How to select an HCP ELISA with sufficient coverage for regulatory documentation

Alphalyse
Aug 25. 2021

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 plays an essential part in biologics development and regulatory documentation.

Since specific HCPs have shown to be 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 both process development, characterization, and validation [2].

(Learn more in this blog post).

Selecting the best ELISA kit: HCP antibody coverage analysis

Before a commercial ELISA kit or a custom-developed 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 followed by 2D gel analysis.

However, both of those 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 have no way of knowing which HCP the spot represents. And perhaps worse, you do not know the coverage of each HCP.

Moreover, column-based methods often need up to 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.

ELISA_MS principle
Illustration of the ELISA-MS™ method

With ELISA-MS, you can perform coverage analysis on an early process sample containing the drug protein. Thus, you do not have to generate a mock cell line with the risk of having another HCP profile than your drug samples. In this way, you can perform coverage analysis without investing time and money in developing the mock cell line.

Finally, when you get the lists of the individual HCPs covered in your process and the HCPs in the purified product, you will know if the HCP-ELISA detects the individual impurities in the product. Therefore, many consider ELISA-MS the best method for determining the coverage of ELISA kits.

You are free to use the best method – both for coverage and HCP analysis

We often get the question: Which coverage method do the regulatory authorities require?

The answer is that you have freedom of choice for the HCP analysis methods as long as you can demonstrate that the method is scientifically sound.

Are you interested to learn more about different HCP coverage methods so you can select the best ELISA kit? Then check out this webinar with HCP expert Rikke Raaen Lund:

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] U. S. Pharmacopeia: “USP 39 Published General Chapter <1132> Residual Host Cell Protein Measurement in Biopharmaceuticals”. 2016

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

[4]          Pilley et al.: “A novel approach to evaluate ELISA antibody coverage of host cell proteins-combining ELISA-based immunocapture and mass spectrometry.” Biotechnology Progress. 2020

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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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