host cell proteins (hcps) enrichment in monoclonal antibody (mab) drug product

Enrichment of Host Cell Proteins in Monoclonal Antibody Drug Products

Host cell proteins (HCPs) are impurities that are released from the cell culture of the host organism and co-purified with monoclonal antibody (mAb)1,2,3,4. Trace levels of HCPs can negatively impact the quality of the drug product5,6,7,8,9,10,11,12,13,14,15, and therefore, a sensitive HCP analysis method is desired to detect HCPs in sub-ppm to ppm levels.
Orthogonal methods can be applied to detect HCPs in low abundance. Enzyme-linked immunosorbent assay (ELISA) is generally used to quantitate overall HCPs, and it can also detect and quantitate individual HCPs if the corresponding antibodies are available16. However, the production of HCP-specific antibodies is time-consuming and labor-intensive. In contrast, liquid chromatography coupled with mass spectrometry (LC-MS) can provide comprehensive information about individual HCPs in mAb drug products and is widely applied for HCP identification4,7,9,10,12,13,14,15,17,18,19,20,21,22,23,24,25,26,27.
Several methods have been developed to detect HCPs with LC-MS/MS, including limited digestion20, filtration17, Protein A deletion21, immunoprecipitation (IP), and ProteoMiner enrichment (PM)18. Most methods aim to reduce the amount of mAb and enrich HCPs prior to LC-MS/MS analysis, thereby decreasing the dynamic range between mAb peptides and HCP peptides. This protocol presents a proteomic sample enrichment method that combines ProteoMiner technology and limited digestion (PMLD)28. The ProteoMiner enrichment principle involves using commercially available proteome enrichment beads containing a diverse library of combinatorial peptide ligands. These ligands specifically bind to proteins on antibody-drug products, allowing for the removal of excess molecules while concentrating low-abundance host cell proteins (HCPs) on their respective affinity ligands. On the other hand, the principle of limited digestion involves using a low concentration of trypsin. This concentration is sufficient to digest low-abundance HCPs but not enough to digest all antibody drug products. This approach enables the recovery and enrichment of digested HCP peptides from the solution.
Compared to filtration methods, the PMLD technique is not limited by the size of the detected HCPs17. Protein A deletion methods are specific to detecting HCPs associated with antibodies21, while immunoprecipitation is restricted to predefined HCPs from a particular cell line (such as the Chinese Hamster Ovary (CHO) cell line), where an anti-HCP antibody was generated4. In contrast, PMLD can be applied to detect HCPs from any drug modules and host cell proteins co-purified with drug products from various cell lines. Additionally, PMLD exhibits better sensitivity compared to the mentioned methods17,18,20,21,24.
This approach can enrich the HCP concentration by 7000-fold and lower the detection limit to 0.002 ppm28. The experimental setup is illustrated in Figure 1.

Author Spotlight: Detecting Low-Abundant Host Cell Proteins in Drug Products Using Enrichment Beads and Limited Digestion 

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

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Host Cell Proteins (HCPs) Enrichment in Monoclonal Antibody (mAb) Drug Product

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