﻿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 

Sınırlı Sindirim ile Birleştirilmiş Zenginleştirme Boncukları Kullanılarak Konak Hücre Protein Analizi

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

Konak Hücre Protein Zenginleştirmesi için Monoklonal Antikor (mAb) Çözeltilerinin Hazırlanması

Konak hücre proteini zenginleştirmesi için monoklonal antikor ilaç ürünlerini uygun şekilde seyrelterek başlayın. İlaç ürünlerini mililitre başına 50 miligramın üzerindeki konsantrasyonlarla seyreltmek için, iki mililitrelik bir mikro santrifüj tüpünde 15 miligram monoklonal antikora deiyonize su ekleyin. Mililitre başına 50 miligramın altındaki konsantrasyonlara sahip ilaç ürünleri için, 20 miligramını 10 kilodaltonluk bir santrifüj filtre cihazına aktarın.İçeriği 14.000 G'de oda sıcaklığında 25 dakika santrifüjleyin ve tüpte yaklaşık 100 mikrolitre çözelti bırakın. Daha sonra, oda sıcaklığında 25 dakika boyunca 14.000 G'de santrifüjlemeden önce tüpü girdap haline getiren, pH'ı altı olan 350 mikrolitre 10 milimolar histidin tamponu ekleyin. Filtreyi ters çevirin ve numune toplama tüpüne yerleştirin.Numuneyi oda sıcaklığında beş dakika boyunca 1000 G'de santrifüjleyin ve tüm çözeltiyi toplama tüpüne alın. Filtreye 200 mikrolitre 10 milimolar histidin tamponu ekleyin ve kalan protein örneğini geri kazanmak için yukarı ve aşağı pipetleyin. Tüm çözeltiyi aynı toplama tüpüne aktarın.Girdap yapın ve daha önce gösterildiği gibi aşağı inin. Numune konsantrasyonunu nano damla ile ölçtükten sonra, histidin tamponunu ekleyerek mililitrede 50 miligrama ayarlayın. Son olarak, numunenin 300 mikrolitresini iki mililitrelik bir mikro santrifüj tüpüne aktarın.

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JoVE Journal

Chemistry

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

270 Görüntüleme.  Regeneron Pharmaceuticals Inc.. Konak hücre proteini zenginleştirmesi için monoklonal antikor ilaç ürünlerini uygun şekilde seyrelterek başlayın. İlaç ürünlerini mililitre başına 50 miligramın üzerindeki konsantrasyonlarla seyreltmek için, iki mililitrelik bir mikro santrifüj tüpünde 15 miligram monoklonal antikora deiyonize su ekleyin. Mililitre başına 50 miligramın altındaki konsantrasyonlara sahip ilaç ürünleri için, 20 miligramını 10 kilodaltonluk bir santrifüj filtre cihazına aktarın.

Video: Konak Hücre Protein Zenginleştirmesi için Monoklonal Antikor (mAb) Çözeltilerinin Hazırlanması

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

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.