What are HCPs and how are they measured?
I recently met the term Host Cell Proteins (HCPs) in relation to process development and production of biopharmaceuticals. What are HCPs, how dangerous are they to patients, and how can HCP levels be measured throughout the purification steps?
In therapeutic protein production of e.g. vaccines, monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs), a wide range of impurities reside along with the biopharmaceutical product itself. Various purification steps are needed before clinical administration of the product. The impurities include host cell proteins (HCPs) as well as nucleic acids, lipids and DNA [1-3].
After purification, the final product may still contain a (reduced) number of HCPs. Importantly, specific HCPs might affect e.g. protein stability or immunogenicity. Thus, HCPs are classified as the most critical process-related impurities in biologics [4-6].
Unwanted immunogenicity, and decreased protein stability
It is well known that a difference between the foreign molecule and the system, where it is introduced (such as a human), is associated with an increased risk of recognition by the immune system.
Due to the genomic variance between commonly applied protein production hosts, such as E. coli, yeast, the mouse myeloma cell line (NS0), and Chinese Hamster Ovary cells (CHO), most HCPs have the potential to generate an immune response in humans. For example, a study showed that a reduction in HCPs correlated with a decline in the release of specific inflammatory cytokines .
There is also a theoretical possibility that some unwanted HCPs may function in humans, if similar enough to human homologs. Thus, we may see sideeffects of introducing these foreign proteins in humans .
To summarize, HCPs might influence:
- the product quality by proteolysis, particle formation, or enzymatic modification
- the process by failure of a specific purification step
- as well as pose a threat to the patient by immunogenicity or decreasing activity of the biologic’s active ingredient.
How to evaluate the risk of HCPs
To reduce the possible damage caused by HCPs or simply evaluate on the risk, it is necessary to both identify and quantify the HCPs during the manufacturing processes and in the final product. It has previously been shown that HCPs found in purified products are a result of interactions with the product itself. This unwanted co-purification is product- and process-condition specific and can thus be reduced with knowledge about the different HCPs .
The draw-backs of using HCP ELISAs
Enzyme Linked Immunosorbent Assay (ELISA) is often utilized for HCP analysis, because of its quick testing time and easy interpretation [1, 7]. Unfortunately, HCP ELISAs have several drawbacks, including the initial time it takes to develop and harvest antibodies, and the antibodies ability to bind to their antigen (antibody specificity) .
When using ELISA, it is worth having in mind that anti-HCP antibodies can only be produced, if immunizations with the HCPs result in an immunogenic response in the research animals. If the individual HCP is not detected by the animal’s immune system, it cannot produce antibodies against it .
The LC-MS solution
Recently, a more comprehensive Host Cell Protein analysis has become available, based on an innovative LC-MS approach. Where current ELISA-based approaches only present approximate information about HCP amount, this HCP analysis is extensive and detailed as none before .
Use of SWATH mass spectromety results in information about the specific HCPs and their quantity in the biopharmaceutical product. Furthermore, the approach can analyze the effect of various purification processes and optimize them to avoid extensive purification matrices.
In addition, there is also a new, ground-breaking HCP coverage analysis on the market that determines the individual HCPs (and coverage percentage) covered by the anti-HCP antibodies in the HCP ELISA kit. The method is based on a unique combination of immunocapture and extremely sensitive LC-MS/MS. You can apply it to evaluate different HCP ELISA kits, in order to choose the most accurate kit.
Check out this poster from the 2019 edition of the world’s biggest HCP conference, BEBPA, where the new analysis was presented for the first time:
 Wohlrab et al: “Tracking Host Cell Proteins During Biopharmaceutical Manufacturing: Advanced Methodologies to Ensure High Product Quality”, American Pharmaceutical Review, 2018
 Dimitrov, Dimiter: “Therapeutic proteins”, Methods in Molecular Biology, 2012
 Goey et al: “Host cell protein removal from biopharmaceutical preparations: Towards the implementation of quality by design“, Biotechnology Advances, 2018
 Wang et al: “Host Cell Proteins in Biologics Development: Identification, Quantitation and Risk Assessment”, Biotechnology and Bioengineering, 2009
 Bracewell et al: “The Future of Host Cell Protein (HCP) Identification During Process Development and Manufacturing Linked to a Risk-Based Management for Their Control” , Biotechnology and Bioengineering, 2015
 Guiochon et al: “Separation science is the key to succesful biopharmaceuticals”, Journal of Chromatography A, 2011
 Zhu-Shimoni et al: “Host Cell Protein Testing by ELISAs and the Use of Orthogonal Methods”, Biotechnology and Bioengineering, 2014
 Heissel et al: “Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein”, Protein Expression and Purification, 2018