Critical reasons why mAbs call for orthogonal HCP analysis techniques

Question:

We produce a monoclonal antibody (mAb) product, and our HCP ELISA shows low ppm levels. Recently, I heard that regulatory authorities might ask for orthogonal data on process-related impurities.

Is this true? Why should we spend time and money on additional host cell protein analysis when our data shows our product is pure?

Answer:

It is correct that mAb products are often relatively pure. This purity is due to the efficient removal of host cell proteins (HCPs) using protein A purification and polishing steps [1].

However, you should be aware that determining the HCP amount to a sparse sum does not rule out that the product may contain ‘problematic’ HCPs at low but critical levels.

‘Problematic’ HCPs are those HCPs that may…

• Affect drug efficacy by reducing protein stability and potency,
  or
• Induce immunotoxic effects and immunogenic reactions in patients

even in sub-ppm amounts.

Most people use HCP ELISAs to demonstrate process consistency and final-drug substance purity. However, fewer know that it merely monitors a subset of the HCP profile. Even with high coverage of the impurities in your sample, it may miss proteins that co-purify with the product or are poor immunogens due to low molecular weight [2].

Also, ELISA does not provide any details about the specific HCPs. Therefore, it is impossible to predict if any HCP (although at a low level) will cause problems later.

There are several examples where specific HCPs have delayed projects due to immunogenic reactions caused by particular HCPs. In other cases, products experienced reduced stability because of HCPs with enzymatic activity [3].

Hence, you need an orthogonal method to determine which HCPs appear in your product. Such a method could, for example, be Mass Spectrometry (MS).

Using mass spectrometry for HCP characterization

So how do you analyze your mAb for problematic HCPs?

Mass spectrometry as an orthogonal method for HCP analysis is very powerful. Mainly because it provides detailed information about the specific HCPs – information you cannot get from ELISA.

I.e., you can analyze the impurities to a level where you get the physical properties like molecular weight (Mw) and isoelectric point (pI). The analysis even provides the accession number to find information about the protein and related proteins. With this information, you are well equipped to evaluate if any of the HCPs are problematic.

The detailed information about each HCP can then be used for data-driven process optimization – to delete any problematic HCP.

Hence, it is crucial to use an orthogonal method to investigate if it contains any problematic HCPs. Even when your HCP ELISA shows low-ppm levels in your monoclonal antibody.

I recommend reading this interview with Denise Krawitz, PhD and principal consultant with CMC Paradigms LLC. It goes further into the managing of risks in HCP assay development.

Orthogonal method for evaluation of problematic HCPs in mAbs

Alphalyse has developed an orthogonal Mass Spectrometry method that lists the individual HCPs with the respective amounts – in any given sample [4].

Moreover, combining this standard method with a native digest optimized for mAbs, substantially lowers the detection limit, even down to sub-ppm levels.

As a unique service, you get a check for specific HCPs known to be of concern in mAb products. The assigned specialist also compares the results to the HCPs found in a study of commercially available mAbs.

You can use this information to remove specific HCPs and thus ensure the stability and efficacy of the drug. Similarly, you can lower the risk of immunogenic reactions in patients while providing regulatory authorities with extensive HCP data.

Learn more on the Alphalyse website