HCP-ELISA coverage analysis without a null cell line

HCP-ELISA coverage analysis using immunocapture and LC-MS

CASE STUDY

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.

Process

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.

The principles of ELISA-MS

Results

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 Host Cell Proteins in an early process sample covered by two different 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.

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


US  Biotech_logo
"Thanks to the ELISA-MS data, we knew which kit to use for the setup and validation of our HCP ELISA assay! Furthermore, we new exactly which individual HCPs were covered by the ELISA antibodies."

US Biotech- Director CMC

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