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

We have developed a highly sensitive method to detect those low-abundant host cell protein from the drug product using the proteome enrichment beads. This approach help us to find the root cause and the risk associated with the drug product. There are multiple ways to improve the detection limit of HCP analysis, the untargeted method including but not limited to protein A depletion, the limited digestion, the molecular weight cutoff, the immunoprecipitation.The targeted way to detect the host cell protein, including liquid chromatography, multiple reaction monitoring, and the non-liquid chromatography parallel reaction monitoring. The major challenge associate with these HCP analysis is the significant dynamic range between antibody drug and host cell proteins. So most method aim to reduce the amount of antibody and the enrich HCP before LCMS analysis to decrease the dynamic range between antibody peptides and HCP peptides.So ProteoMiner technology and the limited digestion protocol significantly improve the detection limit of HCP analysis compared to other methods. It's non-biased compared to immunoprecipitation and can be applied to different drug module compared to protein A depletion method. This protocol is also simple and straightforward.So ProteoMiner technology and limited digestion protocol help us to identify several HCPs that degrade polysorbate and the fragment antibody drug. In combination with the nano LCMPRM method, the enrichment method can also provide quantitative results to find the biological relevant concentration of problematic host cell proteins.

Watch this Scientific Journal Video about Host Cell Proteins HCPs Enrichment in Monoclonal Antibody mAb Drug Product at JoVE.com

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

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Research

JoVE Journal

Chemistry

213 次观看.  Regeneron Pharmaceuticals Inc.. 首先，从商业蛋白质富集试剂盒的离心柱上取下顶盖和底盖，并保留它们以备将来使用。将未加盖的离心柱置于两毫升微量离心管中。将装置在 1000 G 和室温下离心 30 至 60 秒，以取出并丢弃储存溶液。将 200 μL 洗涤缓冲液加入离心柱中的富集珠中，并通过上下移液数次进行混合。如前所述离心离心柱后，丢弃收集的缓冲液。更换底盖，在更换顶盖之前，向离心柱中加入...

视频: 单克隆抗体 （mAb） 药物产品中的宿主细胞蛋白 （HCP） 富集

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks associated with drug products, methods have been developed to identify low-abundance HCPs. A crucial approach for developing a sensitive HCP detection method involves enriching HCPs while simultaneously removing monoclonal antibodies (mAbs) before analysis, utilizing liquid chromatography-mass spectrometry (LC-MS).
This protocol offers detailed instructions for enriching host cell proteins using commercially available proteome enrichment beads. These beads contain a diverse library of hexapeptide ligands with specific affinities for different proteins. The protocol also incorporates limited digestion and subsequent peptide detection using nano LC-MS/MS. By employing these techniques, HCPs with low abundance can be enriched over 7000-fold, resulting in an impressive detection limit as low as 0.002 ppm. Significantly, this protocol enables the detection of 850 HCPs with a high level of confidence using a NIST mAb. Moreover, it is designed to be user-friendly and includes a video demonstration to assist with its implementation. By following these steps, researchers can effectively enrich and detect HCPs, enhancing the sensitivity and accuracy of risk assessment for drug products.

A protocol is presented for enriching host cell proteins (HCPs) from drug products (DP) and detecting peptides using proteome enrichment beads. The method is demonstrated using an in-house manufactured monoclonal antibody (mAb) drug substance (DS), which is a well-characterized reference material for evaluating and comparing different methods in terms of performance.