David P. Chimento, Ph.D. Rockland Immunochemcials
Karin A. Abarca, Ph.D. Rockland Immunochemicals

Designing the appropriate methods to achieve successful bioprocessing reagent generation.

Biopharmaceuticals, Bioprocessing and Host Cell Proteins

Biologic medical products also known as biopharmaceuticals or simply as biologics are one of the fastest growing segments of therapeutic medicines. A steady number of biopharmaceuticals has entered clinical practice in recent years and it is expected that in 2015, more than 50% of the new drug approvals will be biopharmaceuticals [Rader et al]. Examples of biopharmaceuticals include recombinant enzymes, hormones, insulin, peptides and therapeutic monoclonal antibodies [Berkowitz et al]. A new variable in the market is the entry of generic biologics, also referred to as biosimilars [Weise et al]. Biopharmaceuticals are manufactured under meticulously controlled processes. However consider that differences in production processes, for example the use of nonidentical cell lines and growth conditions, may result in subtle differences between two products that potentially have a major impact on the efficacy and safety of those products.

Pharmacovigilance Approaches

An important attribute used to benchmark bioprocessing is the abundance of host cell protein (HCPs) impurities. HCPs are process related impurities derived from the manufacturing method, cell culture or downstream processing that may be left behind during the purification from the expression hosts, such as E. coli, insect, or mammalian cells. HCP content is regulated by the FDA and EMA which require biopharmaceutical products or biologics to be free of contaminating host cell proteins (HCPs) prior to approval (http://1.usa.gov/1igSs6X) [Rader]. Several analytical methods for monitoring the HCPs during bioprocessing are available including mass spectroscopy and the use of process-specific polyclonal antibodies. HCP assays almost always rely on the ability of an anti-HCP antiserum to detect HCP impurities, so the performance of the assay is tightly linked to the quality of this reagent. In some cases it is advisable to use more than one method to fulfill regulatory concerns and considerations.

Figure 1. CHO cells

HCP Polyclonal Antibodies in Bioprocessing

Polyclonal antibodies that detect host specific contaminants are a widely used and accepted method for HCP monitoring although their use is becoming more highly regulated by the FDA and EMA [Purcell et al]. The most common antibody based detection system for host cell protein contamination is enzyme-linked immunosorbent assay (ELISA) [Krawitz et al]. There are a number of commercially available generic HCP ELISA kits on the market supplied by various companies against many cell lines including Chinese hamster ovary cells and E. coli; however it is generally not acceptable to use a generic HCP antibody to demonstrate product purity in late stage bioprocessing. A custom process specific HCP antibody is required.

Development of Custom HCP Antibody

To investigate the presence of residual HCP contamination during the bioprocessing and the final biopharmaceutical product, development of custom polyclonal antibody reagents with maximum coverage against native HCP extracts is required. Coverage is usually measured by assessment of the reactivity of the polyclonal antisera against the HCP sample in Western blot. A one-dimensional Western blot is informative but may not reveal the true activity of the antibody to the HCP sample. To overcome this limitation an analytical method that is becoming more widely utilized for assessment in antibody development is comparison of antibody coverage in a two-dimensional (2D) SDS-PAGE and 2D Western blot. The metric for this comparison is percent coverage of the antibody in 2D Western blot as compared to the total protein demonstrated by SDS-PAGE.

HCP Sample Selection and Preparation and Analysis

In order to prepare an anti-HCP antibody a sample of the HCP extract is required. This sample can be taken from many stages of bioprocessing and the sample selection point can be critical for obtaining the correct antibody reagent. The typical sample point is selected early in bioprocessing but downstream from the crude extract where the extract may come from a number of host organisms including E. coli, a number of mammalian host systems (i.e., CHO, HEK293), and yeast. It is worth noting that mammalian HCP target proteins are not as immunogenic as bacterial host proteins and require a more intensive immunization strategy to achieve reasonable coverage in 2D Western blot. In general the HCP immunogen is a complex mixture of a whole cell lysate or partially purified lysate, for example protein A purified material. In certain cases poorly immunogenic antigens can be chemically modified (i.e., alkylation) to increase the immunogenicity of the HCP extract.

Antibody Generation Method

Anti-HCP antibodies can be, and it is thought should be, generated using a variety of immunization protocols. The use of different immunization strategies offers the advantage of overcoming the limitations found in any of the individual methods.

In the classical approach, the HCP immunogen is immunized as a whole complex mixture in adjuvant. This can be successful, particularly if using bacterial host immunogen. One downside to the classical strategy is that often times the larger immunogens (greater than 50 kDa) tend to dominate and the low molecular weight proteins do not generate a robust immune response leading to insufficient HCP antibody reagent. Using an HCP sample that is fractionated and immunizing distinct animal cohorts allows for the generation of antibodies specific for the high molecular weight and the low molecular weight samples.

Another pitfall is that some immunogens are not immunogenic in all hosts. To overcome this limitation, anti-HCP antibodies should be generated in 2 or 3 different hosts, which could include one or more of the following species: rabbits, goat/sheep/donkey, and chickens. The large animals have the added benefit of a large long term supply of HCP antibody reagent.

The third commonly used method recommended at Rockland Immunochemicals is the cascade immunization. This method uses the antibodies generated after HCP immunization to immunodeplete the immunodominant HCP species. The antibodies are first purified, immobilized, and the starting HCP is reacted with the anti-HCP. The unbound HCP fraction is used to boost the immunization to develop a more comprehensive immune response.

HCP Antibody Validation

Proper validation of the HCP antibody includes Western blot and ELISA. Western blot of the HCP antisera against the HCP material is an indicator of developing coverage. Typically the antibody reagent is considered ready when full coverage is observed in Western blot (see Figure 2). More often the antibody reagent is validated with companion 2D Western blot, considered to be a better indicator of absolute antibody coverage. Polyclonal anti-HCP antibodies are purified—either affinity purified, or protein A purified—to create the finished reagent. The purified antibody is lastly conjugated to a reporter molecule such as alkaline phosphatase or peroxidase. The conjugated antibody is paired with the unconjugated HCP antibody to develop working ELISA for screening HCP contamination in the bioprocessing workflow.

In summary anti-HCP antibodies and 2D Western validation are critical for the rapidly growing field therapeutic biologics.

Figure 2. Western blot showing comprehensive detection of HCP extract by process specific HCP antibody developed at Rockland Immunochemicals.

David P. Chimento, Ph.D., is assistant laboratory director at Rockland Immunochemicals, Boyertown, PA. Karin A. Abarca, Ph.D., is manager of research and development at Rockland Immunochemicals.

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Krawitz DC, Forrest W, Moreno T, Kittleson J and Champion KM, Proteomic studies support the use of multiproduct immunoassays to monitor host cell protein impurities, Proteomics 6(1): pp94-100, 2006
Purcell RT, Lockey RF. Immunologic responses to therapeutic biologic agents. J Investig Allergol Clin Immunol 2008;18(5):335-342.
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Weise M, Bielsky MC, De Smet K, Ehmann F, Ekman N, Narayanan G, et al. Biosimilars-why terminology matters. Nat Biotechnol 2011 Aug 5;29(8):690-693.

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