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In this paper, the authors lay out some commonly accepted HCP analytical methods, the challenges of HCP assay, and provide recommendations on what can easily be accomplished in-house and when it may be better to outsource.
Biologics, especially protein drugs, are often produced in cultured cells. Those cultured cells, called host cells, synthesize the target proteins along with the proteins coded by the cell genome for the cells’ own survival, function, and reproduction. Purifying the target protein from the other host cell proteins (HCPs) is the main task for downstream processing of biologics manufacturing.
HCP removal is crucial, as HCPs are often immunogenic and can affect the potency and stability of biologics. Therefore, detection and quantification of residual HCPs are categorized as obligatory critical quality attributes in the biologics manufacturing process, and HCPs are strictly monitored throughout production to ensure safety and potency of the products.
There are thousands of types of HCPs within one cell. For example, Chinese hamster ovary (CHO) cells, which are often used in biologics production, have more than 24,000 coded genes (1), making HCP detection a challenging technique, as both assay breadth (number of HCPs detected) and sensitivity are required. In this paper, the authors lay out some commonly accepted HCP analytical methods, the challenges of HCP assay, especially during late clinical stages (process characterization and validation stages), and provide recommendations on what can easily be accomplished in-house and when it may be better to outsource, due to time, cost, and expertise factors.
The most common assay method for HCP detection is enzyme-linked immunosorbent assay (ELISA) which utilizes pre-raised HCP antibodies to detect HCPs in samples. As it utilizes enzymes to amplify the signal, ELISA is highly sensitive and can detect HCPs at a level of 1-3ng/mL. ELISA is quality control friendly and is used as a product release assay for nearly all biological products.
The difficult part of HCP ELISA is the coverage assessment of HCP antibodies. HCP antibody coverage data are often requested by regulatory agencies to support the use of an HCP ELISA as early as investigational new drug application (IND) stage. Two-dimensional (2-D) gels followed by Western blot analysis or immunoaffinity purification followed by 2-D gel analysis are often the choices for HCP antibody coverage evaluation.
Two-dimensional gels separate proteins by size and charge, and those proteins are then transferred to a membrane that is incubated with HCP antibodies for Western blot detection (Figure 1a and 1b, courtesy of Xiaoqing Zhu, WuXi Biologics). The technical challenges here are the large size of the gel, which holds thousands of proteins, and the need of transferring those thousands of proteins to a membrane under one voltage and time condition, plus the alignment of each protein on the gel with the corresponding protein on the membrane, making it a task more suitable for experienced and well-equipped labs. There are many experienced contract laboratories available to assist in this more complex analysis.
Coverage evaluation using immunoaffinity purification followed by 2-D gel analysis involves generation of HCP antibody conjugated resin and running 2-D gels for total HCPs and affinity-purified HCPs separately (Figure 1c and 1d, courtesy of Muchen Li, WuXi Biologics). Both the antibody conjugation step and the HCP purification step need multiple experiments for optimization, and often consume mg level of HCP antibodies. This analysis could take a long time and become quite costly. Outsourcing to an experienced lab might be a good option.
The total HCPs needed for coverage evaluation are usually produced in bioreactors using null cell lines (cells transfected with gene of interest [GOI] deleted plasmids) under conditions similar to product manufacturing. Because both plasmid transfection for null cell generation and cell growth in a bioreactor are typically available for most biological drug development labs, those activities could be conducted in-house instead of outsourcing.
Several commercially available ELISA-based kits are on the market for HCP detection. These “off-the-shelf” kits are ideal for preclinical process development support, as most protein analytical labs have the requisite equipment and trained personnel to utilize these kits. The cost of using these kits can potentially be more affordable than outsourcing.
However, as biologics development moves towards late stage (i.e. Phase III and beyond), it is a regulatory expectation that more thorough analytical characterization and assay qualification activities be applied to ensure process robustness and product quality. For HCP detection, the commercial HCP ELISA is expected to be replaced by either a platform HCP ELISA (developed from the use of the same CHO host cell line across multiple products) or process specific HCP ELISA (2). A concern from regulators, as well as empiracal observation from industry, is that the commercially available kits do not have breadth of antibody coverage of the HCPs that are produced by the specific production host cell line in use and therefore may miss the ability to detect certain low level HCPs that may co-purify with the product.
To develop a platform- or process-specific HCP assay, three key reagents are needed: HCP standards, anti-HCP antibodies, and enzyme conjugated HCP antibodies. The process (Figure 2) of making the key reagents can take a year or longer and requires animal facilities, specialized equipment, and expertise. To ensure this process goes smoothly, it is recommended that the activities of developing those specific HCP ELISA assays to be outsourced to the more experienced labs.
Some companies achieve better assay performance of the platform- or process-specific HCP ELISAs. The coverage of HCP antibodies prepared for a specific platform or process could be 20 percentage points higher (i.e. from 60% to 80%) than the coverage from a commercial generic HCP ELISA, and better sample dilution linearity could be observed because more specific antibodies are available for the HCPs to bind in the platform or process HCP ELISA than in commercial ELISA (Figure 3, courtesy of Xingli Gao, WuXi Biologics).
HCP ELISA sometimes fails to detect certain HCPs due to limitations of antibody type and amount. Mass spectrometry-based HCP analysis could serve as an orthogonal method to help identify HCPs. To do this, samples containing HCPs are digested into peptides and analyzed by liquid chromatography–mass spectrometry (LC-MS), and the results are compared with the entire host cell proteome to identify the exact HCPs in the samples. Either hundreds of HCPs as in harvested cell culture fluid (HCCF) or a few HCPs from highly purified product samples could be identified (Figure 4, courtesy of Song Klapoetke, Wuxi Biologics). Proper purification operations could then be designed and protein specific analysis could be applied.
Mass spectrometry is a specialized technique. Though it is powerful to identify unknown proteins, the results are just semi-quantitative and the instruments are costly. One can build a proteomic LC-MS lab if needed often, otherwise outsourcing of HCP analysis by LC-MS could be considered.
Even though HCP detection is a routine assay required for nearly all biologics manufacturing, the full scope of HCP-related activities is not routine and requires expertise, time, and sometimes costly instruments to conduct all assay qualification activities. Thus, outsourcing has become a viable option for many drug developers. The decision as to which activity to outsource depends on the internal resources and expertise, with considerations of instrument cost and time needed to complete the activities. It can be recommended to keep the routine activities in-house and outsource non-routine activities to external expert labs, as shown in Table I. This way, one can concentrate the limited resources on the key activities of biologics development and manufacturing and move the whole process forward more efficiently. It should also be evaluated if performing all activities with a single lab or company would be more efficient and cost effective.
1. Xu, X.; Nagarajan, H.; Lewis,
N. et al. The Genomic Sequence of the Chinese Hamster Ovary (CHO)-K1 Cell Line. Nat Biotechnol. 2011 29, 735–741.
2. USP. <1132> Residual Host Cell Protein Measurement in Biopharmaceuticals. US Pharmacopeia–National Formulary. 2016.
Zhiping Yao is an executive director at WuXi Biologics. Wesley Wang is vice-president at WuXi Biologics. Weichang Zhou is president at WuXi Biologics.
Pharmaceutical Technology Europe
Vol. 35, No. 2
When referring to this article, please cite it as Yao, Z.; Wang, W.; and Zhou, W. Current Trends in Host Cell Protein Detection for Biologics Manufacturing. Pharmaceutical Technology Europe 2023, 35 (2), 31-33.