Case study

HCP-ELISA coverage analysis without a null cell line

HCP-ELISA coverage analysis using immunocapture and LC-MS

Our client, a US-based developer of immuno-oncology biologics, required an HCP-ELISA with sufficient coverage for use during process development. However, 2D-PAGE and Western Blotting were not an option without a null cell line due to the relatively high concentration of drug substance in the production cell line samples obscuring a significant population of HCPs.

Immunocapture combined with mass spectrometry, known as ELISA-MS, does not require a mock sample but works with early and complex process samples. Furthermore, ELISA-MS uses less than 1 mg antibodies rather than methods based on affinity purification columns, which often need 10-15 mg ELISA antibodies.



For ELISA-MS, ELISA antibodies are first immobilized. The early process sample is added as the antigen before washing off unbound proteins, cleaving the bound proteins, and performing LC-MS on the resulting peptides. The coverage analysis hereby mimics the ELISA conditions by immunocapture while using mass spectrometry to identify HCPs recognized by the HCP-ELISA. Also, a negative control with antibodies from another expression system is included as a quality measure.

Principles of ELISA-MS method



LC-MS detected 618 HCPs in the client’s early process sample. ELISA kit A captured 351 HCPs, corresponding to a coverage of 57%. For kit B, 300 HCPs were captured, resulting in a coverage of 50%.

Comparison of two ELISA kits

Comparison of Host Cell Proteins in an early process sample covered by two different ELISA kits


The illustration below lists the 20 most abundant HCPs detected by LC-MS in the early process sample. Kit A detected 14 of them, while kit B only detected 12.

Overview of HCP analysis data

20 most abundant Host Cell Proteins detected in an early process sample bu two different ELISA kits


These data enabled the client to select a suitable HCP-ELISA with the broadest coverage of HCPs for their project. They also plan to use the coverage analysis results for IND filing.

Testimonial Alphalyse

'Thanks to the ELISA-MS data, we knew which kit to use to set up and validate our HCP ELISA assay! Furthermore, we knew exactly which individual HCPs were covered by the ELISA antibodies.'

Director CMC

US Biotech

Talk to us

Whatever protein-related challenge or question you may have, we would love to help. Our experts can help you decide on the best analytical approach for your project by email or online meeting - providing advice without obligation.

I would like an
This field is for validation purposes and should be left unchanged.