What are Host Cell Proteins (HCPs)?

Question:

What are Host Cell Proteins, and how are they measured?

I recently met the term Host Cell Proteins (HCPs) in relation to process development and production of biopharmaceuticals. How dangerous are HCPs to patients, and how can HCP levels be measured throughout the purification steps?

Answer:

In therapeutic protein production of, e.g., vaccines, monoclonal antibodies (mAbs), and antibody-drug-conjugates (ADCs), a wide range of impurities resides along with the biopharmaceutical product itself. The contaminants include host cell proteins (HCPs), nucleic acids, lipids, and DNA [1-3]. Various purification steps are needed before the clinical administration of the product.

After purification, the final product may still contain a (reduced) number of HCPs. Notably, 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 [4].

There is also a theoretical possibility that some unwanted HCPs may function in humans if similar enough to human homologs. Thus, we may see side effects of introducing these foreign proteins in humans [4].

To summarize, HCPs might influence:

• the product quality by proteolysis, particle formation, or enzymatic modification
• the process by the 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 Host Cell Proteins

To reduce the possible damage caused by HCPs or evaluate the risk, it is necessary to 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 result from 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 [5].

The drawbacks of using ELISA

Enzyme-Linked Immunosorbent Assay (ELISA) is often utilized for HCP analysis because of its quick testing time and straightforward interpretation [1, 7]. Unfortunately, HCP ELISAs have several drawbacks, including the initial time it takes to develop and harvest antibodies and their ability to bind to their antigen (antibody specificity) [7].

When using ELISA, it is worth considering that anti-HCP antibodies can only be produced if immunizations with the HCPs result in an immunogenic response in the research animals. If the animal’s immune system does not detect the individual HCP, it cannot produce antibodies against it [7].

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 methods only present approximate information about HCP amount, this HCP analysis is as extensive and detailed as none before [8].

Using SWATH mass spectrometry 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, a new, ground-breaking HCP coverage analysis on the market 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 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: