My company uses a commercial HCP ELISA kit for host cell protein analysis of clinical phase 1 material. We hear that we must develop a process-specific ELISA for clinical phase 2 and 3. Developing a process-specific ELISA could take 2 years, cost us a fortune and might not be good enough anyway.
Is there a better method or a way to use commercial HCP ELISAs for impurity analysis and obtain FDA approval of all clinical stage trials?
The short answer is – yes.
Many people still believe that the tiresome development of process-specific ELISA antibodies is required and unavoidable. This is probably since commercial HCP ELISA kits sometimes have a low HCP coverage. The term coverage comprises the percentage of host cell proteins (HCPs) recognized by the antibodies.
However, recent recommendations from U.S. Food and Drug Administration (FDA) officials suggest that the chosen HCP assay should have sufficient coverage with demonstrated ability to detect most HCPs present in the product.
Based on this, a suitable HCP coverage assay could be any ELISA documented to cover enough HCPs – also commercial HCP ELISAs (reference https://www.alphalyse.com/news/bebpa-2019-hcp-conference-review/).
Comparing HCP coverage methods
Typically, coverage determination is by protein spot counting using 2D SDS-PAGE and Western Blotting or Immunoaffinity binding and 1D/2D SDS-PAGE.
The list of disadvantages of this method is long and includes high variability, low reproducibility, as well as a high risk of not detecting abundant HCPs of low MW. Furthermore, the HCP antigens denature during 2D PAGE analysis whereas the method detects antigens in native form in ELISA.
If a biopharmaceutical company wishes to completely avoid process-specific antibody development, it needs an improved coverage analysis to determine the precise coverage percent. In this way, the company can select the ELISA with the best coverage, whether it is commercial or process specific.
The ideal coverage method should include a complete list of HCPs covered by the assay, in order to select the best HCP ELISA. Fortunately, a method that can achieve this is now available and described below:
In this method, antibodies are first immobilized to the 96-well plate like the sandwich ELISA. Subsequently, process samples are added, and antigens bind to their respective antibodies, if the ELISA antibody kit contain them. Finally, the bound antigens are digested by proteolytic enzymes before mass spectrometry (MS) sample prep and LC-MS/MS analysis.
Proteins covered by the ELISA are found with information-dependent acquisition (IDA) liquid chromatography (LC) tandem mass spectrometry (MS/MS), also denoted IDA LC-MS/MS or SWATH LC-MS/MS, using the Sciex TripleTOF 6600 system, and searches in relevant protein databases.
The analysis provides you with:
- A list of proteins recognized by the ELISA antibodies
- A list of all proteins in mock or process sample
- The HCP coverage percentage
- The specific coverage of HCPs in the drug product
Curious to learn more?
The method is described in this poster from the BEBPA 2019 conference, the largest HCP conference in the world, where the new analysis was introduced:
More details about the coverage analysis can be found on the Alphalyse website