Host Cell Proteins: the Uninvited Guests in your Biologics Preparation

The use of biological systems to synthesize valuable biotherapeutic product has been the backbone of the biotech industry (C.E. Hogwood et al., 2014). Recombinant biotherapeutic proteins are usually produced by genetically-modified prokaryotic or eukaryotic host cells using cell culture and fermentation technology. For this reason, Chinese hamster ovary (CHO) cells and E.coli have become the predominant choice. During the recombinant protein production, host cells co-produce proteins related to the normal cell functions such as cell growth, proliferation, survival, gene transcription, protein synthesis, etc. Other non-essential proteins may also be released to the cell culture/fermentation as a result of cell apoptosis/death/lysis. Apart from the therapeutic protein of interest, all endogenous proteins co-expressed by the host cells are called host-cell proteins (G. Guiochon ., 2011).

Host cell proteins (HCPs) constitute a major part of process-related impurities during biologics production. HCPs are complex mixtures with diverse physiochemical properties and the primary risk associated with HCPs is immunogenicity. So, regulatory guidelines mandate that they are identified and quantified in order to protect patient safety. Some HCPs can act as adjuvants to enhance immune response to a drug product (L.C. Eaton, 1995). In view of this, HCPs can cause loss of efficacy and alter pharmacokinetics and stability if not adequately removed or inactivated (F. Robert et al., 2009). Consequently, they represent a major risk for product failures and recalls. As the patents for the first generation of approved biopharmaceuticals have either expired or are about to expire, giving way to emerging biosimilar therapeutics, more biopharmaceutical companies are interested in HCPs analysis in recent years.

Some of the challenges of HCP detection include low levels of residual HCPs present in large excess of product protein (C.E. Hogwood et al., 2014). The assay must also be able to measure a large number of different protein analytes. The population of HCP species may change during process development. All analytical methods for measuring HCPs face significant challenges due to the wide dynamic range of the protein concentration. Immunoassay, commonly in the form of sandwich enzyme-linked immunosorbent assay (ELISA) remains the industry gold standard for HCP measurement due to its high sensitivity and high throughput capabilities (J. Zhu-Shimoni et al., 2014). Since HCPs are potentially immunogenic, a commonly accepted method to evaluate the presence of HCPs is through an immunoassay. In theory, an HCP mixture injected into an animal, such as a rabbit, goat, or chicken, will elicit an immune response, and the animals will generate anti-HCP antibodies against these foreign proteins. Although the identities of all HCPs are not known, the polyclonal antibodies raised in animals recognize a majority of the proteins contained in the HCP mixture. Using these polyclonal antibodies, a multi-analyte sandwich ELISA can be developed.

Commercially available ELISA kits offer the advantage of eliminating lengthy assay development time. These kits are produced by injecting animal models with an HCP mixture to raise antibodies. The HCP mixture is commonly the null cell line, containing an empty vector. The biotechnology industry employs both commercially available HCP ELISA kits and customized in-house designed assays. This makes the kit useful in early phase development. Upon qualification and validation, HCP ELISA can serve as a QC release assay for drug substance. ELISA can be used in conjunction with other techniques such as 1D- and 2-D polyacrylamide gel electrophoresis to obtain a broader picture of HCP dynamics at various stages of recombinant protein production workflow.

Enzo Life Sciences offers a comprehensive bioprocess portfolio for contamination monitoring, including HCP ELISAs, protein aggregation assay and Protein A ELISA to optimize and monitor product integrity. In addition, Enzo will be attending BioProcess International 2015 in Boston, October 26-29. Stop by booth 220 to learn more about our bioprocess product range.

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