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J Pharm Biomed Anal. 2017 Jun 5;140:98-104. doi: 10.1016/j.jpba.2017.03.027. Epub 2017 Mar 18.

Identification and characterization of a thermally cleaved fragment of monoclonal antibody-A detected by sodium dodecyl sulfate-capillary gel electrophoresis.

Author information

1
Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan; Analytical and Quality Evaluation Research Laboratories, Daiichi Sankyo Co., Ltd., 1-12-1 Shinomiya, Hiratsuka-shi, Kanagawa 254-0014, Japan. Electronic address: kubota.kei.c7@daiichisankyo.co.jp.
2
Analytical and Quality Evaluation Research Laboratories, Daiichi Sankyo Co., Ltd., 1-12-1 Shinomiya, Hiratsuka-shi, Kanagawa 254-0014, Japan.
3
Biologics Technology Research Laboratories, Daiichi Sankyo Co., Ltd., 2716-1 Aza Kurakake, Oaza Akaiwa, Chiyoda-machi, Oura-gun, Gunma 370-0503, Japan.
4
Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.

Abstract

This report describes a novel, comprehensive approach to identifying a fragment peak of monoclonal antibody-A (mAb-A), detected by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-cGE). The fragment migrated close to the internal standard (10kDa marker) of SDS-cGE and increased about 0.5% under a 25°C condition for 6 months. Generally, identification of fragments observed in SDS-cGE is challenging to carry out due to the difficulty of collecting analytical amounts of fractionations from the capillary. In this study, in-gel digestion peptide mapping and reversed phase liquid chromatography-mass spectrometry (RPLC-MS) were employed to elucidate the structure of the fragment. In addition, a Gelfree 8100 fractionation system was newly introduced to collect the fragment and the fraction was applied to the structural analysis of a mAb for the first time. These three analytical methods showed comparable results, proving that the fragment was a fraction of heavy chain HC1-104. The fragment contained complementarity determining regions (CDRs), which are significant to antigen binding, and thus would affect the efficacy of mAb-A. In addition, SDS-cGE without the 10kDa marker was demonstrated to clarify the increased amount of the fragment, and the experiment revealed that the fragment increases 0.2% per year in storage at 5°C. The combination of the three analytical methodologies successfully identified the impurity peak detected by SDS-cGE, providing information critical to assuring the quality and stability of the biotherapeutics.

KEYWORDS:

Fragment characterization; Gelfree fractionation system; In-gel digestion peptide mapping; Monoclonal antibody; Reversed phase liquid chromatography-mass spectrometry; Sodium dodecyl sulfate-capillary gel electrophoresis

PMID:
28346883
DOI:
10.1016/j.jpba.2017.03.027
[Indexed for MEDLINE]

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