HCPs: The Inevitable Impurity in Biopharma
A lion’s share of biotherapeutics today is a fruit of the recombinant DNA technology. In biopharmaceutical productions, the protein drugs are expressed at high concentrations in carefully selected and engineered host cell systems, such as mammalian cells (CHO), E. coli, yeasts or plant cells.
The products then undergo sophisticated purification steps to remove all cellular components of the host systems. However, a large number of other proteins expressed by the host cells during production can easily contaminate the drug and be left behind during the purification process, making host cell proteins (HCPs) a residual process-related impurity.
Presence of HCP contaminants in the final product, even at very low levels (1-100 ppm), may elicit unpredictable and serious immunogenic response in humans. The accurate detection, identification and quantification of HCPs during the biotherapeutic development is thus critical in ensuring patient safety, and is a regulatory requirement. To be compliant with such stipulations, the application of advanced purification and analytical methods is a must.
Importance and Challenges of HCP Analysis
Presence of HCPs in biological products is considered a Critical Quality Attribute (CQA),1 thus making their clearance from the products a benchmark to demonstrate a robust bioprocess.
This major group of process-derived impurity constitutes complex mixtures with diverse physiochemical and immunological characteristics. HCP in a drug product threatens patient safety and efficacy of the product in three major ways:
a) HCPs are immunogenic even in minimal concentration as they are foreign to the human body.
b) HCPs with proteolytic activity cause fragmentation of the protein product and significantly reduces product yield, stability and efficacy, if not adequately removed or inactivated.
c) Aggregation of HCPs to form soluble or insoluble aggregates of various sizes is a potential problem as the aggregates may cause adverse effects when administered and act as adjuvants that exacerbate the immune response to a drug product.2
According to Professor Annie De Groot of the Institute for Immunology and Informatics of University of Rhode Island, the unique challenges associated with HCP clearance are:
a) The heterogeneity and complexity of HCPs as an impurity, which sets it apart from other single analyte impurities. While there are potentially thousands of proteins that need to be monitored, it is not clear how many of these proteins are expressed and which ones tend to co-purify with a product as they differ in physical properties such as molecular weight, isoelectric point and hydrophobicity, and can undergo different post-translational modifications, such as glycosylation, phosphorylation and truncations.3
b) Co-purification of HCP with products driven by similar biochemical properties or direct interaction of HCP and the product. This “hitchhiking” tendency of HCPs is product and/or process specific. HCPs co-purifying with the protein product in the capture step of protein A Chromatography typically range from 200 ppm to 3,000 ppm, but values as high as 70,000 ppm have been reported.4 Other interactions between HCP and drug product have not been fully characterized yet. Assays must be capable of casting a very wide net sampling hundreds to thousands of analytes simultaneously based on the starting population of HCP that enters the purification stage.
There is no guideline that specifies the exact acceptable limit of HCP in a product. Dr Keshavamurthy Prakash, a methods development expert in a multinational biopharmaceutical company, explains that setting an industry-wide numerical threshold is challenging, if not impossible, with the currently available analytical technologies and given the complex and variable nature of this impurity as well as the assays.
Typically, biotechnology products are expected to contain less than 100ng HCP/mg of product. Health authorities evaluate the acceptable HCP levels on case-by-case basis, and base the decision of factors such as phase of clinical development, patient population, route of administration, dose and mode of action of the product. The use of a risk assessment is critical for setting HCP limits for every product.4
Sections Q6B (Specifications for Biotechnology Products) and Q11 (Development and Manufacture of Drug Substances) of the International Conference on Harmonization guidelines5 address process‐related impurities, specifying that HCP impurities must be monitored and reduced to acceptable levels to ensure product quality and patient safety. Both the US6 and European Pharmacopeia7 have published general chapters outlining the best practices for the development and validation of HCP assays. The FDA8 expects the contaminants introduced by the recovery and purification process to be below detectable levels using a highly sensitive analytical method, wherever possible. The European Medicines Agency (EMA) guideline CPMP/BWP/382/979 summarizes that residual HCP must be tested for and monitored routinely using suitable analytical assays, such that results are consistent and meet specification limits. Regulatory authorities of other nations have their own clauses on HCP control, but it is generally accepted that a sensitive, validated method is required to monitor residual HCPs in accordance with ICH guidelines.
