Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US

Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
1

á1132ñ RESIDUAL HOST CELL PROTEIN MEASUREMENT IN

BIOPHARMACEUTICALS
1. INTRODUCTION AND SCOPE

Many medicinal products are produced through recombinant technology via a host cell (e.g., bacteria, yeast, or mammalian,
insect, or plant cell lines). During the manufacture of such products, some amount of non-product, host cell-derived material
will inevitably be introduced into the process stream. This process results in a mixture of the desired product and host cell-derived
impurities, including host cell proteins (HCPs), and other process-related impurities that will be targeted for clearance through
bioprocessing.
Residual HCPs have the potential to affect product quality, safety, and efficacy; therefore, the quantity of HCPs should be
low. The product purification processes must be optimized to consistently remove as many HCPs as feasible, with the goal of
making the product as pure as possible.
The primary concern with HCPs in biopharmaceutical products is their potential to induce anti-HCP antibodies that could
induce a clinical effect in patients. In addition, HCPs may possibly act as adjuvants, which can induce anti-drug antibodies that
can affect the safety or efficacy of the drug. A more extensive discussion of immunogenicity and its effect on preclinical and
clinical studies is described in USP general chapter Immunogenicity Assays—Design and Validation of Immunoassays to Detect
Anti-Drug Antibodies á1106ñ. HCPs can also have a direct effect on the quality of the product itself. For example, proteolytic
HCPs, even in minute quantities, can cleave the desired protein product over time, reducing or eliminating biological potency

al
or altering stability.
This chapter focuses on HCP immunoassays for recombinant therapeutic products. It does not address products such as
vaccines or gene-, cell-, or tissue-based therapies, although the general principles discussed may apply to the measurement of
HCPs in these products. The design and validation of immunoassays for HCPs involve unique and significant challenges due to:
1) the wide variety of possible HCPs in medicinal products; 2) the general use of polyclonal antibody reagents to detect them;
3) the lack of exactly matched standards for quantitation; 4) in some cases, a considerable effect from sample dilution effects;
and 5) inherent limitations to measure single HCP species.
ci
The chapter includes assay development strategies throughout the product and process development lifecycle, and it
describes approaches to demonstrate that the assay is fit for use (e.g., illustrates unit operation clearance of HCPs, lot release).
Because of the complexity of HCP immunoassays, careful development and characterization of critical reagents are required,
particularly for the immunogen used to elicit the anti-HCP antibodies, the antibody reagent(s), and the assay HCP standard.
ffi
Because HCP testing is an essential part of process development and product quality control, HCP testing is also discussed in
conjunction with regulatory requirements and other considerations for guidance on an overall control strategy for HCPs. A brief
outline of the general chapter follows:
1. Introduction and Scope
1.1 Considerations for Manufacturing, Characterization, and Consistency
2. Terminology
O

3. HCP Immunoassay Methods

3.1 The Assay Development Cycle
3.2 Development and Characterization of HCP Reagents
3.3 Immunoassay Method Development and Qualifying as Fit for Use
4. HCP Immunoassay Method Validation
4.1 Accuracy
4.2 Sensitivity and Assay Range
4.3 Sample Linearity
4.4 Specificity
5. Supporting Technologies for Residual HCP Detection, Identification, and Measurement
5.1 Considerations for Electrophoretic Methods
5.2 Considerations for Western Blot Methods
5.3 Considerations for Chromatographic and Proteomic Methods
5.4 Concluding Remarks on Supporting Technologies for HCPs
6. Use of HCP Immunoassays for Process Development, Characterization, and Validation
6.1 Assays for Individual HCPs
6.2. Control Strategy
7. Summary and Conclusions
8. Bibliography

1.1 Considerations for Manufacturing, Characterization, and Consistency

Different cell-based expression systems are used to manufacture medicinal products, such as bacteria (Escherichia coli,
Pseudomonas fluorescens), yeast (Saccharomyces cerevisae, Pichia pastoris), mammalian cells (e.g., Chinese hamster ovary (CHO),
mouse myeloma cell line NSO, and others), insect cells (baculovirus-infected Spodoptera frugiperda cells), and plant cells
(tobacco, Arabidopsis, rice). The particular HCP profile is unique and specific to the particular host cells under specific culture
conditions and manufacturing processes. HCPs can vary in pI (~3–11) and hydrophobicity, and HCPs display a wide range of
molecular weights (from ~5 kDa to at least ~250 kDa), depending on the host cell and manufacturing process used. The number
of HCPs in upstream samples can run anywhere from several hundred to more than one thousand proteins, depending on the
host cell and culture conditions. Although many cellular hosts have been used in biopharmaceutical manufacturing historically,

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 1/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
2

the most experience has been gained using E. coli and the mammalian cells CHO, NSO, SP2/0, and human embryonic kidney
cell line HEK293. The guidance in this chapter draws most heavily from the experience with these expression systems; however,
general principles apply broadly to any host cell system.
In mammalian cells, the recombinant protein is typically secreted from the cells into the cell culture fluid (CCF), along with
many of the HCPs. However, it has been observed that intracellular protein trafficking may not proceed in a normal fashion in
production cultures. For example, proteins usually associated with intracellular organelles, such as lysosomes, may be found in
the CCF of largely viable cell cultures, because the clones have been selected for maximum protein export. In addition, as some
of the cells die, their soluble, intracellular proteins are released into the CCF. Some harvest operations also lyse cells; therefore,
the resulting harvested CCF typically contains both secreted and intracellular HCPs. While this mixture of proteins incubates in
the fermenter, additional changes in the HCP population may occur, for example, as the result of enzymatic activity (e.g.,
proteinases or sialidases).
HCP assays provide important information about the composition of the material entering the downstream recovery process
and how each purification step affects HCP clearance. In some cases, HCPs can even bind to, and co-purify with, certain
products. Process characterization and validation studies are needed to show which process steps remove HCPs and also to
demonstrate the robustness of these steps for consistently removing HCPs. As such, HCP assays are an essential part of
purification process development and help ensure manufacturing consistency. Lastly, reproducible and reliable HCP assays may
be required to measure residual HCPs remaining in the drug substance (DS) used to make drug product (DP) that is delivered
to the patient. HCP levels should be measured in: 1) preclinical lots used in toxicology assessment, 2) all lots during clinical
development, and 3) process validation samples from the final manufacturing process. After approval, HCP monitoring may be
required as an element of the control system. Subsequent sections of this chapter discuss in more detail the use of HCP assays
in process validation and in a good manufacturing practices (GMP) control system.

al
2. TERMINOLOGY

To help establish a common nomenclature in the literature and with regulatory agencies, Table 1 lists common terms with
their definitions (indicating how they are used in this chapter) in addition to synonyms that have been used historically. Note
that the term “platform” indicates that the same set of standards and reagents is used within a company to test a variety of
ci
products made from the same type of expression system (e.g., CHO cells) grown under similar upstream conditions. In the case
of platform HCP assays, the antibodies to HCP are obtained from animals immunized with HCP antigens generated from a
common upstream process that is applicable to many products, even if the downstream purifications are different. This approach
allows the knowledge from prior products to be leveraged. Justification that an assay is suitable for a new product, using the
same expression system and common upstream conditions, is therefore often relatively straightforward.
ffi
The HCP immunogen used to generate platform anti-HCP antibodies and used often as the assay calibration standard is, by
design, comprised of a broad set of HCPs. In contrast, the qualifier “process-specific” indicates that the immunogen/standard
has been prepared from a set of HCPs unique to a given process (either a unique upstream cell culture process or a unique
downstream purification process). Process-specific assays are, therefore, limited in their utility, and each must be fully qualified
for each process. Process-specific immunogens and calibration standards are, by intent, more narrow and specific to a given
process. “Commercially available” assays produced by vendors are often derived from a combination of strains and harvest/
O

purification procedures, and these assays are intended to have a broad application; but these commercially available assays are
not specifically designed for a given manufacturer’s proprietary cell line, and users do not have control over reagent availability
and lot-to-lot consistency.

Table 1. HCP-associated Terminology

Term Definition Historical Synonyms
Commercially Available to the public for commercial sale; typically a combination of upstream isolates and Generic
available corresponding antibodies made by the vendor and sold as reagents or kits.

Platform The same set of an HCP standard and antibodies is developed with a company’s proprietary host Custom, in-house, proprietary
cell strain and used broadly within a type (e.g., CHO) across several products when the up-
stream conditions are similar.

Upstream An assay designed from material where the upstream culture process deviates significantly from Custom, in-house, product-specific,
process spe- the platform. proprietary
cific This is generally before any purification and may be applied to more than one product if these
parameters are similar.

Downstream An assay designed from materials where the downstream unit operations are used to enrich the Custom, in-house, product-specific,
process spe- HCP population. proprietary
cific This may be applied to more than one product if these parameters are similar. This is rarely used
today and is not recommended except for certain products with exceptional downstream
processing.

Assay for an An assay using a standard composed of an identified, single, known HCP and its specific anti- Single analyte assay or Custom,
individu- body/antibodies. HCP-specific
al HCP

Coverage Describes the assessment of how completely a population of polyclonal antibodies recognize the
population of HCPs.
The coverage assessment may be made on the HCP population used as the HCP antigen or
from the product production culture.

Qualification Demonstration of suitability of analytical methods (including reagents used in these methods)
for their intended application to a given process and in-process samples.

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 2/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
3

Table 1. HCP-associated Terminology (continued)

Term Definition Historical Synonyms
Protein A/ Affinity purification of antibodies with immobilized Protein A or G a Affinity chromatography
G-affinity pu-
rification

HCP-affinity Affinity purification of antibodies using immobilized HCP (antigen)a Affinity chromatography, immunoaffinity chro-
purification matography

Null cell The cell strain used for production that does not contain the product-specific genetic elements; Parental, blank, or mock-transfected cell
includes untransfected parental cells and cells transfected with the expression vector but with-
out the product gene.

ng/mg The numerical quantity (ratio) of HCP per product, where ng represents HCP mass and mg ppmb
represents the product mass.
It is calculated by dividing the HCP concentration (ng/mL) by the product protein concentration
(mg/mL).

a In some cases, both purifications are performed, typically the protein A/G first, then the HCP affinity.
b Although ppm has been used historically, it is not advised because this term is used to reflect mass per unit volume for other types of tests. It is recognized that
ng is used conventionally as a value derived from interpolation from an HCP standard curve (in units of ng/mL), where the signal is reflective of antibody binding
and, unlike the therapeutic protein concentration measurement, does not strictly reflect the mass of HCP that may be present.

3. HCP IMMUNOASSAY METHODS

al
Immunoassay methods rely on antibodies that recognize, as broadly as possible, the population of HCPs entering the
downstream purification process; therefore, the sandwich immunoassay, designed with polyclonal antibodies, is the workhorse
of HCP monitoring and quantitation. This assay format offers a combination of high sensitivity, specificity, throughput,
automation potential, rapid turnaround, quantitative results, and low cost per assay that is unmatched by any other currently
available assay technology. Other immunoassay formats (e.g., competitive immunoassays) may or may not be suitable, because
ci
they lack either the specificity or the sensitivity afforded by the sandwich format. Although these methods result in a single HCP
value for a given lot, the number can give greater weight to HCPs for which high-affinity antibodies are present in the reagent(s)
—and no or low weight to HCPs which are either not recognized or recognized by low-affinity antibodies in the assay. For these
reasons, orthogonal measures of product purity are often needed. More details on these methods can be found in 5. Supporting
Technologies for Residual HCP Detection, Identification, and Measurement.
ffi
The basic principles and design of immunoassays are discussed in USP chapter Immunological Test Methods—Enzyme-Linked
Immunosorbent Assay (ELISA) á1103ñ. The format that is most commonly used for HCP testing is the sandwich immunoassay
with detection systems such as colorimetric, electrochemiluminescent (ECL), chemiluminescent, radioactive, or others.
Homogeneous immunoassays, including competitive assays, where all of the reagents are combined at once and the binding
occurs in a single step without washing, may be problematic due to antigen excess leading to antibody insufficiency issues
(discussed later in the chapter); therefore, these formats should be used with caution. The heterogeneous sandwich
O

immunoassay format described in chapter á1103ñ is generally preferred, because the dynamic range and sensitivity may be
reduced in the homogeneous format. The formats, with their advantages and disadvantages, are discussed further in chapter
Immunological Test Methods—General Considerations á1102ñ. Data analysis is typically performed with a nonlinear fit of the
sigmoidal curve generated by a wide range of standard concentrations, although some analyses may focus on the low end of
the curve for greater sensitivity.

