﻿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

Watch this Scientific Journal Video about ?Preparation of Monoclonal Antibody mAb Solutions for Host Cell Protein Enrichment at JoVE.com

숙주 세포 단백질 농축을 위한 단클론 항체(mAb) 용액 준비

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, 실온에 25 분 동안 14, 000 G에 관에서 남아 있는 해결책의 대략 100개 마이크로리터를 남겨두기 위하여 내용을 분리한다. 그 때 10 밀리몰 히스티딘 완충액의 350 마이크로리터를, 14에 그것을 분리하기 전에 와동하는 여과기로 6의 PH가 있는 350 마이크로리터를, 실내 온도에 25 분 동안 000 G 추가하십시오. 필터를 뒤집어 샘플 수집 튜브에 넣습니다.샘플을 실온에서 1000G에서 5분 동안 원심분리하여 수집 튜브에서 전체 용액을 회수합니다. 200 마이크로리터의 10밀리몰 히스티딘 완충액을 필터에 추가하고 피펫을 위아래로 움직여 나머지 단백질 샘플을 회수합니다. 전체 용액을 동일한 수집 튜브에 옮깁니다.이전에 설명한 대로 소용돌이 및 스핀 다운. 나노 드롭으로 샘플 농도를 측정한 후 히스티딘 완충액을 첨가하여 밀리리터당 50밀리그램으로 조정합니다. 마지막으로 샘플 300마이크로리터를 2밀리리터 마이크로 원심분리기 튜브로 옮깁니다.

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JoVE Journal

Chemistry

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

278 조회수. 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?...

비디오: 숙주 세포 단백질 농축을 위한 단클론 항체(mAb) 용액 준비

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.