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 for host cell protein analysis of clinical phase 1 material. We hear that we must develop a process-specific ELISA for clinical phases 2 and 3. Development of a process-specific ELISA could take 2 years, cost us a fortune, and might not even have good coverage.

Is there a way to ensure that we pick commercial HCP ELISAs for impurity analysis 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. This is probably because 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 [1]).

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. Because they just 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, lets 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].

The list of disadvantages of this method is long: It 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 [4].

So what if a biopharmaceutical company wishes to skip process-specific antibody development? Then it needs an better coverage analysis that determines the precise coverage percent of the ELISA for specific HCPs. In this way, the company can compare and select the HCP ELISA with the best coverage of all HCPs. Whether it is commercial or process specific [3, 4, 5].

The ideal coverage method could include a complete list of HCPs covered by the assay, in order 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.

Proteins covered by the HCP ELISA are found with information-dependent acquisition (IDA) liquid chromatography (LC) tandem mass spectrometry (MS/MS). This method is denoted IDA LC-MS/MS or SWATH LC-MS/MS, and uses the Sciex TripleTOF 6600 system. It also includes searches 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

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 now 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

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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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