3.1 The Assay Development Cycle

Figure 1 illustrates common assay development plans, depending on the reagents available at various stages. Fewer bridging
studies are required when: 1) platform reagents are available, and 2) upstream processes are historically consistent. Figure 1A
illustrates a scenario where platform methods are not available, and a commercially available assay is used up to the stage of
process validation with appropriate assay qualification. For phase III and beyond, either a platform or upstream, process-specific
method is preferred. A bridging study should be performed to support assay replacement. If the commercial assay is intended
for phase III and post-approval, care must be taken to fully demonstrate that the assay reagents are applicable to the process
HCPs. An additional consideration is that the reagents are from an outside vendor over whom the biopharmaceutical
manufacturer has less control.

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 3/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
4

al
Figure 1A. If there is no platform assay before product development starts. Figure 1B. If there is a platform assay before
product development starts.
ci
Figure 1B suggests that a platform assay can be used through all stages of product development if already available when
product development starts. The platform assay should be qualified for each new product. Switching to an upstream,
process-specific assay may be necessary for phase III or process validation and beyond, if the cell culture process is significantly
changed from the platform process, and may introduce significantly different HCP populations. A bridging study should be
ffi
performed to support assay replacement.
The limitations of a downstream-specific HCP assay should be considered, because it may be tempting to think that HCP
assays are improved by taking the HCPs from null expression through the first column(s) and immunizing animals with a
downstream column pool. Historically, the logic was that the immunogen and standard would be enriched in those HCPs most
likely to enter the recovery process and be in the final product. This strategy was based on the assumption that, rather than
having thousands of irrelevant proteins in the immunogen, only those most likely to be in the process will be present in the
O

immunogen; thus, the resulting assay will be focused on those HCPs of greatest interest. Potential concerns that may arise as a
result of this approach are:
1. If only HCPs from a null cell run pool from the first column are used, then later changes during process development
(e.g., changes in column run conditions) could invalidate the HCP assay. Thus, process development becomes very
restricted and involves the risk of needing to develop multiple HCP assays for slight process changes (or the need to
manage the uncertainty).
2. HCPs that co-purify with a product may do so because they bind directly or indirectly to the product protein. A null cell
run of a column without the product protein would miss these HCPs.
3. Nonspecific adsorption to chromatography resins is not uncommon and, often, is not a reproducible phenomenon.
Compared to the first passage, subsequent passage of the null cell run material over a column will likely produce a different
set of HCPs after passage over a new column resin.

3.2 Development and Characterization of HCP Reagents

3.2.1 PREPARATION OF ANTIGEN/HCP STANDARD
As described in 1.1 Considerations for Manufacturing, Characterization, and Consistency, the total HCP “antigen” is a complex
population of proteins; therefore, when generating the HCP antigen/standard reagent, it is important to ensure that: 1) the
calibration standard is representative of the cell line and manufacturing process, 2) its protein concentration is accurately
quantified, and 3) the immunogen is administered in a way that generates polyclonal antibodies with reactivity to as many
different HCPs as possible. The HCP antigen composition should also be comprehensive enough to tolerate normal process
manufacturing changes during the life cycle of the product(s).
In addition to the uses above, the HCP antigen may also be needed to prepare the affinity column for purification of the
antisera. If affinity purification is used, the quantity of HCP antigen needed should be carefully planned. The amount produced
should be large enough to provide sufficient inventory for many years (often 10–20 years); ideally, for the whole life span of
the product. However, the antigen preparation process should be performed and documented in a way that facilitates a
potential resupply.

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 4/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
5

Because the HCP assay is used to test DS samples that contain trace HCP impurities, any cross-reactivity of the anti-HCP
antibodies with the product may compromise the test method and yield biased results. Therefore, any contamination of the
HCP antigen with product must be avoided to prevent the generation of anti-product antibodies.
3.2.1.1 Preparation of HCP antigen from mammalian cells: The majority of biopharmaceutical products produced today are
expressed in mammalian cells, e.g., CHO cells. A typical process for the preparation of CHO HCP antigen is outlined in Figure 2.

al
ci
Figure 2. Example of mammalian HCP antigen preparation.
ffi
The first step is to establish a null cell that does not express the product gene, either by using non-transfected (i.e., parental)
cells with a common origin to those used to make product alone, or to transfect them with the vector used to create the
production cell line without the product coding gene (historically described as a “mock” transfection). The main advantage to
the latter is that selective markers (e.g., dihydrofolate reductase or glutamine synthetase) are expressed. Alternatively, antibodies
can be raised to selective markers independently. Another advantage is that mock-transfected null cells express selection markers
and may be grown in cell culture conditions more closely resembling the manufacturing process. Using pools of mock
O

transfected null lines which have not been cloned allows for the maximum potential genetic diversity and therefore gives the
highest potential of generating a broad host cell protein population. However, these cultures, which are seeded with cell pools
(not cloned), may demonstrate more variability from run to run. Both approaches [non-transfected (parental) and mock
transfected] are commonly used. Both are also subject to the following issues: the cell culture process optimized for the
production clone may not lead to similar viability and cell density of the null cells. In addition, in the absence of the stresses
experienced by cells producing large quantities of product protein, the null cell lines may exhibit a different HCP profile. These
changes in viability, cellular metabolism, and cell density may alter the HCP profile of the antigen, making it less representative
of the manufacturing process. More about choosing appropriate HCP antigen preparation conditions is discussed below.
Once the null cells have been established, the HCP antigen is prepared in a bioreactor, cell culture flask, or cell culture bag
reflective of the product or process cell culture conditions. A platform cell culture process may be applied, which represents a
common manufacturing process for several products (e.g., CHO-derived monoclonal antibodies). Typically, the resulting HCP
antigen is minimally processed to ensure a broad HCP spectrum, making the antigen suitable for HCP monitoring of multiple
products generated from the platform process. A platform HCP antigen also may be produced by combining several null cell
runs with slightly different cell culture conditions (e.g., allowing the culture to remain in the bioreactor for longer periods of
time to vary the cell viability). This approach is used to obtain a broader HCP spectrum, and antisera raised against this
immunogen may be suitable for HCP detection in products generated from a variety of slightly different processes. These
reagents also may increase the robustness of the HCP immunoassay toward process changes.
Alternatively, the HCP antigen may be prepared using an upstream, process-specific approach in which the cell culture
process is tailored to mimic the production process for a unique product or specific process. The advantage of this approach
may be the high relevance of the resulting HCP antigen to a particular upstream process. As with platform reagents, because
no additional processing purification steps are included, the antigen contains a broad range of HCPs, and the antigen will often
remain suitable for HCP monitoring after downstream production-process changes. The disadvantage of this approach is that
the use of the reagents typically will be limited to a single (or few) product(s) or process(es).
The HCP antigen can be produced from a representative small scale, pilot scale, or production scale run. There are pros and
cons for each approach. Pilot or production scale may mimic the actual process best; however, normal variation between cell
culture runs may not be reflected if only one run at large scale is applied. Conversely, several small-scale preparations can be
pooled and may better reflect the run-to-run variability of the cell culture process. Regardless of the scale, the presence of
product should be aggressively avoided, because its presence in the HCP immunogen will compromise the quality of the
resulting immunoassay antisera.

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 5/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
6

After performing the null cell run, the HCP antigen can be prepared from the cell lysate, the concentrated CCF, or as a mixture
of both as shown in Figure 2. If the antigen is prepared from CCF, the cells may be harvested at a time when production line
harvest is performed or some days later, to allow for some of the cells to lyse and release HCPs into CCF and thereby broaden
the HCP spectrum.
To prepare immunogen from cell lysate, the cells are harvested by centrifugation, and the cell pellets are washed and lysed
(e.g., by using repeated cycles of freeze/thaw, or high pressure homogenization). Conditions are typically selected to mimic
the production process (i.e., to prepare immunogen from CCF, cells and debris are removed by centrifugation, and the CCF is
then treated by ultrafiltration/diafiltration). The buffer is exchanged (e.g., into PBS or HEPES), and the CCF is concentrated. The
cut-off for filtration should be chosen to minimize loss of HCP, e.g., 10 kDa or less.
3.2.1.2 Preparation of HCP antigen from bacterial cells: Many of the above-described principles used in the preparation of
mammalian HCP antigens also apply to prokaryotic cell culture. However, some unique challenges should be considered. In
particular, manufacturing processes for bacterial expression system come in more varieties, such as those yielding concentrated
protein deposits known as inclusion bodies, which can represent up to 95% of the total cell protein. HCP impurities are also
present in the inclusion bodies, including bacterial membrane proteins, ribosomal subunit proteins, and cytoplasmic proteins,
such as small heat shock proteins or chaperones. The formation of inclusion bodies in the production strain makes it more
difficult to generate a representative HCP preparation from a null cell fermentation process. The null cell may not have the same
HCP profile, and it will not generate inclusion bodies with which specific HCPs may co-purify, as described above. In other cases,
HCPs have been created from periplasmic secretion systems, if the production process can be replicated in the null cell.
Because there is no obvious approach for overcoming these limitations, in practice the bacterial HCP antigen is usually
generated from the lysates of washed cells, using null cell fermentations, that are grown and induced under conditions
representing the upstream manufacturing process as closely as possible. As for any HCP antigen, it is necessary to demonstrate
that the HCP profile is representative of the manufacturing process before use in assay development. This approach yields the
full complement of HCPs expressed and hence is very broad.

al
Because most animals have been exposed to bacterial antigens and may have pre-existing antibodies to many bacterial cell
proteins, it is important to characterize the pre-existing antibody responses, and if appropriate, consider the use of specific
pathogen-free (SPF) animals. The presence of significant levels of pre-existing anti-HCP antibodies may confound the analysis
of the antibody responses; therefore, it is important to screen preimmune sera from animals for pre-existing antibodies prior to
immunization.
ci
Null bacterial cells transfected with the expression vector contain chaperone genes that may be expressed at high levels
(e.g., 5% of the total HCPs). Such overexpression of particular HCP impurities might necessitate the development of
single-analyte assays for these particular impurities (see 6.1 Assays for Individual HCPs).
3.2.1.3 Characterization of the HCP antigen: Several analyses are recommended before immunization: 1) protein content
(total protein assay); 2) absence of product (as shown by Western blotting, immunoassay, and/or MS analysis); and 3)
ffi
characterization by 1-D or 2-D-polyacrylamide gel electrophoresis (PAGE). Protein concentration is measured to establish the
standard concentration for future use and to determine the overall amount of the prepared antigen. The most commonly used
methods are the bicinchoninic assay (BCA), the Bradford assay, and amino acid analysis (AAA), although the colorimetric
methods require a standard (e.g., BSA) that is not well matched to the (HCP) analyte. Absorbance at 280 nm (A280) may also
be used although it is less specific for protein (e.g., nucleic acids will also be measured). These methodologies often provide
similar results, but some methods may be more significantly affected by the presence of interfering substances than others.
O

