Bridging ELISA and Mass Spectrometry: Expert Panel Discussion on Host Cell Protein Analysis

This summary accompanies a recent expert forum discussion organised by BioQC. We appreciate the panellists’ time and expertise. The views expressed by the panellists are their personal interpretations and do not represent the positions of their respective organisations.

This panel discussion explored the limitations of ELISA-based host cell protein (HCP) testing and the growing role of mass spectrometry in characterising and controlling process-related impurities in biotherapeutics.

Bridging Between ELISA Kits: Why a Higher Number Does Not Mean a Better Assay Journey

A recurring challenge for many biopharmaceutical companies arises when existing ELISA reagents are discontinued, and a new kit must be introduced to replace them. This bridging exercise frequently produces different HCP results between the old and new assays, and panellists were clear that a higher reading from the newer kit should not be interpreted as evidence of improved sensitivity. It simply indicates that the two assays measure different things.

This difference stems from the polyclonal antibodies in each ELISA kit being generated in different animals, each mounting a distinct immune response against the HCP mixture used for immunisation. As a result, the antibody populations in the two kits recognise different subsets of host cell proteins, and the overall composition of each antibody pool will vary considerably. Highly immunogenic HCPs tend to provoke a disproportionately strong immune response, meaning they can dominate the antibody pool and, in turn, dominate the assay signal. A newer kit reading substantially higher than an older one, for example fifty parts per million against ten, cannot therefore be assumed superior. It may simply be more heavily influenced by these high-responder proteins, whilst still failing to capture other HCPs that are genuinely relevant to the process.

Panellists noted that kit standards must also be scrutinised during any bridging exercise, since a standard with a composition that diverges from the actual manufacturing process can distort the calibration curve independently of any differences in antibody coverage. Reference was made to recent comments from European regulators, including remarks attributed to a representative from the European Medicines Agency at a recent host cell protein conference, indicating that authorities now expect coverage analysis, dilutional linearity studies, characterisation of critical reagents, and side-by-side testing of historical batches with both the old and new ELISA kits, rather than simple adoption of a replacement kit off the shelf.

ELISA as a Black Box

A central theme running throughout the discussion was that ELISA testing, used in isolation, functions essentially as a black box. The technique provides a reproducible, relative measurement tied to a specific antibody pool and a specific standard, rather than an absolute quantification of host cell protein content. 

Whilst batch-to-batch comparisons remain possible, the underlying meaning of a rising or falling number cannot be established without further characterisation.

One panellist drew on prior experience leading quality control within a cell therapy setting where mass spectrometry was not available, describing the resulting situation as one of limited confidence. Results could be compared numerically from batch to batch, but there was no way of knowing whether any change reflected something of genuine concern to patient safety. 

Older orthogonal techniques such as two-dimensional gel electrophoresis and Western blotting were discussed as partial solutions used prior to the availability of mass spectrometry, though these were characterised as offering only a subjective assessment of coverage rather than the detailed, protein-level resolution that mass spectrometry can provide.

High-Responder HCPs and Mismatched Kit Standards

Drawing on experience across a large number of client projects, panellists described specific and recurring problems uncovered once ELISA kits were characterised using mass spectrometry. In one case, five individual HCPs were found to account for more than seventy per cent of the antibody signal within a kit, effectively masking the presence of other, potentially more relevant, host cell proteins. 

Kit standards were also found on several occasions to have a skewed composition relative to the actual drug substance, leading to miscalibration and misleading HCP quantification. 

A further recurring bias was reduced ELISA sensitivity towards low molecular weight host cell proteins, which tend to be less immunogenic and are therefore underrepresented within the antibody pool, despite the fact that some low molecular weight proteins carry disproportionately high biological activity and clinical relevance.

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