The selection of appropriate methods for identification and quantification of HCP have been a concern during process development and manufacturing. Throughout the biotechnology industry, immunospecific assays such as ELISA, western blotting as well as non-specific methods like 1D and 2D electrophoresis, 2D chromatography and MS have been the workhorses for HCP monitoring. More recently, evaluation techniques like surface plasmon resonance (SPR) has offered a more sensitive, efficient and simplified process of HCP analysis.10
Although the starting population of HCPs is often relatively consistent across products produced by the same platform cell culture process, the composition and quantity of those proteins present at each step of the purification procedure may differ greatly. The value reported by the assays, representing the aggregate HCP content, is highly reagent (e.g., antibody preparation) and method (ELISA versus MS) specific. Moreover, as assay standards vary from lab to lab, the same sample tested in different assays/lab will likely produce different HCP values.
Due to its ease, speed, and sensitivity anti-HCP enzyme-linked immunosorbent assay (ELISA) is considered the gold standard to analyse HCPs.1 The assay is developed with polyclonal antibodies raised against the host cells and provides a relatively high throughput and sensitive quantification. HCP-ELISA may take up to several months to develop and are typically capable of detecting the majority of HCPs, particularly the highly abundant or highly immunogenic proteins. However, ‘the majority’ isn’t sufficient for guaranteeing the safety of the final product, as undetected immunogenic proteins can induce a serious reaction.
Western blot analysis using the polyclonal antibody mixture generated for the HCP‐ELISA can demonstrate an abundance of HCPs in different downstream process pools and may also highlight the differences between samples. One‐dimensional western blots are also very useful to further characterize HCPs in a sample when the HCP-ELISA indicates HCPs are present. Western blots can be used to indicate whether one HCP is present in great abundance or 10 HCPs are present in low abundance.11
These process specific immunoassays, while widely-used, require prior knowledge regarding the nature of HCP contaminants, are time consuming and expensive to develop. As such they cannot be readily adapted to fully evaluate biopharmaceutical products from different cell types and purification schemes.12
Orthogonal detection and monitoring techniques such as LC-MS/MS and 2D-DIGE have become popular to supplement ELISA methodologies. These non-specific methods enable complete detection and characterization HCPs in a biologic preparation, and may give individual protein identity information directly from the acquired data, rather than simply a number for the total sum of HCP, which is a current limitation of ELISA based methods.
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) based methods used to assess HCP impurities are straightforward and fast. SDS‐PAGE gels can be overloaded with 10–50 µg of product and combined with sensitive total protein stain, such as SyproRuby, silver stain, or another comparable technique. This powerful orthogonal method can detect HCPs that are not reactive in the HCP ELISA, though sensitivity is limited to ~100 ng per each HCP impurity/mg of product.1 Nonetheless, this is a relatively simple check of final purification pools to ensure the purification process is performed as expected. Furthermore, 1D SDS‐PAGE is compatible with MS techniques that are very powerful for HCP characterization.
In a 2D-polyacrylamide gel electrophoresis (2D-PAGE) assay, complex protein mixtures are separated according to their physiochemical properties, stained and visualized. However, quantifying HCPs with 2D-PAGE is limited by its narrow dynamic range. Low-abundance HCPs cannot be detected, and the presence of recombinant proteins often masks the visibility, hence, compromise the detectability of these HCPs.12 Moreover, HCPs that possess physical properties like that of mAb could not be easily separated with 2D-gel, even in cases when it was the main impurity in the final drug substance when quantified with mass spectrometry.4
Two-dimensional difference gel electrophoresis (2D DIGE), an extension of 2D-gel electrophoresis can resolve multiple samples on the same gel using spectrally resolvable, size- and charge-matched fluorescent dyes. The capability of sample multiplexing and the inclusion of internal standards provide 2D-DIGE with enhanced accuracy in protein quantification for comparative analysis. However, these methods are only semi-quantitative, has limited dynamic range (two to three orders of magnitude) and requires additional techniques (e.g., mass spectrometry) for HCP identification.3
Mass spectrometry (MS) emerges as the leading proteomic analytical technology to detect and quantify low-abundance HCPs with high confidence in the presence of the recombinant product.13,14,15 Tandem mass spectrometry (MS/MS) is often coupled with liquid chromatography (LC) to rapidly monitor and identify multiple protein analytes in a high throughput manner. Immunogenic and problematic HCPs can also be targeted in highly purified samples once the MS protocol is set up.11 Therefore, many previously overlooked HCPs in commercial biologic products may be characterized as this technology continues to improve.16 Nonetheless, highly skilled personnel are required to operate LC-MS/MS. The equipment is expensive, and absolute quantification of individual HCPs requires the use of synthetic peptides that are cost- and labor-intensive. Subsequent peptide validation work also remains a challenge.17
To develop a complete picture of the HCP impurities throughout the process and to implement a robust HCP removal strategy, the use of multi-dimensional HCP analysis is imperative. MS continues to evolve in terms of new capabilities, more intuitive software, and ease of use, and is expected to play an increasing role in HCP analysis. As the amount of data acquired through these techniques increases, the ability to measure and monitor thousands of proteins grows.