One should consider using two orthogonal methods to exclude a gross over- or underestimation of the protein concentration
by one particular method. For more information, see general chapter Biotechnology-Derived Articles—Total Protein Assay á1057ñ,
which discusses the advantages and disadvantages of various protein analysis methods. The HCP concentration must be
assigned with a scientifically sound approach that is used consistently for its lifetime. The lack of product can be assessed with
gels, anti-product Western blots, or other suitable methods. Lastly, the 1-D and 2-D gels help characterize the pattern of HCPs,
show that a broad spectrum of proteins is present, and show that a reasonable match to production is present. However, this
can be challenging because of the presence of large amounts of the product, which can obscure HCP detection. Comparisons
may be made between the specific populations of host proteins produced in the null cultures and production cultures using
the orthogonal methods discussed in 5. Supporting Technologies for Residual HCP Detection, Identification, and Measurement.
3.2.1.4 HCP standard reference reagents: Frozen HCP material is usually stable for a very long time, but stability of the HCP
standard reagents should be monitored over time because the assay lifetime can be very long. HCP standard stability can be
confirmed by monitoring the HCP standard performance in the HCP immunoassay as well as in orthogonal methods.
Appropriate controls within the assay range may be established to monitor assay performance. Controls may be prepared
from independent dilutions of the HCP standard, product samples or intermediate pools, or spiked product samples. However,
there is a slight risk that control samples prepared as a dilution from the same HCP standard material can degrade at the same
rate as undiluted HCP standard, and degradation of the standard will not be detected. To mitigate the risk, data from several
standard curve parameters (e.g., signal, background, slope, coefficient of determination) are often assessed for each assay and
used to support HCP standard stability. Using material different from the reference to prepare controls will help ensure that the
degradation rate will be independent, and HCP standard degradation can be detected easily.
Assay control charts can be established and used to record reference curve parameters, control sample values, and lot-specific
information for the critical reagents, such as labeled HCP antibodies. Acceptable ranges for the controls should be established
on the basis of multiple runs (usually 20–30) and may be used as part of routine assay acceptance criteria. In addition, the values
for % coefficient of variation (CV) of standard and control replicates are often a part of the assay acceptance criteria. Differences
over time in HCP standard curve performance or HCP control levels, or an increase in assay variability, may indicate a lack of
HCP standard stability.

3.2.2 PREPARATION AND CHARACTERIZATION OF ANTIBODIES

3.2.2.1 Preparation of antibodies to HCPs: The anti-HCP antibodies prepared are often used for many applications (Western
blots, immunoassay) over a long period of time (e.g. the anticipated life of a product or platform). Therefore, material needs

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 6/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
7

should not be underestimated. The recommended antisera quantity will depend on many factors including the intended use,
the predicted quantity purified from a given volume of antisera, the amount coated on microtiter plates, and many other
considerations. The choice of animal species used to generate anti-HCP antibodies is driven by the immunogen, the
requirements for the particular method, and the preferences of the individual investigator. Rabbits, sheep, and goats are often
chosen for immunization programs. In some cases, multiple animal species have been used. In such cases, using species more
phylogenetically distant from mammals (e.g., chickens) have helped to generate anti-HCP antibodies to conserved mammalian
HCP proteins. The choice of animal species may also be driven by a desire to either increase the amount of antisera available
from one or several animals (goats or sheep), or to use a strategy to obtain a greater diversity of responses provided by the use
of many more individual animals from a single species (e.g., rabbits). The number of animals chosen to generate the antiserum
pool(s) depends on the species used and the duration of the immunization protocol. If smaller animals are used to generate
the antiserum, more individual animals are generally needed (e.g., 10–20 rabbits). In contrast, if larger animals (goats or sheep)
are used, investigators usually will immunize 3–10 animals per protocol. Typical immunization protocols provide approximately
100–150 mL of antiserum per rabbit or approximately 500 mL of antiserum per goat, and the total yield of Protein A or Protein G
purified antibody is approximately 1 gram per rabbit or 5 grams per goat. Animals should be screened for pre-existing antibodies
against drug substance prior to immunization. If positive results are found, then these animals should be excluded from
immunization.
A portion of the prepared HCP antigen is mixed with an appropriate adjuvant (most commonly, Freund’s adjuvant or in
combination with incomplete adjuvant) and used for the immunization of animals. Each animal receives an initial priming
immunization, followed by multiple (often 4 to 8) booster immunizations, to mature the immune response and generate
high-titer, high-affinity antibody responses. Antiserum is collected from each animal before the initial priming immunization
(time 0) and at appropriate intervals (usually 10–14 days) after booster doses. Four to six bleeds are usually collected from each
animal. However, the duration of the immunization protocol may be extended by increasing the number of booster
immunizations, making it possible to collect additional high-titer antisera. Other immunization strategies may also be helpful

al
(e.g., cascade immunization or size fractionation of the HCP immunogen).
Investigators should perform titer or Western blot analyses of the individual test bleeds before antiserum pooling to screen
for and remove sera that 1) have low titer, 2) display an immunodominant immune response to a small group of HCPs, or 3)
have nonspecific binding characteristics or anti-product reactivity. This evaluation should also be performed on the final
antibody pools to demonstrate broad coverage of HCPs and a lack of binding to the therapeutic product. In addition, some
ci
laboratories have found it useful to evaluate antibodies for aggregates using size exclusion chromatography as described below.
Acid elution conditions may cause some antibodies to aggregate, and multimers of this type, particularly when labeled with
either biotin or horseradish peroxidase, may lead to increased assay background. In addition, some laboratories include a final
process-scale size-exclusion chromatography step to reduce aggregate levels to low levels (e.g., <5%).
Multiple approaches to the purification of antibodies from antiserum pools have successfully generated antibodies suitable
ffi
for use in HCP immunoassays. Antibody purification is often performed by Protein A, Protein G, or HCP column affinity
chromatography. The application of coated magnetic beads can also provide benefits in some scenarios. The choice of Protein A
or Protein G is driven by which animal species was used to generate the antibody. Manufacturers provide standard protocols
for these routine antibody purifications. When affinity purifying anti-HCP antibodies, materials such as chromatography resins,
which may have been used previously for other products, should be avoided, and additional cleaning cycles should be in place
to minimize carryover. Ideally, manufacturers should use a resin that has never been exposed to a DP. Before the affinity
O

purification step, some laboratories also include an initial 50% ammonium sulfate precipitation of the crude antiserum to enrich
and concentrate the antibody fraction and thereby extend the performance and duration of use of affinity columns. The use of
Protein A or Protein G purification strategies generates reagents in which the anti-HCP-specific antibodies compose a smaller
proportion of the total antibody. In contrast, HCP affinity purification strategies selectively purify anti-HCP-specific antibodies
from the antisera or antibody-enriched preparations. In this case, the HCP antigens are coupled to an activated resin, and the
antiserum is purified over the column following established protocols or the resin manufacturer’s instructions. The purified
antibodies may be further purified by size-exclusion chromatography to remove aggregates, then dialyzed, concentrated,
divided into aliquots, and stored frozen (typically −70° or colder). Neat antisera should also be stored frozen (see á1106ñ for
additional information regarding storage of serum samples).
Affinity purification of specific antibodies using HCP immobilized on a column requires careful management of the column
preparation and use so that it can be reproduced reliably for future purifications. In addition, if reused, appropriate resin storage
and regeneration procedures should be evaluated and controlled to avoid degradation during storage. Done properly, it is a
reliable, reproducible process that has led to consistent anti-HCP antibodies and HCP immunoassays. After loading the column
with antisera, an optimized wash procedure is critical. Because the preparation is a mixture of antibodies with differing affinities,
extensive washing may remove low-affinity antibodies. Similarly, loading large amounts of anti-HCP antisera onto the column
may result in high-affinity antibodies, displacing low-affinity antibodies if they recognize epitopes that are in close proximity.
In either case, the low-affinity antibodies may be removed. In theory, higher affinity antibodies may also be more challenging
to elute. Whatever load and wash conditions are initially selected for the antigen-specific affinity purification, these conditions
need to be maintained in future production lots of the antibodies. A comparison between these two purification strategies is
shown in Table 2. Both approaches are commonly used.

Table 2. Antibody Purification Strategies

Type of Purification Pros Cons
Protein A or G Affinity Column Chromatography Robust and reproducible for quickly separating IgG Lower proportion of antibodies specific to HCPs and
fraction from other serum proteins may have a higher amount of low affinity antibod-
ies

HCP Affinity Column Chromatography Enriches for specific high affinity anti-HCP antibod- More skill and documentation required to make and
ies; excludes nonspecific antibodies and serum pro- maintain resins consistently. Some high affinity an-
teins ti-HCP antibodies may not be eluted in usable form
and may not be recovered

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 7/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
8

3.2.2.2 Characterization of antibodies to HCPs: Antibodies prepared for capture and detection are assessed independently
and as part of the sandwich immunoassay pair. The concentration of the unmodified antibody is determined most commonly
by absorbance at 280 nm, AAA, or BCA. Each approach is acceptable, provided it is applied consistently and (if colorimetric) is
standardized similarly. It is important to determine the concentration of the capture and detection antibodies in each batch
because they will be diluted to a certain concentration in the optimized immunoassay method. The detection and capture
antibodies are characterized by their performance in the immunoassay method(s). The best label to antibody conjugation ratio
should also be empirically evaluated primarily based on the best performance in the assay. This optimal ratio should be targeted
in the future when new labeled antibodies are made. Finally, although antibodies are generally very stable when stored frozen
(e.g., −70°), investigators should ensure storage integrity and determine a defined period of use. This can be accomplished by
continuous monitoring with trending charts or by requalification experiments at specified time intervals.
The quality of the antibody pair (capture and detector) is often evaluated in two ways:
1. Show that the antibody pairs are specific and sensitive in an immunoassay format for the HCPs present in a series of
samples taken from the unit operations of a given purification scheme, including the DS. This topic is also addressed in
more detail in 4. HCP Immunoassay Method Validation. In concept, the ability to detect and measure HCPs
(immunoreactivity) in a series of actual process samples from a given process for a product in development is
demonstrated with data showing: 1) sequential clearance as the product is purified, 2) sample linearity throughout a
dilution series, and 3) lack of cross-reactivity to product or matrix. The reduction of HCPs during the purification process
is determined by clearance factors where the HCP content (in ng/mg) at each step in the process is divided by the amount
in the prior step. Typically, starting samples from the cell culture may have HCP levels ranging from several hundred
thousand to several million ng/mg, and final products may have HCP levels ranging from <1 to 100 ng/mg (showing
many logs of clearance). When these trends are observed, they suggest that the process and the assay are functioning
effectively, and the antibodies used in the immunoassay have suitable quality attributes. If these trends are not observed,

al
it may not necessarily reflect a problem with the antibodies, because it is also possible that the purification process is not
effective.
2. Show that a broad range of HCPs in the calibration standard is recognized (i.e., that the coverage of the HCP population
is adequate). Coverage is evaluated for at least the capture antibodies using 2-D gel Western blots or immunoaffinity
fractionation of the total HCP population by immobilizing the anti-HCP antibodies on a column. In addition it is
ci
recommended to test the coverage of the detection antibody if different coating and detection antibodies are used. The
benefits and limitations of using either 2-D gels or immunoaffinity fractionation to demonstrate coverage is discussed
more fully below. Whichever method is used, the extent of coverage must be addressed in qualifying the
immunochemical reagents.
HCP coverage evaluations help assess the ability of the antibodies to recognize a wide range of HCPs in the calibration
standard and those present in in-process and DS samples. Two methods (2-D gels followed by Western blot analysis and
ffi
immunoaffinity purification followed by 2-D gel analysis) are in fairly common usage, and both have the limitation that they
tend to underestimate the true antigen binding in immunoassay methods but may have value in comparing two preparations
head-to-head (or a platform to a commercial reagent). Both procedures use reducing 2-D gel electrophoresis to separate HCPs
in the HCP standard or in early process sample(s) (e.g., harvest). Numerical coverage comparisons should be used with caution
because of the many method variables and the art required to reproduce results, even with the same reagents in the same
laboratory. Because of this variability, the results are best evaluated qualitatively. Comparisons of batches (or sources) of
O

antibodies should be done side by side as much as possible to determine if antibody lots are comparable or if one is superior
to another. One approach, the SDS-PAGE/Western, is essentially a comparison of the number of spots present in the
immunologically stained membrane versus the number in a duplicate gel (or blot) stained for total protein (e.g., by fluorescent
dye or silver; see also USP chapter Immunological Test Methods—Immunoblot Analysis á1104ñ). Differential staining of each
antibody preparation may power the analysis, because it can be used to analyze the same 2-D gel. The second approach,
immunoaffinity binding/SDS-PAGE, involves comparing the flow through and eluate to the load from the HCP calibration
standard (or early process sample) passed over a resin to which the capture antibodies have been covalently immobilized. The
resin is washed after loading and before elution to remove nonspecifically bound HCPs. For the purpose of sample comparison,
difference gel electrophoresis (DIGE) technology could be helpful. Table 3 lists the advantages and disadvantages of these two
coverage methods.

