Formation of an Unprecedented Impurity during CE-SDS Analysis of a Recombinant Protein
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RESEARCH PAPER
Formation of an Unprecedented Impurity during CE-SDS Analysis of a Recombinant Protein Bin-Bin Shen 1,2 & Zhongwei Zhang 1,2 & Jun-Jie Yuan 3 & Aiping Zheng 4 & Su Zeng 1 & Jian-Qing Gao 5 & Wenhan Bao 6 & James Barnard 7 & Haibin Wang 3 & Wei-Jie Fang 1,2
Received: 8 August 2020 / Accepted: 5 October 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
ABSTRACT Purposes The main purposes of this article are to describe an unprecedented phenomenon in which significant amount of a shoulder peak impurity was observed during normal non-reducing capillary electrophoresissodium dodecyl sulfate (CE-SDS) analysis of a recombinant fusion protein X, and to evaluate the root cause for this phenomenon. Methods A series of experiments were conducted to study the nature of this degradation. Effects of iodoacetamide (IAM), heating temperature, duration, and SDS on the formation of this specific impurity were evaluated using a variety of characterization techniques. Results The formation of the impurity as observed in CESDS was actually due to alkylation of lysine and serine residues with IAM, as confirmed by peptide mapping and LCMS/MS, which increased the molecular weight and therefore decreased the electrophoretic mobility. The amount of
impurity was also strongly dependent on sample preparation conditions including the presence or absence of SDS. Conclusions Our study clearly suggested that even though IAM has been used extensively as an alkylation reagent in the traditional non-reducing CE-SDS analysis of monoclonal antibodies and other proteins, alkylation with IAM could potentially lead to additional impurity peak, and therefore complicating analysis. Therefore, before performing CE-SDS and other analyses, the effects of sample preparation procedures on analytical results must be evaluated. For protein X, IAM should be excluded for CE-SDS analysis.
KEY WORDS alkylation . CE-SDS . impurity . LC-MS . peptide mapping . sample preparation
ABBREVIATIONS * Wei-Jie Fang [email protected]
CD CE-SDS IAM icIEF LC-MS
1
Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
2
Hangzhou Institute of Innovative Medicine, Zhejiang University, Hangzhou 310016, China
3
Zhejiang Hisun Bioray Biopharmaceutical Co., Ltd, Taizhou Zhejiang 318000, China
4
Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China
5
Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
SDS-PAGE
6
School of Life Sciences, Shandong University, Qingdao 266237, China
SE-HPLC
7
Drug Product Development, Biological, Allergan, Irvine California 92612, USA
mAb MALDI-TOFMS RP-HPLC
Circular dichroism Capillary electrophoresis-sodium dodecyl sulfate Iodoacetamide Imaged capillary isoelectric focusing Liquid chromatography coupled with mass spectrometry Monoclonal antibody Matrix-assisted laser desorption/ionization time of flight mass sp
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