The future of HCP analysis involves development of improved risk management and assessment approaches by exploiting the information to determine critical HCPs and the cumulative knowledge of multiple risk factors.18 A web tool that uses such a database to predict the immunogenicity of HCP from CHO-based productions is currently under development.19 It would be interesting to see how these tools can be utilized for individual therapeutic programs, particularly if the presence of some HCPs in therapeutics, that are characterized by a favorable clinical safety profile, could be investigated.
1. Krawitz, Denise, et al. "Characterization of Residual Host Cell Protein Impurities in Biotherapeutics." Analytical Characterization of Biotherapeutics (2017): 211-237.
2. Vanderlaan, Martin, et al. "Experience with host cell protein impurities in biopharmaceuticals." Biotechnology progress (2018).
3. Jin, Mi, et al. "Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development." Biotechnology and bioengineering 105.2 (2010): 306-316.
4. Wang, Fengqiang, Daisy Richardson, and Mohammed Shameem. "Host-cell protein measurement and control." Biopharm Int 28.6 (2015): 32-38.
5. ICH harmonised tripartite guideline, “Specifications: test procedures and acceptance criteria for biotechnological/biological products-Q6B”, (1999) http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q6B/Step4/Q6B_Guideline.pdf
6. The United States Pharmacopoeia 39, National Formulary 34, “<1132> Residual Host Cell Protein Measurement in Biopharmaceuticals”, (2016) https://www.usp.org/sites/default/files/usp/document/our-work/biologics/USPNF810G-GC-1132-2017-01.pdf
7. European Pharmacopoeia, Issue 27.2, “2.6.34. Host Cell Protein Assays”, (2015) http://www.e-gmp.pl/wp-content/uploads/2015/05/2.6.34.-Host-cell-protein-assays-draft.pdf
8. FDA, “Points to Consider in the Manufacture & Testing of Monoclonal Products for Human Use” (1997), www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatory-Information/OtherRecommendationsfor-Manufacturers/UCM153182.pd
9. The European Agency for the Evaluation of Medicinal Products Human Medicines Evaluation Unit, London, June 10, 1997, CPMP/BWP/382/97, www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003322.pdf
10. Lundström, Christine Sund, et al. "Sensitive methods for evaluation of antibodies for host cell protein analysis and screening of impurities in a vaccine process." Vaccine 32.24 (2014): 2911-2915.
11. Tscheliessnig, Anne Luise, et al. “Host Cell Protein Analysis in Therapeutic Protein Bioprocessing – Methods and Applications.” Freshwater Biology, Wiley/Blackwell (10.1111), 22 Feb. 2013, onlinelibrary.wiley.com/doi/abs/10.1002/biot.201200018.
12. Zhu‐Shimoni, Judith, et al. "Host cell protein testing by ELISAs and the use of orthogonal methods." Biotechnology and bioengineering 111.12 (2014): 2367-2379.
13. Ahluwalia, Deepti, et al. "Identification of a host cell protein impurity in therapeutic protein, P1." Journal of pharmaceutical and biomedical analysis 141 (2017): 32-38.
14. Levy, Nicholas E., et al. "Identification and characterization of host cell protein product‐associated impurities in monoclonal antibody bioprocessing." Biotechnology and bioengineering111.5 (2014): 904-912
15. Zhang, Qingchun, et al. "Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry." MAbs. Vol. 6. No. 3. Taylor & Francis, 2014.
16. Madsen, James A., et al. "Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions, LC-MS/MS, and multivariate analysis." MAbs. Vol. 7. No. 6. Taylor & Francis, 2015.
17. Doneanu, Catalin E., et al. "Enhanced detection of low-abundance host cell protein impurities in high-purity monoclonal antibodies down to 1 ppm using ion mobility mass spectrometry coupled with multidimensional liquid chromatography." Analytical chemistry 87.20 (2015): 10283-10291
18. Bracewell, Daniel G., et al. “The Future of Host Cell Protein (HCP) Identification during Process Development and Manufacturing Linked to a Risk‐Based Management for Their Control.” Freshwater Biology, Wiley/Blackwell (10.1111), 14 July 2015, onlinelibrary.wiley.com/doi/abs/10.1002/bit.25628.
19. Bailey‐Kellogg, Chris, et al. "CHOPPI: A web tool for the analysis of immunogenicity risk from host cell proteins in CHO‐based protein production." Biotechnology and bioengineering 111.11 (2014): 2170-2182.