Table 3. Comparison of Coverage Methods

Pros Cons
Good separation of individual HCPs allows for individual spot HCP antigens are denatured, may not represent what is seen in
counting. immunological assays (e.g., ELISA).

High variability—numerical “percent coverage” values vary

widely with the same material tested within a single lab and
between different laboratories.
2-D SDS-PAGE/Western blot Transfer efficiency of a broad range of HCPs difficult to opti-
mize, leading to underestimates due to over-transfer through
the membrane or failure to transfer from the gel, dependent
on molecular weight.

Matching spots between blots and gels are difficult and not
standardized.

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 8/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
9

Table 3. Comparison of Coverage Methods (continued)

Pros Cons
HCPs bind to antibody resin in solution under native conditions May underrepresent some HCPs if they are bound too tightly
similar to the sandwich immunoassay. and do not elute, resulting in an underestimation of coverage.

Analysis of spots in gels does not require immunoblotting and Preparation of the anti-HCP resin must be done carefully and
Immunoaffinity binding/
is simpler because the problems of transfer are avoided. may be difficult to reproduce. Results can be dependent on
1- or 2-D SDS-PAGE
resin loading and elution conditions.

Proteins at low concentration that only show up in Westerns will

not be detected.

More information about the appropriate use of SDS-PAGE/Western blot methods and ways to minimize the disadvantages
above can be found in the later section 5.2 Considerations for Western Blot Methods.
An example of a suitable use of this approach is shown in Figure 3.

al
ci
ffi
O

Figure 3. Left panel: 2-D IEF/SDS-PAGE analysis of representative CHO HCP calibration standard stained with a sensitive
fluorescent stain. Right panel: Western blot analysis of the same gel shown in the left panel.

3.2.3 GENERATION AND QUALIFICATION OF REPLACEMENT REAGENTS

When the supply of HCP reagents are depleted or if their performance declines, new reagents may be needed. Often, new
HCP antigens and new HCP antibodies are both generated at the same time. In theory, the new set of reagents should be
created by following the same protocol that was used for the previous set of reagents to match the performance of the new
and old assays as closely as possible and produce consistent data for a given program. In practice, however, it is not always
possible (nor advisable) to generate the assay reagents exactly the same way that they were originally made. For example, if
the manufacturing process has been optimized over time and the original platform assay reagents are no longer relevant, a
careful assessment should be made. When such changes are required, the ideal way to confirm the quality of the replaced
reagents is through characterization of antibodies (see below) and demonstrating that they can detect similar or greater HCP
log reduction, from harvest to DS, using the same samples tested head to head with the original assay reagents.
When replacing an HCP antigen in which the current production process is essentially the same as the old process, the new
HCP antigen should be prepared as similarly to the old as possible. It is important to compare the protein concentrations of the
old and new HCP antigens side by side, using the same method to correctly assign the protein concentration for the new antigen
and to ensure that the protein concentrations are comparable in the new and old HCP calibration standard. Characterization
by 1-D and 2-D SDS-PAGE should also be performed as a side-by-side comparison of the old and new HCP antigens (see above)
to be sure that gross changes in protein composition have not occurred.
For antibody replacement, the same animal species should be used, if possible. In cases where a better antibody response is
desired, antibodies produced by different species may be evaluated. The selection of the species should take into account all
the points discussed in this chapter. A side-by-side comparison of results from process samples using old and new antibodies
in a sandwich immunoassay format should be included to demonstrate suitability of the replacement antibodies. Additional
characterization by orthogonal methods (e.g., coverage using 1-D and 2-D SDS-PAGE) is recommended, as described above.
Assay qualification or validation should also be considered after changing reagents.
To fully understand the effect of replacing HCP critical reagents on assay results requires testing of process samples in bridging
studies using multiple samples from harvest, process intermediates from all appropriate purification steps, and the final DS.
These studies can be very resource intensive, which underscores the importance of making sufficient quantities of HCP critical
reagents at the outset (and minimizing replacement).

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 9/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
10

3.3 Immunoassay Method Development and Qualifying as Fit for Use

As noted in 1. Introduction and Scope, HCP immunoassays serve two important functions in the development and control of
biopharmaceutical manufacturing processes. HCP immunoassays serve as a measure of product purity, and therefore are
potentially related to patient safety. They also serve as a measure of manufacturing consistency, and therefore they are reflective
of process control. To meet these requirements, HCP assays are often validated and incorporated into the cGMP control system,
which includes specific reject limits (in the DS as a Certificate of Analysis (C of A) method or as an in-process control). If the
final DS is a conjugated protein, the testing should be done on the protein intermediate (before conjugation). In addition, HCP
assays are used to guide process development and are often used to demonstrate robust process performance in the context
of formalized process validation activities.
HCP immunoassays are often formatted as a sandwich ELISA, and multiple antibody labeling options are available (see
á1103ñ). Depending on the label chosen, appropriate conjugates and substrates are selected; examples are described in
á1103ñ. Most proteins can be biotinylated because of their lysine content and, because biotin is small, it is less likely to affect
the protein’s binding activity. Because antibodies are large and have many lysine residues, the conjugation ratio of biotin to
antibody can be higher than that of smaller proteins. However, it is important to not over- or under-label the antibody (e.g.,
with biotin or HRP), because this might lead to either less antigen binding or lower signal/sensitivity, respectively. Controlling
and defining the labeling stoichiometry (e.g., mole of biotin to mole of IgG) is useful for making future batches consistently.
Multiple ratios of any antibody label should be tested and optimized for performance in the immunoassay. Excess unconjugated
reagents should be removed, either by dialysis, affinity purification, or by using a suitable desalting column.
Often, the standard is first screened to see whether a dose-response curve can be generated, and if so, in which concentration
range of analyte. The results can also reveal a starting point for initial reagent concentrations that can be optimized further. If
more than one antibody pair has been prepared, then each is tested to find the best performance (see USP chapter á1103ñ for

al
more information). The signal (low calibration standard) to noise (background) ratios generated with each antibody candidate
can be used to select the best pair. Typically, HCP immunoassays do not always possess a full dose-response curve with both
asymptotes; therefore, for the purposes of residual HCP detection, the assay for DS release should focus on the low end of the
curve.

3.3.1 QUALIFYING A NEW MANUFACTURING PROCESS WITH A PLATFORM HCP ASSAY

ci
As noted earlier, if the upstream/isolation procedure is matched, then a new product produced in that system can benefit
from a platform assay approach that can decrease qualification time and effort. However, in cases where there are variations
that occur in optimizing the production cultures and the cell culture design space is not completely defined, it is important to
understand whether these variations are significant enough to render the platform assay unsuitable. In these cases, the platform
ffi
assay should be qualified experimentally to demonstrate that it is suitable for measuring HCPs from the new process. Assay
accuracy, precision, sensitivity, linearity, range, and specificity must be evaluated (see 4. HCP Immunoassay Method Validation).
In addition, the range of HCPs in the platform HCP standard should be compared to those present in the new process. The
antibodies should also be characterized using samples from the new process or product using approaches discussed previously.
The following approaches and concepts may be useful in determining if a platform assay is suitable for a product made with a
new process:
O

1. Conduct a representative, small-scale null cell run grown under conditions used in the new culture process, and determine
the protein concentration of the null cell run CCF. Assay the null cell run CCF at the same nominal concentration as the
assay HCP standard using the HCP sandwich immunoassay, and compare the dilution curve from the null cell run CCF
to the standard used in the assay. If the standard and new null cell run CCF curves are similar (e.g., in shape and amplitude,
and the calculated HCP concentrations are within a factor of two), then the new process is considered not different from
the platform, and a process-specific HCP assay is not usually needed.
2. With the CCF from the above null cell run from the new process, make a comparison to the HCP calibration standard
using 2-D gel electrophoresis. This will compare the diversity of proteins produced by the two processes. However, be
aware that new “spots” may appear in the 2-D gels that may not reflect immunochemical differences in the assay, for
example, post-translational modifications or limited proteolysis. As such, the appearance of a few new spots or
up-regulated or down-regulated spots in the process-specific null cell run is not a basis for invalidating the application
of the platform assay. A qualitative assessment (e.g., versus a Western blot) is recommended.
3. Comparison of HCP levels at harvest in actual production runs (CCF samples) with the same product produced in the
original versus the “new” process is also valuable. In general, immunoassay results within a factor of two are often
considered similar, thereby confirming the platform HCP reagents.
4. HCPs in CCF from production cultures may also be estimated on the basis of total protein measurements. The total protein
in the CCF is the sum of product (measured using a product-specific titer assay) and HCPs. Samples from harvests of the
manufacturing run cell culture can be dialyzed, as long as they are sufficiently concentrated to make membrane losses
negligible, to remove small peptides and amino acids, and then the total protein is determined (e.g., by amino acid
analysis). Subtraction of the product concentration from the total protein provides an estimate of the non-product
protein, HCP, in mg/mL. This value may be compared with the HCP concentration from the immunoassay. Comparable
values (e.g., within 2×) indicate that the HCP platform is confirmed for the new process.

3.3.2 NEED FOR NEW REAGENTS AFTER PROCESS CHANGES

Major changes, such as substantial and unambiguous changes in cell culture conditions (e.g., moving from a
serum-containing process to a serum-free production process), typically require production of new reagents and development
and validation of a new HCP assay. In this example, the HCP reagents created using the old process recognize a number of
serum proteins but may not recognize many new HCP proteins in the serum-free process. Because of both the process change

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 10/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
11

and the assay change, different HCP levels will be observed when the same samples are measured for HCP in the old and new
assays (i.e., the samples from the serum-containing process may have a high level of HCP measured by the old assay and low
level of HCP measured by the new assay, whereas the samples from the serum-free process may have much lower HCP results
by the old assay but more with the new assay).
If the assay was developed as a downstream, process-specific assay, changes in the purification steps can also require replacing
the assay with a new one that is relevant to the new downstream process. This is the biggest problem with these very specialized
assays and why platform-based assays are usually preferred, because they often do not require a new assay when the
downstream steps are changed.

3.3.3. SAMPLE STABILITY

It is important to demonstrate that the sample handling procedures do not affect the assay results. This is particularly true
when comparing results for various intermediate process pools. Occasionally, HCPs can precipitate during freezing or when
the sample pH is adjusted from acidic to neutral. In addition, storage at 2°–8° or multiple freeze-thaws may result in loss of
reactivity. The analyst may need to add specific stabilizing components (e.g., divalent cations) to the samples to ensure stability.

