Can I use commercial HCP ELISA kits in clinical trial documentation?

ELISA coverage
Alphalyse
Jul 10. 2019

Question:

Does our generic HCP ELISA kit have sufficient coverage?

My company uses a commercial HCP ELISA kit to analyze clinical phase 1 material host cell protein. We hear that we must develop a process-specific ELISA for clinical phases 2 and 3. Developing a process-specific ELISA could take two years, cost a fortune, and might not even have good coverage.

Is there a way to ensure that we pick a commercial HCP ELISA with high coverage? And thus increase the chances of using it for approval of clinical-stage trials?

 

Answer:

 

The short answer is – yes.

Many people still believe clinical trials require the tiresome development of process-specific ELISA antibodies – and is unavoidable. It is probably because commercial HCP ELISA kits sometimes have a low HCP coverage*. (*the percentage of host cell proteins (HCPs) recognized by the antibodies [1]).

ELISA HCP coverage

A suitable assay could be any HCP ELISA documented to cover enough HCPs

However, recent recommendations from government officials at FDA and EMA suggest otherwise. They only say that the chosen HCP assay should have sufficient coverage with demonstrated ability to detect most HCPs present in the product [2].

Based on this, a suitable HCP coverage assay could be any ELISA documented to cover enough HCPs. And thus, even a commercial HCP ELISA with proper validation of its coverage [3], like CHO, E. coli, and HEK 293.

 

hcp-coverage

 

Now, let’s compare methods for evaluation of HCP ELISA kits:

Typically, you evaluate the coverage by protein spot counting, using 2D SDS-PAGE and Western Blotting, or Immunoaffinity binding and 1D/2D SDS-PAGE [3].

This method’s disadvantages are long: It includes high variability, low reproducibility, and a high risk of not detecting abundant HCPs of low MW. Furthermore, the HCP antigens denature during 2D PAGE analysis. In contrast, the method detects antigens in their native form in ELISA [4].

 

So what if a biopharmaceutical company wishes to skip process-specific antibody development? Then it needs a better coverage analysis to determine the precise coverage percent of the ELISA for specific HCPs. It enables comparing and selecting the ELISA with the best coverage of all HCPs,  whether commercial or process-specific [3, 4, 5].

The ideal coverage method includes a complete list of HCPs covered by the assay to select the best HCP ELISA [3, 4].

Fortunately, a method that can achieve this is now available and described below:

 

What is ELISA-based immunocapture?

This method first immobilizes the antibodies to the 96-well plate, like the sandwich ELISA. Subsequently, process samples are added, and antigens bind to their respective antibodies – if the HCP ELISA antibody kit contains them. Finally, the process digests the bound antigens with proteolytic enzymes before mass spectrometry (MS) sample prep and LC-MS/MS analysis.

Information-dependent acquisition (IDA) liquid chromatography (LC) tandem mass spectrometry (MS/MS) can find the proteins covered by the HCP ELISA. This method is denoted IDA LC-MS/MS or SWATH LC-MS/MS and uses the Sciex TripleTOF 6600 system. It also includes searches of 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

Coverage analysis - a method for selecting the best ELISA kit

What else should you know about evaluating the coverage of HCP ELISA kits?

The new analysis was first introduced at BEBPA 2019 conference, the largest HCP conference in the world. This poster describes the method in more detail:

HCP COVERAGE POSTER

More information about the coverage analysis is available on the Alphalyse website.

 

References

[1]          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 Bioengineering2015

[2]          The United States Pharmacopeia: “Residual Host Cell Protein Measurement in Biopharmaceuticals,” USP 39 – NF 34, 2016 

[3]          Alphalyse: “BEBPA 2019 review – key topics and take-home messages“, 2019

[4]          Zhu-Shimoni et al.: “Host Cell Protein Testing by ELISAs and the Use of Orthogonal Methods,” Biotechnology and Bioengineering2014

[5]          Wang et al.: “Host Cell Proteins in Biologics Development: Identification, Quantitation and Risk Assessment,” Biotechnology and Bioengineering, 2009

More posts about Analysis of Host Cell Protein
  • Host Cell Protein monitoring in process development – using mass spec

  • Identification of small host cell proteins (small Mw HCPs)

  • What is SWATH mass spectrometry and its advantages?

  • How do ‘Jackpot HCPs’ and a ‘Lucky Goat’ influence my ELISA results?

  • Simple way to perform label-free quantification of Host Cell Proteins

About Alphalyse

Alphalyse uses expertise in the field of protein chemistry and bioinformatics, combined with top-of-the-line mass spectrometry equipment, to provide protein analysis services globally.

Alphalyse aims to deliver high quality data and customer service to all of our clients in a fast and convenient way.

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