How to select an HCP ELISA with sufficient coverage

Question:
How do I choose the best ELISA kit for my biologic? In other words, I would like to select the impurity assay with the highest coverage of the process HCPs, not only for harvest samples but also for the HCP impurities in the purified drug substance.

Answer:
Host Cell Protein (HCP) analysis is essential in development and regulatory documentation of biologics.

Since specific HCPs are problematic, either as a potential risk factor for immunogenicity in patients or by affecting drug stability, biologics should be as free of residual HCPs as possible [1, 2].

Historically, most companies performed HCP analysis using anti-HCP ELISAs raised by immunizing animals with a protein lysate from the host cell line [1, 2].

Today, most companies also use orthogonal methods for HCP characterization, including liquid chromatography mass spectrometry (LC-MS), to quantify individual and potential problematic HCPs. Orthogonal methods are especially beneficial when there is no commercial kit on the market, or it is impossible to develop a good HCP ELISA.

Still, HCP ELISAs are valuable during process development, characterization, and validation [2].

Selecting the best ELISA kit: HCP antibody coverage analysis

Before a commercial HCP ELISA kit or a custom-developed HCP ELISA is considered sufficient and fit-for-purpose, you must perform an HCP antibody coverage analysis. This analysis determines how completely a population of polyclonal antibodies recognizes the population of HCPs, as stated in USP 39 [2].

Thus, comparing the coverage of various ELISAs – kits or custom – allows you to pick the best and most fitting ELISA for your drug.

Traditionally, you had to choose between two methods for HCP coverage evaluation. These were 2D gels followed by Western Blotting or immunoaffinity purification and 2D gel analysis.

However, both methods have widely known limitations, including manual and variable spot counting in gels [2, 3]. This led to improved coverage evaluation methods, such as ELISA-MS, which provides the name and identity of each HCP covered by the ELISA kit [4].

Coverage analysis with only 0.5 mg antibody – without a mock cell line

ELISA-MS combines ELISA-based immunocapture in the 96-well plate with LC-MS. The result is a list of individual HCPs in a drug sample recognized by the specific ELISA antibodies [4].

Where 2D gel-based and immunoaffinity chromatography-based coverage methods evaluate the antibodies outside of the ELISA plate, ELISA-MS mimics the conditions of the HCP-ELISA. Thus ELISA-MS captures the HCPs by the anti-HCP antibodies under the same conditions as the ELISA in the 96-well plate.

2D gel-based methods rely on spots in a gel to provide a total number of covered HCPs. If you only utilize this method, you cannot know which HCP the spot represents. And perhaps worse, you do not see the coverage of each HCP.

Moreover, column-based methods often need 10-15 mg ELISA antibodies. Whereas ELISA-MS only requires 0.5 mg antibody for running HCP coverage analysis and thus saves your valuable ELISA antibodies.