4. HCP IMMUNOASSAY METHOD VALIDATION

HCP immunoassays for product purity assessment typically are part of the cGMP control system, either as an in-process test
or on the C of A, and need to be validated appropriately. The validation approach depends in part on the types of samples that
will be tested with the method. All validation parameters for a quantitative impurity test are needed when used for the final
DS, whereas validation for in-process samples usually focuses on dilution linearity, interference, and precision. Regardless of

al
whether the assay is an in-process or release assay (DS), action limits are valuable as a part of an overall control strategy. Readers
are referred to ICH Q2(R1) guidelines and chapter Validation of Compendial Procedures á1225ñ for general expectations for assay
validation, particularly those requirements for cGMP assay validation, and to chapter á1103ñ for sandwich immunoassays.
Although the main focus of this section is on cGMP assay validation, many of the same principles apply to the assays used to
demonstrate that process purification steps are suitable for their intended purpose.
ci
This section will focus on those aspects of HCP immunoassays that are unique or require special attention and documentation.
HCP immunoassays differ from more conventional immunoassays in that they are multi-analyte assays. There are potentially
thousands of proteins in the immunogen and standard, and a correspondingly diverse set of antibodies is produced, yet only
upstream CCF will contain a diversity of proteins that approximate the HCP calibration standard. Intermediate pools and final
products typically contain progressively fewer of the HCP proteins that are present in the standard. As discussed previously, this
ffi
is the aspect of accuracy that is compromised by HCP immunoassays, and why the mass unit ratios (ng of HCP per mg of DS)
are not a literal “mass” relationship. However, below are common industry approaches that address these limitations as much
as possible.
The validation summary below is for the final DS or a penultimate process step, whichever is chosen for the C of A method.
It should be noted that for in-process testing, the focus is on method accuracy (spike recovery), dilution linearity, and precision.
In the case of accuracy, some samples will have more potential interfering substances (other process impurities or additives)
O

that may need to be confirmed as non-interfering (e.g., residual DNA, leached protein A, virus inactivators, chaotropes, and
low pH buffers).

4.1 Accuracy
To assess HCP assay accuracy in the DS, at a minimum the analytical assay standard should be added to appropriate samples
and accurate spike recovery demonstrated over the range of the assay. Matrix interference can come from buffer components
and product, and the minimum dilution required for acceptable spike recovery (typically between 70% and 130%) needs to
be determined for each sample type. Typically, spike recovery experiments are conducted with spikes using at least three and
potentially as many as five different levels. Because samples are assayed at multiple dilutions, the investigator may spike and
then dilute the sample, or dilute the sample and then spike the diluted samples. Spikes near the quantitation limit (QL) help to
evaluate the accuracy and repeatability of the assay near the QL, which is where the measurement is often the most variable. A
spike recovery of 50%–200% may be acceptable for a spike at or near the QL.
Although these are minimum requirements for assay validation, they should not be interpreted as demonstrating accuracy
for any one specific HCP that may co-purify with the product. To accomplish that, comparison to a standard of that particular
HCP species is needed; however, because this HCP is rarely known, this may not be possible. Tips for discerning these situations
are provided below in the dilution linearity section and in 5. Supporting Technologies for Residual HCP Detection, Identification,
and Measurement.

4.2 Sensitivity and Assay Range

HCP sandwich immunoassays often achieve standard curve sensitivity in the low single digits of ng/mL. For sample protein
concentrations where the test dilution is 10 mg/mL, this implies that a sensitivity of about 1 ng of HCP per mg of product is
possible. However, this sensitivity may be difficult to achieve for products with lower protein concentrations. It is important to
highlight that the reported HCP ratio (ng of residual HCP relative to mg of product) is not really the ratio of masses implied by
the unit ng/mg but rather “immunological equivalents” per mg of product.
The first aspect of determining QL is typically determined experimentally for each product in a given sample buffer matrix
in spike recovery studies. The minimum recommended dilution (MRD) for the DS should be established by spiking the HCP
standard in a sample dilution series. For example, an HCP spike of 10 ng/mL added to each of a series of solutions containing
undiluted (e.g., 10 mg/mL) protein and serially diluted DS. Those dilutions in which the spike recovery is 70%–130% (50%–

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 11/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
12

150% is also commonly used for levels near the QL) are considered acceptable. Typically, a proposed DS MRD is tested further
to ensure that spike recovery is consistently achieved.
For the second part of the QL determination, the DS is tested at the MRD (if the DS samples have high levels of detectable
HCP, a formulation buffer may be used). The QL is generally determined by the analysis of spiked concentrations of HCP and
by establishing the minimum level at which the HCP can be determined with acceptable accuracy and precision. This study is
typically performed at least three times, preferably by different analysts or on different days. For example, a spike of 3 ng/mL
of HCP in a product protein concentration of 5 mg/mL has a QL of 0.6 ng/mg or 3 ng/mL if spike recovery is achieved in, e.g.,
at least four of six tests or if mean spike recovery criteria are met.
Typically, assays are set up to measure the range from a few ng/mL to >100,000 ng/mL. This range allows the analyst to
test a variety of samples at multiple dilutions (see discussion of 4.3 Sample Linearity) and allows for a practical assay that can
accommodate in-process pools with highly differing HCP levels. For example, upstream samples may contain >100,000 ng/mL
of HCPs and require large dilutions to obtain results in range of the standard curve.
For routine commercial DS manufacture where the protein product concentration is known and the process impurities are
well understood, testing at a single dilution may be used for release. The results are reported as the ratio of measured HCP (ng/
mL) to the product concentration (mg/mL) resulting in units of ng/mg. When the DS has undetectable levels the results are
reported as “less than” the assay QL (ng/mL) divided by the product concentration (mg/mL; e.g., <0.6 ng/mg in the example
above). Before setting this target concentration for testing, however, the dilution linearity of the samples should be well
understood and a robust manufacturing process established. In the event that the level or species of HCPs vary run-to-run it
may be necessary to test each sample at multiple dilutions (see below).

4.3 Sample Linearity

al
HCP assay nonlinearity is not uncommon for some samples and should be assessed for multiple batches of a given sample
type (e.g., from several clinical DS lots or process validation batches) to ensure consistency and proper reporting of results.
Because a single HCP (or a few individual HCPs) may co-purify with product, it is possible that particular HCP(s) will be present
in excess of the available antibodies in the HCP immunoassay. This is possible in multi-analyte assays where only a limited surface
area is available on assay microtiter plates or beads, and because thousands of different anti-HCPs are needed, antibodies to
ci
any one HCP will, of necessity, be limited. Furthermore, the relative amount of antibody to each separate HCP is also not
controlled, so the binding capacity for each separate HCP differs. For samples exhibiting this behavior, the analyst must dilute
the sample into the range of the assay, i.e., to a point where nonlinear behavior is no longer observed because the HCP has
been diluted to a concentration where it no longer exceeds the available antibody. In some cases, the sensitivity of the assay
becomes limiting, and one never reaches a dilution where the assay result is independent of the sample dilution. In such cases,
the highest HCP ratio value (corrected for sample dilution) within the validated assay range should be reported. A detailed
ffi
example of how to handle nonlinear dilutions arising as a result of antibody insufficiency follows.
Table 4 and Figure 4 show data for three different samples tested in an ELISA that has a range of 1–100 ng/mL. In this example,
all samples were diluted initially to 10 mg/mL (the MRD, where spike recovery had been previously confirmed), and then serially
diluted using a twofold dilution series to below the assay QL of 1 ng/mL. Whereas Sample 1 (diamonds) dilutes linearly over
the full range tested, both Sample 2 (squares) and Sample 3 (triangles) HCP results plateau at higher concentrations of the
sample. Saturation of the antibodies reflects antigen excess, and determination of the HCP value is made by diluting the sample
O

into the range of linear dilution; visually estimated as <2 mg/mL for the squares and <1 mg/mL for the triangles. [NOTE—If large
sample dilutions are required to get into the range of the HCP assay, consider making intermediate dilutions to limit
dilution-related errors.]

Table 4. Raw Data for the Graph in Figure 4a

Product Sample 1 Sample 2 Sample 3

Conc. HCP conc. HCP ratio % max ratio HCP conc. HCP ratio % max ratio HCP conc. HCP ratio % max ratio
(mg/mL) (ng/mL) (ng/mg) value (ng/mL) (ng/mg) value (ng/mL) (ng/mg) value
10.00 (neat) 49 (neat) 4.90 83% 20 (neat) 2.00 <20 3.2 (neat) 0.32 22%

5.00 28.5 5.70 96% 16.5 3.30 54% 2.5 0.50 35%

2.50 12 4.80 81% 10 4.00 66% 1.5 0.60 42%

1.25 7.4 5.92 100% 7.4 5.92 97% 1.1 0.88 61%

0.625 3.1 4.96 84% 3.3 5.28 87% 0.9 1.44 100%

0.3125 1.6 5.12 86% 1.9 6.08 100% <1 <6 NA

0.15625 <1 <6 NA <1 <6 NA <1 <6 NA

Reported HCP
ratio value
with guide Guide 1 = 5.2 (n = 6); Guide 1 = 5.8 (n = 3); Guide 1 = 1.2 (n = 2);
and n Guide 2 = 5.2 (n = 6) Guide 2 = 5.3 (n = 4) Guide 2 = 1.4 (n = 1)

a Numerical data for three samples (also graphed in Figure 4) show differing degrees of saturation of the response at higher concentrations of product (and HCP
impurity) tested. Two guides on how to interpret these data (guides 1 and 2) illustrate how the “linear range” of the assay is limited to the most dilute data points
shown in Table 4 and Figure 4. Note—Samples are always diluted until the HCP concentration is below the QL of the assay (in this example the QL is 1 ng/mL).
Guide 1: All values within 20% of maximum value are averaged. Guide 2: Values are averaged as long as the CV of the values is <20%, removing less diluted samples
first. A third guide might also be used that reports the highest value measured above the QL but the suitability of this approach should be very well demonstrated
by method validation.

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 12/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
13

al
ci
Figure 4. Twofold serial dilution of the three samples listed in Table 4. HCP concentration results are plotted against the
product concentration in the samples, which serves as a measure of sample dilution.
ffi
Because measurements near the QL may have higher variability and less reproducibility than measurements farther from the
assay limit, usually (if possible) values within the sample linear HCP concentration response range are averaged. The procedure
for which data points may be averaged must be specified in advance of running the assay and is typically documented as part
of the test procedure. In the example shown in Table 4, two different guidelines are illustrated. Guide 1 averages all values
within 20%–25% of the maximum HCP ratio value. Guide 2 averages values only if the CV for the resulting averaged values is
<20%–25%. Although their data sets are not identical, both rules are justified, based on the argument that variations of 20%–
O

25% are within the variation seen in assay replicates and reflect the underlying reproducibility of immunoassay methods. In
this example, both methods give very similar results. In contrast, the third potential guide, which reports the highest value
measured above the QL, would yield results that are at least 10% higher. In addition, the validity of the third guide would
depend highly on the quality of the method development.
Homogeneous (one-step incubations without washes between reagent additions) sandwich immunoassays are more prone
to antigen excess and resulting dilution nonlinearity problems and may exhibit “high-dose hook effects” in which samples with
large amounts of antigen give inaccurately low readings, because the antigen excess prevents formation of the complete
sandwich. Suitable use of this format should be confirmed experimentally for each sample type and could be acceptable if the
samples are diluted to assay QL and lack of hook effects is confirmed.
Historically, some companies have used spike-recovery data produced in the validation of accuracy to demonstrate linearity.
Although this demonstrates the linear dilution of the standard, it does not demonstrate the linear dilution of the HCPs in the
samples. For the reasons of antigen excess discussed above, the linear dilution of the standard is a necessary condition but not
sufficient to demonstrate linearity of the assay for different sample types.
Finally, although antibody insufficiency is generally believed to be the primary cause of a lack of dilution linearity, other
potential causes are: 1) reactivity of the antibodies with product protein, 2) the presence of non-specific antibodies (“sticky”
antibodies), 3) reactivity of the antibodies to non-HCP proteins present in samples (such as peptones or BSA), 4) HCP
aggregation, 5) HCP standard dilution profile that is not representative of the in-process or final product HCP, and 6) sample
matrix interference. Cross-reactivity and matrix interference are discussed below.

