may reduce individual variations in
immune competence and provide additional response diversity resulting in maximised antibody coverage against
the HCP antigens. 67
An immune response to a limited number of HCP antigens may be obtained rapidly, particularly when adjuvants
are used to boost the immune response. However, in complex mixtures, differential enhancement of the immune
response towards weaker antigens or those at lower concentrations may be necessary. 68
It usually takes several immunisations to reach a maximum immunological response, and depending on the
frequency of immunisation, the process can take 3-6 months to complete. 69
The progress of the immune response against HCPs for a given immunisation scheme must be monitored by
determining the antibody titre using, for example, an ELISA, and by comparing the results of 1D or 2D
electrophoresis (after protein staining) with a western blot, where the polyclonal anti-HCP antibodies are used as
primary antibody. In practice, some minor proteins that elicit a strong immune response may not be visible in the
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implementing a manufacturing process change that may impact the suitability of the HCP reagent, or when a new
lot of antibody reagent is purified from the initial serum. 86
Accuracy 87
Accuracy is demonstrated by spike/recovery analysis of the HCP reference standard in a relevant background
matrix (e.g. the active substance or a sample from a relevant purification step). 88
Specificity 89
Specificity is demonstrated by the absence of interference from the matrix background (including the active
substance). For instance, data from the accuracy study can be used to assess specificity. 90
Precision 91
As for any other quantitative assay, repeatability, intermediate precision and reproducibility are appropriately
addressed. 92
Quantitation and detection limits 93
Sensitivity is usually in the ppm range and is normally described through the quantitation limit (QL). QL is typically
determined by HCP spike recovery studies in the active substance or an appropriate sample matrix, and is
calculated from the minimal spike providing a response with predefined accuracy and precision from replicate
analyses. 94
Detection limit (DL) is often not determined (optional validation parameter). 95
Linearity 96
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Depleted reagents 115
114
Process change 117
HCP reference standard
119
118
Anti-HCP antibody 121
116
120
The protein concentration
of the new reference
standard is determined
using the same method as
for the current reference
standard to ensure that the
protein concentrations are
comparable. 122
Total protein concentration
of the new antibody is
determined. The final
assay concentration must
be titrated for the new lot
in order to achieve a
similar standard curve as
for the current lot. 123
The effects of process
change on the HCP
composition of relevant
process steps are
analysed by suitable
methods (e.g.
1D-/2D-PAGE, western
blot, HCP assay). 124
Reagent
Using suitable methods
characterisation (e.g. 1D-/2D-PAGE,
125
2D-DIGE), the similarity in
protein composition
between the new and
For biotinylated detection
antibodies, the
biotin:protein
stoichiometry is controlled
and ensured to be similar
If the process change
does not lead to a relevant
change in HCP
composition, the current
HCP reagents are also
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Standard curves obtained
with the new versus the
current reference standard
are assessed for
similarity. 136
A bridging study is
performed with testing of
relevant process samples
(e.g. purification steps
from harvest to the final
active substance). In a
side-by-side experiment,
new antibodies must
detect HCP levels at
different process steps
with the same or better
sensitivity as current
antibodies. 137
Relevant process samples
(e.g. purification steps
from harvest to the final
active substance) from the
new and the previous
process are tested
side-by-side. 138
If reagent characterisation
and ELISA testing
demonstrate suitability of
the new HCP reference
standard, the current
reference standard can be
If reagent characterisation
and ELISA testing
demonstrate that the new
antibody is suitable, the
current antibody can be
replaced. No revalidation
If for the new process, the
antibody shows similar or
higher immunoreactivity
compared to the previous
process, and the HCP
assay shows adequate
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