Recombinant proteins, especially monoclonal antibodies open up new therapies for prior barely treatable diseases. Their importance in the pharma market is therefore growing rapidly. Expression hosts used for generation of recombinant proteins for therapeutics include insect cells, yeast, and bacteria. In order to emulate human protein expression conditions, the focus is shifting from classic hosts like E. coli to cells of mammalian origin, especially Chinese hamster ovary (CHO) cell lines.1 Major advantages are similar or identical posttranslational processing (for example, glycosylation patterns) and codon bias to that of humans.2
What are HCPs
Host cell proteins (HCPs) are proteins produced or encoded by the host organisms used to produce recombinant therapeutic proteins. Recombinant therapeutic proteins are usually produced by genetically-modified prokaryotic or eukaryotic host cells using cell culture/fermentation technology.3,4
Biopharmaceuticals require products to be free of process-related impurities to meet clinical application.5 A cellular proteome is the collection of proteins found in a particular cell type under a particular set of environmental conditions. With regard to protein production all proteins present in the expression system or substrate beside the protein of interest itself are regarded as impurities, commonly summarized under the term host cell proteins (HCP).6
Removal of HCP is one of the biggest challenges for the production of biopharmaceuticals. Due to the inherent variability in biological systems, the amount and composition of residual HCP are unique to their respective host and the manufacturing process used for biologics production.
The choice of host has the biggest impact on the number of unwanted proteins. For example, E. coli has ~4,300 genes, whereas CHO cells have ~30,000 genes.3 Although not every host gene will be transcribed and translated to protein, the complexity of host genome and the post-translational modification present in mammalian cells make it almost impossible to understand the complete HCP composition in a given manufacturing process.
HCP in final Drug
HCPs, as the predominant class of process related impurities are undesirable in the purified protein product as they are suspected to have an impact on patient’s safety. HCPs may be biologically active in the human body or in case of HCPs with protease activity impact product stability by degradation either of the therapeutic protein itself or product-stabilizing additives. In extreme cases residual HCPs have the potential to provoke adverse events such as immune response or the formation of anti-drug antibodies.6 Consequently, regulatory guidelines mandate the setting of HCP specifications.
Host Cell Protein Detection and Quantification
Development of HCP detection methods is a substantial step to examine the presence of residual contamination during the bioprocessing and the final biopharmaceutical product. Several approaches have been established over time, mainly differing in sensitivity and total coverage. Industry thoroughly follows the development and combines different methods to evaluate all potential HCPs which contaminate the final product during production.
To detect such small total quantities of HCPs against the background of protein product being present at huge excess, a highly sensitive and specific analytical method with a wide dynamic range is required. Enzyme-linked Immunosorbent Assays (ELISA), are simple to use in routine analysis and can quantify low levels of HCPs.7 Prior development of custom polyclonal antibody reagents with maximum coverage and sensitivity against native HCP extracts is required.
HCP Specific Antibodies
Polyclonal antibodies against native HCP extracts are the most commonly used tool for detecting and assessing HCP, since they can be used for identification, detection, and quantification. Development, evaluation and validation of anti-HCP antibodies is a crucial step in effective residual host cell protein monitoring. In the case of an immunoassay, a polyclonal antibody used in the test is generated by immunization with a preparation of a production cell minus the product-coding gene, fusion partners, or other appropriate cell lines7. It is important to mimic the workflow of the later production process closely; as mentioned above, the manufacturing process impacts HCP composition.
With ongoing research, scientists observed that proteins, based on their properties, differ in separation capability. Hard to separate proteins are summarized under the term “Bad Actor” Proteins. There are various reasons for a protein to be included, for example enzymatic acitivity, high amount of membran parts or omnipresence of the protein itself. Optimizations for media and process help to reduce, or even eliminate nascent critical HCPs and improve separation efficiency.7
Multi-analyte assays, commonly in the form of sandwich enzyme-linked immunosorbent assay (ELISA), capable of detecting the majority of protein impurities are routinely used to quantify and monitor HCPs. They combine sensitivity with high sample throughput – necessary requirement for industrial scaling.
However, generic HCP ELISA kits do not offer complete coverage for all process-specific HCPs and process-specific ELISAs might be not qualified to evaluate the HCP content after process changes.8,9 HCP ELISAs are not expected to deliver 100% coverage, however they are currently still indispensable for high sample throughput process consistency monitoring and they provide a suitable means to serve in a routine quality control laboratory.
Orthogonal HCP Detection Methods
Non-specific methods, orthogonal methods, such as 2D-DIGE and 2D-HPLC combined with MS are more complex; however, they provide a holistic view of the HCP profile and qualitative information of the composition of HCP in the sample. Despite higher costs and time investement, orthogonal methods are irreplaceable as they ensure product purity in the clinical development phase.9
- Florian M. Wurm (2004). “Production of recombinant protein therapeutics in cultivated mammalian cells. in Nature Biotechnology. 22: 1393–1398. PubMed
- Simon Fischer, René Handrick, Kerstin Otte (2015). The art of CHO cell engineering: A comprehensive retrospect and future perspectives in Biotechnology Advances, Volume 33, Issue 8, December 2015, Pages 1878-1896
- F. Wang, D. Richardson, and M. Shameem, (2015) “Host-Cell Protein Measurement and Control” BioPharm International” 28 (6). PubMed
- M. Jin, et al., (2010) . “Profiling of host cell proteins by two-dimensional difference gel electrophoresis (2D-DIGE): Implications for downstream process development.” in Biotechnol. Bioeng. 105 (2), pp. 306-16,
- FDA, (2018). “Establishing Impurity Acceptance Criteria As Part of Specifications for NDAs,
ANDAs, and BLAs Based on Clinical Relevance” in MANUAL OF POLICIES AND PROCEDURES, Link
- Stefanie Wohlrab (2018) “Tracking Host Cell Proteins During Biopharmaceutical Manufacturing: Advanced Methodologies to Ensure High Product Quality” in APR
- Martin Kornecki, (2017) “Host Cell Proteins in Biologics Manufacturing:
The Good, the Bad, and the Ugly” in Antibodies, 6(3), 13, DOI
- Kesh Prakash, (2015). “Analytical Methods for the Measurement of Host Cell Proteins and Other Process-Related Impurities” in ACS Symposium Series, Chapter 9, pp 387-404 DOI
- DG Bracewell (2015). “The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk-based management for their control.” in Biotechnol Bioeng 112(9):1727-37 PubMed