4.4 Specificity
Assessment of potential matrix interference in samples is addressed in section 4.1 Accuracy and is primarily used to establish
the lack of formulation interference (in the DS) and other impurities that are normally present (for upstream process samples).
Another specificity issue to evaluate is the potential cross-reactivity of the anti-HCP antibodies with the product itself. Although
this is possible, antibodies to HCPs that cross-react with product epitopes are rarely observed.
In those cases where cross-reactivity is observed, more false-positive assay signals are risks that must be managed. If
cross-reactivity is suspected, it should be carefully and scientifically established. In the vast majority of cases, signals in HCP
immunoassays are real, indicating the presence of HCP impurities. In particular, instances where the HCP(s) are noncovalently
associated with product, and they co-purify, may give the appearance of cross-reactivity. However, because of the tremendous

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 13/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
14

variety of antibodies in a polyclonal anti-HCP pool, when measuring residual HCP in the presence of excessive levels of product,
some antibodies may still bind product, yielding a false-positive signal. In this case, it may be necessary to modify the assay or
the anti-HCP reagent. If a platform HCP assay is used to test multiple batches (for the same program), and different levels of
HCP between batches are observed, this is strong evidence that the HCP-positive signal is real and not due to cross-reactivity.
Evidence for cross-reactivity is often seen in a product that has been purified in a sequence of steps and where the HCP levels
are not reduced by typical purification processes. The HCP/product ratio (ng/mg) should be used so that changes in the HCP/
product ratio during purification can be compared. First, Western blots of DS are usually probed with anti-HCP antibodies to
determine if HCP(s) are noncovalently associated with product. If bands are detected that are unrelated to the product, this
suggests that cross-reactivity is not occurring, and further process development may be required if lower HCP levels are sought.
Alternatively, if the anti-HCP is recognizing a product-associated band, this suggests cross-reactivity. Caution should be
exercised, however, because the product is denatured (by SDS-PAGE), and binding may be to epitopes not accessible when in
the native, solution phase (i.e., in the immunoassay). In this case, a positive Western blot could be an artifact (conversely, a
negative Western blot could also miss cross-reactivity, but this is less probable). Regardless, if cross-reactivity is suspected, it
can be confirmed and corrected by either: 1) addition of a high-purity product (or related structures, such as human IgG from a
different source, for a monoclonal antibody) to block the cross-reacting antibodies in the assay, or 2) depletion of the anti-HCP
in the reagents using a product-affinity column made from highly purified product. In either case, it is important to ensure that
the antigen used to block or deplete the serum is itself free of residual HCPs. This information is available from the Western
blots probed with anti-HCP. If HCP bands are present, this indicates that a higher-purity product may be needed and can be
prepared with orthogonal, lab-scale purifications. Sometimes reversed-phase HPLC can be effective in making this material,
although this is not useful during product production because of its denaturing properties.

5. SUPPORTING TECHNOLOGIES FOR RESIDUAL HCP DETECTION, IDENTIFICATION, AND

al
MEASUREMENT

Biopharmaceutical manufacturers need an integrated control system that offers assurance of overall product purity and
manufacturing consistency. Typically, product purity is demonstrated by using a combination of methods, and this is very much
ci
the case for HCPs. In addition to the most common sandwich immunoassay format, other assay types provide valuable
complementary information. Tables 5, 6, and 7 provide overviews for commonly used methods for HCP detection, quantitation,
and characterization. Table 5 discusses electrophoretic methods; Table 6 discusses Western blot methods; and Table 7 discusses
chromatographic and proteomic methods. Gel electrophoresis and immunoblots are also discussed in 3.2.2.2 Characterization
of antibodies to HCPs.
Orthogonal assay methods used to provide additional assurance of purity and the lack of HCPs may be used in any of three
ffi
modes: “detection”, “identification”, or “quantitation”. In the case of detection, orthogonal assays address the question, “Is
there any protein other than a form of the product that the immunoassay may have missed?” If this is the case, as knowledge
accumulates regarding the identity of the HCP(s), it becomes possible to perform a specific protein risk assessment and, if
necessary, generate a standard and separate test for that particular HCP impurity. The orthogonal method may then be used
in a “quantitative” mode by comparing the product sample with the appropriate HCP standard for the known HCP. For
example, a quantitative HPLC-MS/MS method may be developed, based on monitoring peptides that ionize well from digests
O

of the analytical standard protein and the sample.

5.1 Considerations for Electrophoretic Methods

Three electrophoretic methods that are useful in impurity analysis are 1-D and 2-D SDS-PAGE and SDS-capillary
electrophoresis using non-gel sieving (CE-SDS). In all these methods, excess sample is loaded to ensure detection of trace protein
impurities. The drawback is that excess product obscures impurities that migrate close to the product and cause interference.
However, 1-D gels or CE-SDS may already be part of the GMP control system for monitoring product-related fragments;
therefore, extending these methods for HCP monitoring does not involve additional testing. 2-D gels have been used to
investigate the purity of the final product and to identify minor spots in the gels associated with either product variants or HCP
impurities. The presence of a new band in the gel, or a new peak in the CE-SDS electropherogram, may represent either a new
product-related substance/impurity or may reflect the presence of an HCP impurity (“process related”). In either case, an
investigation is warranted. If not part of the routine control system, a 1-D gel is still an excellent method for extended
characterization of important lots, such as registration lots, and for manufacturing investigations. Gels and CE-SDS are useful
for confirming the absence of gross levels of HCPs (should they be present and missed in the immunoassay). Gels are typically
stained with a high sensitivity stain, such as a fluorophore or silver. Ideally, the stain should be compatible with in-gel proteolytic
digestion and subsequent analysis by mass spectrometry to identify the protein.
Proteins differ in their staining intensity with silver or fluorescent dyes; therefore, only semiquantitative estimates of the
amount of protein in a band or spot are possible (e.g., glycosylated forms may not stain as intensely). Nonetheless, because
these methods do not rely on anti-HCP antibodies, they provide an important orthogonal measure of purity.
Some proteins may appear in gels as multiple bands or spots that result from post-translational modifications or proteolytic
clipping. To the extent that a single gene product is distributed to multiple locations in the gel, this further reduces the sensitivity
of gels to detect impurities.
As discussed following Table 7 on proteomic methods, it is often possible to excise bands or spots from the gels. These excised
gel fragments may then be incubated with proteases to provide in situ digestion of the protein into peptides. These peptides
may then be fractionated and sequenced using LC-MS/MS and compared with databases to identify the origin of the peptides
as either product-related or HCPs.

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 14/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
15

Table 5. Analytical and Characterization Electrophoretic Methods for HCP Testing

Approximate
Sensitivity for HCPs and Abil-
Method Use Advantages Disadvantages ity to Quantify
1-D gel, either reduced or Screening a large number of High resolution separation; Large excess of product pro- 100 ng/mg.
nonreduced, stained with sil- samples for consistency and provides approximate molec- tein may obscure HCP bands. Semiquantitative unless used
ver or high-sensitivity fluores- presence of nonproduct pro- ular weight (MW) informa- Different proteins show dif- with an analytical standard
cent dye. tein bands. May be used as tion; relatively simple to run. ferences in staining intensities for a known HCP.
part of GMP control system. Side-by-side analyses of sam- so only semi-quantitative.
ples provide powerful com- Limited capacity of gel lanes
parisons for manufacturing for total protein load.
consistency.

Large-format 2-D gel (e.g., 20 Screening a small number of High-resolution separation of Large excess of product pro- 100 ng/mg of total pro-
× 25 cm) stained with silver samples for consistency and trace HCP impurities from tein may obscure HCP spots. tein load).
or a high-sensitivity fluores- presence of unknown protein product. Provides approxi- Different proteins show dif- Semiquantitative unless used
cent dye. spots. Not suitable for routine mate MW and pI information ferences in staining intensities with an analytical standard
use in lot release but may be on protein spots. so only semiquantitative. for a known HCP.
used for characterization of Highly technique-dependent
specific lots or HCP stand- results requiring skilled staff
ards. and sophisticated equip-
ment.

CE-SDS with laser-induced flu- Screening a large number of Rapid, high-resolution meth- Sensitivity may not be as high 1000 ng/mg (0.1% of total
orescence (LIF) detection. samples for consistency and od to separate fluorescently as a 1-D gel with a highly sen- protein load).
Fluorescently labeled samples presence of unknown protein labeled proteins. sitive stain. Fluorescent labels Semiquantitative unless used

al
are separated as either re- peaks. May be used as part of are not specific for HCP pro- with an analytical standard
duced or nonreduced sam- GMP control system. teins, so there is no distinc- for a known HCP.
ples. tion between HCP and prod-
uct-related peptides. Difficult
to extend to other measures,
ci such as Western blots, or to
identify protein peaks.

5.2 Considerations for Western Blot Methods

The Western blot technique is complementary to the sandwich immunoassay for demonstrating product purity and for
ffi
characterizing the immunochemical reagents used in the immunoassay (see 3.2.2.2 Characterization of antibodies to HCPs).
When the anti-HCP antibody is used as a probe, it may be useful to: 1) detect HCPs that are noncovalently associated with the
product and (potentially) missed in immunoassay, or 2) monitor an individual HCP. In other cases, a protein that reacts well in
the immunoassay may not be detected in Western blot because a conformational epitope was lost following denaturation.
Immunoblots require only one antibody to a given protein, whereas sandwich immunoassays require two. Previous work has
shown that high-affinity antibodies that react with a given HCP can be more sensitive than silver or fluorescent dye staining
O

of gels.
1-D Western blots of final product provide a simple reliable orthogonal method for demonstrating product purity. Although
the same antibody preparation may be used in both the immunoassay and Western blot, for the reasons noted above, the blot
may provide additional information on trace proteins (and miss others), hence its orthogonal nature. Table 6 presents the
advantages and disadvantages of 1-D and 2-D Western blot methods for these purposes.
Below is a list of the major considerations for optimization of SDS-PAGE/Western methods:
• 2-D-gel format and size: The larger formats (18-cm length or longer) resolve proteins better and come in a variety of
isoelectric focusing (IEF) application modes and molecular weight dimensions (e.g., gradients). Total protein staining of
various formats can be studied to optimize the separation in both dimensions and thereby find the balance that separates
most (or the maximum number) of HCPs.
• The load on 2-D gels should be optimized to balance sensitivity needed (higher load) versus resolution desired (lower
load). The protein load, ionic content of the sample, and speed of separation need to be balanced to avoid over-heating
and to optimize resolution. The stains or substrates to be used will influence this choice. Typically, more is loaded for the
immunoblot (e.g., 200 µg) than for the total protein-stained gel or blot (e.g., 50 µg for silver stains). The difference in
load may confound the separation to some extent; therefore, finding the right balance will require experimentation.
• Signal development for both the total protein (gel or blot) and the Western blot requires significant experimentation (e.g.,
dye types, exposure times, buffer conditions) to optimize. For example, because it is important to detect as many bands
as possible, sometimes the signal is amplified so much that the most abundant species obscure the others, or the
background increases and contrast is lost. A related issue is standardization and calibration of the densitometer or imager,
which must be fine-tuned to achieve consistency under experimentally determined conditions. Both reagent and
instrument optimization can require significant time to reproducibly establish the optimized conditions where signal is
maximized and background is minimized (see also á1104ñ).
• The transfer and blocking reagents require experimentation to identify the best conditions (such as buffers, instrument
power/time, temperature, and blockers).

