﻿preparation of monoclonal antibody (mab) solutions for host cell protein enrichment

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.

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﻿Preparation of Monoclonal Antibody (mAb) Solutions for Host Cell Protein Enrichment

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265 조회수.  Regeneron Pharmaceuticals Inc.. 숙주 세포 단백질 농축을 위해 단클론 항체 의약품을 적절하게 희석하는 것으로 시작합니다. 농도가 밀리리터당 50밀리그램을 초과하는 의약품을 희석하려면 2밀리리터 마이크로 원심분리기 튜브에 있는 단클론 항체 15밀리그램에 탈이온수를 첨가합니다. 농도가 밀리리터당 50밀리그램 미만인 의약품의 경우 20밀리그램을 10킬로달톤 원심 필터 장치로 옮깁니다.14, 실온?...

﻿Preparation of Monoclonal Antibody (mAb) Solutions for Host Cell Protein Enrichment

Preparation of Monoclonal Antibody (mAb) Solutions for Host Cell Protein Enrichment