Residual protein analysis of complex products

Unlike traditional biologics, which are typically manufactured by expressing one recombinant protein in a single cell line, advanced therapies, viral vaccines, and oligonucleotide-based medicines contain highly heterogeneous protein mixtures from multiple sources and organisms.

Commercial HCP ELISA kits, typically used for monitoring Host Cell Proteins (HCPs) from CHO and E.coli expression systems, cannot simultaneously detect proteins from different species and often demonstrate low coverage of, e.g., HCPs from non-immunogenic human cell lines. As such, classical impurity assays often fail to detect a substantial proportion of the residual proteins in complex biotherapeutics.

Liquid chromatography-mass spectrometry (LC-MS) is a robust and reproducible orthogonal method to ELISA, which has high specificity, sensitivity, and dynamic range to detect highly heterogeneous HCPs. Using Data Independent Acquisition (DIA), LC-MS can quantify and characterize all protein impurities in a sample and provide thorough, precise, and robust HCP coverage.

Regulatory authorities often fast-track novel therapies and vaccines that have the potential to treat severe, life-threatening conditions. Furthermore, since they can be one-shot cures, being first to market can provide a competitive advantage. However, developing a process-specific ELISA for complex products requires 1 - 2 years for antibody development and assay validation. Even then, there is a risk of delayed regulatory approval due to inadequate impurity detection and clearance.  

LC-MS assays require only 6-8 weeks to be established and optimized for any biologic product. This can significantly accelerate the development, marketing approval, and commercial biomanufacturing of complex biologics. 

Manufacturing changes commonly happen for complex biologics, and comparability studies of residual proteins are frequently necessary for product development and regulatory filings. For instance, when improving the process consistency and product quality, upscaling, or transferring to GMP facilities for commercial release. However, demonstrating comparability in complex biologics is often challenging by still-evolving process knowledge, small numbers of batches, and material constraints for each batch.   

The LC-MS assays at Alphalyse have been developed for high robustness and tolerability toward different sample types, complexity, and proteins from multiple sources. It is a flexible method allowing for process changes and the use of various buffers and matrices. The use of the same intact internal protein standards enables comparable results between samples, across process changes, and for comparability between GMP batches.