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 15/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
16

Table 6. Analytical and Characterization Western Blot Methods for HCP Testing
Approximate
Sensitivity and Ability to
Method Use Advantages Disadvantages Quantify HCPs
1-D gel, Western blot Screening a large number of High-resolution separation; Requires high-quality an- Not quantitative for HCPs un-
samples for consistency and provides approximate MW ti-HCP antibody source. less a specific protein stand-
presence of unknown protein information; relatively simple Large excess of product in the ard is used. Sensitivity de-
bands that react with an- to run. Side-by-side analyses gel can lead to nonspecific pends entirely on the quality
ti-HCP antibodies. May be of samples provide powerful staining of product bands. of the antibody.
used as part of GMP control comparisons of lots. SDS-induced denaturation of
system. proteins leads to loss of con-
formational epitopes.

Large-format 2-D gel (e.g., 20 Screening a small number of High-resolution separation of Requires high-quality an- Not quantitative for HCPs un-
× 25 cm) Western Blot with samples for consistency and trace HCP impurities from ti-HCP antibody source. less a specific protein stand-
chemiluminescence or similar presence of unknown protein product. Provides approxi- Large excess of product pro- ard is used.
high-sensitivity detection. spots reacting with HCP anti- mate MW and pI information tein may obscure HCP spots. Sensitivity depends entirely
bodies. Not suitable for rou- on protein spots. SDS-denaturation of proteins on the quality of the anti-
tine use but may be used for leads to loss of conformation- body, but 100 ng/mg is pos-
characterization of specific al epitopes. Produces highly sible.
lots of product or characteri- technique-dependent results
zation of antibody reagents. requiring skilled staff and so-
phisticated equipment.

5.3 Considerations for Chromatographic and Proteomic Methods

al
Mass spectrometric techniques for the detection, identification, and quantitation of individual HCPs are rapidly evolving and
leverage much of the technology developed for proteomics. Furthermore, as new genomic databases become available (e.g.,
the CHO genome), mass spectrometric approaches are more useful. These methods (see also Applications of Mass Spectrometry
á1736ñ for additional information) often combine sample preparation such as reduction, alkylation, and proteolytic digestion,
ci
followed by separation (e.g., with reversed-phase chromatography RP-HPLC) before introduction into a mass spectrometer that
fragments all proteins thus providing an amino acid sequence for each peptide. The resulting sequence information is compared
to the product sequence to identify product-related fragments and to a database related to the host (e.g., E. coli, CHO) to
identify HCPs.
A challenge for MS analysis stems from the overwhelming number of product-derived peptides relative to impurity peptides.
ffi
One approach to address this issue of competitive ionization of the peptides (also called ion suppression of the HCP peptides
by the product peptides), is to apply LC-MS/MS analysis on partially resolved HCP preparations (e.g., HPLC fractions). This
purification reduces the product contribution to total ions in the mass spectrometer. Other approaches are in development.
Recent technological improvements such as 1) chromatography resins able to resolve effectively using MS-friendly mobile
phases, 2) improved interfaces to front end LC and/or IEF separation systems, and 3) mass spectrometers with higher mass
resolution, accuracy, and faster scan rates, now make it possible to identify and quantify specific HCPs in DS with a high degree
O

of confidence. Major challenges in terms of sensitivity and quantitation of sufficiently large sets of heterogeneous HCPs, cost,
and QC-related issues remain to be met before this technology can replace immunoassays for the control of HCPs in DS.
As a characterization method orthogonal to the HCP immunoassay, LC-MS/MS data may be used in two ways. First, if the
MS-based method does not find HCPs in samples that were also below QL in the immunoassay, then these orthogonal
techniques can increase confidence that the HCP ELISA did not miss an HCP that has co-purified. The detection limit for many
LC-MS/MS methods is currently in the range of about 10 to 100 ng of HCP per mg of product. It is therefore sufficiently sensitive
to rule out a single HCP being present at a high level and is more sensitive than gels. Second, if an impurity is identified at a
high level or one that is a safety concern, then one can assess whether the purification process needs to be improved to remove
it, whether the current control system is adequate, and whether development of a specific assay is required. Absence of signal
does not prove absence of HCPs, but once an HCP is identified, the sensitivity of LC-MS/MS can be enhanced. LC-MS/MS is
complementary to other HCP technologies and is increasingly valuable as an orthogonal method. Detailed discussion of all the
proteomic approaches to the analysis of HCPs is beyond the scope of this chapter, in part because the field is evolving so rapidly
that any review would become out of date quickly.
Intact HCPs also may be separated using HPLC with UV detection. The profile has been used for comparing different lots of
null cell runs, for example. Readers are referred to Chromatography á621ñ for the core science behind HPLC chromatographic
methods. Because HCPs differ in abundance, and the chromatography may not provide unique resolution of all proteins, an
individual HCP must constitute about 1% (or less with good resolution) of the total protein population for possible detection
(even then, a given HCP may be obscured by product, depending on its retention time). Nonetheless, investigation of unknown
peaks as a general practice and comparisons of the chromatographic profiles of null cell run HCPs may be useful in determining
the similarity of HCPs produced under different culture conditions.
As with the electrophoretic methods discussed in this chapter, reversed-phase separation of proteolytically cleaved product
samples with UV detection may already be included in a GMP control system as part of product testing. The presence of new
or unexpected peaks in the UV chromatogram may indicate the presence of an HCP impurity, although this method is not
overly sensitive (~1% impurity if not obscured by other peaks). However, because no new testing or sample preparation is
required, this opportunity to use an orthogonal method for determining purity should be considered.

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 16/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
17

Table 7. Analytical and Characterization Chromatographic and Proteomic Methods for HCP Testing
Approximate
Sensitivity and Ability to
Method Use Advantages Disadvantages Quantify
Mass Spectrometry Identification of individual Allows identification and mon- Low-throughput method re- 100 ng/mg and semi-quanti-
– HCPs. May be applied to the itoring of individual HCPs. quiring significant sample tative if used in the discovery
HPLC/electrospray ionization characterization of a purified May be combined with gels preparation, skilled staff, and mode for unknown HCPs.
(ESI)-MS/MS or cIEF/ product lot, or null cell run to identify the individual pro- sophisticated instrumenta- 10 ng/mg (or better may be
ESI-MS/MS or MALDI-TOF of HCP standards. teins seen in gels. tion. possible) and quantitative if
protein digests used with an analytical stand-
ard for a known HCP.

RP-HPLC/UV or RP-UHPLC/UV. Suitable for comparing null High resolution, separates Difficult to find a stationary 10,000 ng/mg (1%).
May be applied to intact pro- cell run standards if proteins HCPs based on hydrophobic- phase and mobile phase Semi-quantitative unless used
teins or proteolytic digests are well separated. Larger ity and size; direct compari- combination able to resolve with an analytical standard
proteins may need limited son of null strain and product and elute all HCPs. Co-elution for a known HCP.
proteolysis or reduction to strain is possible via overlays. may hinder quantitation of
improve resolution. specific proteins. High con-
centration of product peak(s)
may interfere with detection
of trace HCPs.

5.4 Concluding Remarks on Supporting Technologies for HCPs

al
A number of various hybrid analytical techniques have been attempted over the years. One example is fractionation of HCP
standards by ion exchange, followed by RP chromatography, and then an immunoassay or MS analysis (2-D MS) on the proteins
in the final fractions. Alternatively, “product subtraction” has been evaluated to remove the bulk of the total protein from final
product samples before gel electrophoresis of LC-MS/MS analysis. These hybrid methods for coverage determination are very
labor intensive, and they may introduce artifacts or biases of their own (e.g., antibodies to product do not typically remove all
ci
the myriad of heterogeneous forms). As such, they have not found widespread application, although in individual cases they
may provide insight into the nature of product impurities.
Electrophoresis, Western blotting, and immunoassay remain the most widely used methods for HCP reagent characterization
and for demonstration of HCP clearance by purification processes, because they are robust, sensitive, and can cover a broad
range of protein impurities. Immunoassay and (increasingly) mass spectrometry are highly complementary and the most
powerful methods for monitoring residual HCP levels in samples and confirming their absence in final DSs.
ffi
6. USE OF HCP IMMUNOASSAYS FOR PROCESS DEVELOPMENT, CHARACTERIZATION, AND
VALIDATION
O

As described previously, although HCP testing has limitations as quantitative assays, if the immunoassay can rapidly and
robustly detect most of the HCPs, then it is extremely valuable during process development, characterization, and validation.
HCP clearance at each intermediate process step under a variety of conditions is valuable data. Application to process validation
requires that the assay be demonstrated to be scientifically sound, suitable for its intended purpose, and appropriately
documented. As stated earlier, process pools can differ widely in terms of buffer components, product concentration, and HCP
composition. At a minimum, acceptable spike recovery of the HCP standard into in-process pool samples is necessary to qualify
test dilutions of the various pools in the assay. In addition, testing each sample type at multiple dilutions is helpful during
development to verify that: 1) results are within the range of the assay, and 2) nonlinearity in sample dilution is considered. In
cases where the process is shown to clear HCPs robustly, process validation may also be used to justify not having a routine
HCP test as part of the cGMP control system.
Figure 5 illustrates data gathered during process development and validation and shows the dilution dependence for a
number of in-process pools (Pool 1 is after the first CCF purification step, Pool 2 the next, and so on) through the process. Figure
5 shows HCP ratio (in ng/mg) on the y-axis plotted against the product dilution in the test well on the x-axis. Each sample is
serially diluted through three or four twofold dilutions. The starting dilution is determined by both the need to dilute samples
with high levels of HCP into the range of the assay and the need to test only at dilutions where acceptable spike recovery has
already been demonstrated. In Figure 5, the CCF has slightly more than 1 million ng of HCP per mg of product, meaning that a
little less than one-half of the total protein in the CCF is product. The first column pool reduces the HCP level to just over
10,000 ng/mg. The second column has minimal HCP clearance; the third column reduces the HCP ratio to 1,000 ng/mg. The
final column in the process reduces the HCP level by an additional 2 logs, resulting in a final HCP ratio of 10 ng/mg. All of the
intermediate pools show HCP ratio values that are independent of the dilution of the sample.

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 17/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
18

al
Figure 5. Example of HCP clearance by each purification step. Within a sample set, the first value is the most dilute in its
ci
dilution series.

Figure 6 presents a set of data for a different recovery process. As in Figure 5, the HCP ratio data for a serial twofold dilution
series of each in-process pool sample are plotted. For CCF and the column 1 and column 2 pools, the HCP ratio (ng/mg) is
independent of sample dilution (dilution linearity is achieved as in Figure 5). But after processing over the third column, distinct
ffi
dilution dependence is apparent in the assay. This nonlinear dilution is likely associated with antigen excess relative to available
antibody for the particular set of HCP(s) in the sample (i.e., a single or limited number of HCP species co-purify with the product).
With product purification, more product must be put in the assay well for residual HCPs to be detectable in immunoassay. The
product concentration in the assay may be increased with each purification step, and thus there is a similar increase in the
concentration of the co-purifying HCP impurity. By column 3, the amount of the impurity likely exceeds the capacity of the
available antibodies. This phenomenon is repeated in the final pool, which involves no HCP clearance but only buffer exchange
O

by UF/DF. The reported value for these pools is the one obtained by diluting the sample to near the QL of the assay (the most
dilute sample in the series) using a method described in Table 4, which is about 300 ng/mg in this case. Additional discussion
of assay nonlinearity is found in the 4.3 Sample Linearity subsection of 4. HCP Immunoassay Method Validation.

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 18/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
19

al
ci
ffi
Figure 6. Example of HCP clearance with a purification process. Within a sample set, the first value is the most dilute in its
dilution series.
O

6.1 Assays for Individual HCPs

In some cases, a manufacturing process might result in production of relatively high levels of a single HCP that can result
in a bias in the reported results. As discussed above, in this situation the concentration of antibodies present in the total HCP
assay may not be sufficient to capture all of that particular HCP present. In such cases, where a single, known HCP is present,
another assay for that single HCP may be needed. For example, in the case of E. coli production cultures, co-expressing
chaperones or other processing enzymes that aid in folding or disulfide formation can be produced in very high levels. In
addition, these chaperones may be present in the immunogen used to develop the HCP assay reagents, in a conformation that
is different from the way the protein is presented in samples. Thus, antibodies produced to chaperones not bound to product
may not recognize, or may poorly recognize, product-bound HCPs. In this case, separate assays using appropriate antibodies
may need to be developed. The existence of high levels of a single HCP is not unique to bacterial cell cultures and is possible
with any cellular expression system. Another example is when a protein is intentionally added as part of the processing, then
it is treated as a single HCP, and a specific assay will provide better data (than the total HCP immunoassay).

6.2 Control Strategy

6.2.1 TARGETS, ALERT LIMITS, AND REJECT LIMITS
In both United States and European Union regulations, and generally in other markets, HCPs are described as process-related
impurities, and guidances state that their presence should be minimized by the use of appropriate, well-controlled
manufacturing processes. U.S. 21 CFR 610.13 requires biological products to be “...free of extraneous material except that
which is unavoidable...”. ICH Q6B indicates that biologic product manufacturers should assess impurities that may be present
and develop acceptance criteria for the impurities based on their preclinical and clinical experience. However, regulatory
guidance also recognizes the inherent challenges involved in using assay-specific data and product- and process-specific
reagents. Guidance states “The absolute purity of biotechnological and biological products is difficult to determine and the
results are method-dependent” (ICH Q6B), and, “In the same manner, standardization of the analytical methods would be
problematic since the reagents used are product and production system-related” [European Agency for the Evaluation of
Medicinal Products (EMEA) 1997].

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 19/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
20

Although FDA guidance describes the need to test for HCPs, it also allows, through validation, removal of the release testing
requirement, once clearance has been shown to be consistent. An expectation exists that, whenever possible, impurities that
were introduced by manufacturing processes and remain in products should be below detectable levels based on a highly
sensitive analytical method. However, an appropriately conducted clearance study performed as part of process validation can
be an acceptable substitute for lot-to-lot (C of A) testing (FDA 1997), even when low levels of HCPs are present in the DS. In
addition, “If impurities are qualitatively and quantitatively (i.e., relative amounts and/or concentrations) the same as in the DS,
testing of the DP is not necessary” (ICH Q6B). This is the case for HCPs, the concentration of which in the DP may be inferred
from the DS, because the manufacturing process does not introduce, or clear, HCPs when the DS is converted to the DP. Thus,
in these instances, HCP testing of the DP is generally not required.
The overarching goal is to develop processes that minimize the quantities of residual HCPs in DSs. When using HCP
immunoassays as part of a manufacturing control system, limits for acceptable results need to be established. The “target” for
HCP in final product gives the purification development group an objective for the process, which may or may not be achievable.
Antibody processes with highly selective affinity chromatography steps (e.g., using Protein A) are sometimes able to achieve a
sensitivity of 1 ng of HCP per mg of protein. Target HCP levels determine the design criteria that are then used to develop the
process.
Preclinical toxicology lots are the first lots for which official HCP values should be determined, because at this point in product
development, the investigator is building the data set that will be used to support the safety of subsequent clinical studies. As
the molecule advances through clinical development, the preceding studies are used to support the projected safety (and risk)
associated with subsequent studies. Ideally, changes in HCP level and thus clinical exposure should decrease as the product
moves through clinical development.
As part of an overall control strategy, the “reject limits” are those levels which, if exceeded, render the product no longer fit
for use. During product development, knowledge about the process and the product increases; therefore, it is expected that
reject limits can be tightened as one moves from toxicology material to phase I/II material, to phase III, and finally to commercial

al
material. Each drug candidate profile and its clinical program are unique; hence, no single numerical limit is applicable to all.
Reject limits are primarily concerned with patient safety. Typically, one does not have specific data documenting “unsafe”
levels of HCPs. Rather, acceptable levels for early stage products should be determined through a risk assessment (including
non-clinical data, available literature, previous experience with products manufactured using the same or a similar cell line,
etc.). For commercial products, the acceptable levels are also based on the experience gathered during clinical trials. Because
ci
the HCP impurity profiles may be influenced by the purification process, the registration batches used in the pivotal clinical
trials must be representative of the to-be-marketed process. Reject limits for commercial product should be set to exclude HCP
values that are outside the range of clinical experience upon which the risk/benefit decision was made for the biopharmaceutical.
Alert limits, trend limits, or action limits (different companies use different terms) reflect considerations of manufacturing
consistency. In a commercial process, there should be sufficient manufacturing history to be able to define out-of-trend results.
ffi
However, early in product development, where there is little or no manufacturing history, action limits may be used to set upper
limits on the expected HCPs, based on experience with similar products for which there is a history, or in comparison to the
target value. Exceeding these limits should trigger an investigation to determine whether the lot is releasable. Orthogonal
measures of product purity are valuable in such investigations.
With the emergence of orthogonal measures of purity, any non-product related atypical signals should be evaluated. Efforts
should be made to revise the purification process to remove any unwanted HCPs present at higher than desirable levels (based
O

on, for example, clinical and non-clinical data, available literature, etc.), or a specific justification of its acceptability must be
provided. This is accomplished as part of a risk analysis that considers the clinical exposure similar to that described above for
immunoassays.
In cases where HCP assays are improved or changed due to unforeseen events, the reject limits may need to be modified,
even increased in some cases (if the new assay has broader coverage). However, it is essential that samples be tested in bridging
studies to ensure that no changes in quality would be missed in the new, improved assay. In some cases, improved assays give
higher values, so bridging studies (head-to-head with original versus improved assay) will be needed to justify the new
specification limit.

6.2.2 SAFETY CONSIDERATIONS IN SETTING LIMITS

HCP specification limits in units of ng/mg are typically set using process capabilities and also on the basis of clinical experience
(i.e., the levels used in trials where the clinical outcomes are known). Although there are several theoretical safety concerns
about HCP impurities, the immunogenicity of the impurity is a primary consideration. An immune response may include
formation of antibodies directed against the HCP or directed against the therapeutic protein, where the HCP is believed to have
acted as an adjuvant. Readers are referred to á1106ñ for a more in-depth discussion of factors influencing immunogenicity.
When patients receive more than one biotherapeutic protein with their associated impurities, immunogenicity is an even greater
concern. Although high-dose products may contain a higher mass of residual HCPs per dose and this should be considered
during risk assessments, the potential effect is not necessarily predictable of a clinical outcome. In some cases, very low levels
of certain HCPs have been shown to have clinical effects, whereas high levels of other HCPs have had none.
Different limits may be set, depending on whether the product has only one or a very limited number of co-purifying HCPs
or it has a multiplicity of HCPs, each present as a small fraction of the total HCP level in the product. This could lead to a separate
assay and specification for individual HCP(s) that cannot be cleared from the process and are expected to be present in the DS
and may have bioactivity.
The host cell origin can also be a consideration. It is possible that HCPs from human cell lines (e.g., HEK293 cells) may pose
less immunogenicity risk than HCPs from more distantly related species because of sequence homology. However, if these
proteins are normally intracellular, then they might still be immunogenic and seen as “foreign” by the patient’s immune system.
If such antibodies develop, then they could cross-react with endogenous human proteins and neutralize or render ineffective
one or more necessary biological systems. As a consequence, arguments can be made on both sides; hence, the risks based on

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 20/21
Printed on: Tue Apr 11 2023, 08:56:57 AM(EST) Status: Currently Official on 11-Apr-2023 DocId: GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US
Printed by: Raoxu Wang Official Date: Official as of 01-Dec-2015 Document Type: GENERAL CHAPTER @2023 USPC
Do Not Distribute DOI Ref: 4dw5w DOI: https://doi.org/10.31003/USPNF_M8647_01_01
21

production cell line (microbial versus mammalian) are generally believed to be the same and cannot be practically informative
a priori.
In addition to considering immunogenicity, the HCP may be bioactive and a risk. For example, a hamster homolog of a
therapeutic protein expressed in CHO cells could be sufficiently similar to the product to co-purify. Alternatively, an anti-human
cytokine antibody product expressed in CHO cells could bind to the homologous hamster cytokine, and the complex could
co-purify with the antibody product. In both cases, the homologous hamster proteins could be biologically active when
administered to patients.

7. SUMMARY AND CONCLUSIONS

Biopharmaceutical products should be as free of residual HCPs as reasonably possible. The industry standard for measuring
HCPs is a sandwich immunoassay using polyclonal antibodies directed against the broadest possible spectrum of potential
impurity proteins. Because the diversity and proportion of HCPs in the calibration standard is never matched exactly to the
HCPs in any sample and due to the broad range of antibodies in the immunochemical reagents, different HCP assays will quantify
individual HCPs to a differing extent. Thus, the single numerical value readout of the HCP immunoassay is “immunologically
weighted”. Highly immunogenic proteins will overquantify relative to the average population, and weakly immunogenic
proteins will underquantify. Some proteins may not induce an immune response in the animal species used to generate
antibodies, and the HCP immunoassay will be blind to those proteins. Because of this possibility, orthogonal methods for judging
product purity also should be included in the control system. The best strategy is to use the HCP immunoassay in conjunction
with other analytical tools.
Finally, one should use caution in extrapolating safe HCP values across products. No single specification limit will suffice.
Differences in the biological properties of particular HCPs that co-purify with product, the route of administration, the patient

al
population, and product/HCP dose all contribute to possible factors influencing patient risk. Over the history of recombinant
biologic therapeutics, there have been examples where individual co-purifying proteins have been immunogenic, acted as
adjuvants to increase the patient immune response to the therapeutic protein, or had biological activities themselves. Although
these examples exist, they are rare, and overall there is an excellent safety record for biologics. In spite of the limitations and
complexities of HCP assays, careful and diligent development and application of these assays have helped to ensure patient
ci
safety for millions of doses. HCP analysis continues to be a complex problem demanding time, resources, and careful thought
by biopharmaceutical manufacturers with the goal of ensuring that products consistently meet the standard of having as low
an HCP impurity level as can reasonably be achieved.

8. BIBLIOGRAPHY
ffi
EMEA. 1997. CPMP position statement on DNA and host cell proteins (HCP) impurities, routine testing versus validation
studies. London: European Medicines Agency. CPMP/BWP/382/97.
FDA. 1997. Points to consider in the manufacture and testing of monoclonal antibody products for human use.
www.fda.gov/CBER/gdlns/ ptc_mab.pdf. http://www.fda.gov/downloads/BiologicsBloodVaccines/
O

GuidanceComplianceRegulatoryInformation/OtherRecommendationsforManufacturers/UCM153182.pdf. Accessed
2013-11-01.
ICH. 1999. Guidance for Industry Q6B Specifications: Test procedures and acceptance criteria for biotechnological/
biological products. http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html. Accessed 2013-11-
01.
ICH. 2005. Validation of analytical procedures: Text and methodologies. Q2(R1). http://www.ich.org/fileadmin/
Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf. Accessed 2014-02-26.

https://online.uspnf.com/uspnf/document/1_GUID-636A76B7-028E-4F4F-B1C2-4592B3EAF8D9_1_en-US 21/21