Specific Racemization of Heavy-Chain Cysteine-220 in the Hinge Region of Immunoglobulin Gamma 1 as a Possible Cause of Degradation during Storage

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• 作者：Masato Amano ; Jun Hasegawa ; Naoki Kobayashi ; Naoyuki Kishi ; Takashi Nakazawa ; Susumu Uchiyama ; Kiichi Fukui
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：May 15, 2011
• 年：2011
• 卷：83
• 期：10
• 页码：3857-3864
• 全文大小：951K
• 年卷期：v.83,no.10(May 15, 2011)
• ISSN：1520-6882

文摘

Therapeutic antibodies often suffer from degradation due to various modifications during storage. We detected a degradation of immunoglobulin gamma 1 (IgG1) stored for 6 month at 40 掳C, and identified the modification as the racemization of Cys220 in the hinge sequence S²¹⁹CDKTHT²²⁵ of the heavy chain by tryptic peptide mapping and tandem mass spectrometry. The rate of racemization at Cys220 was enhanced deliberately by incubating the protein at 50 掳C and pH 9.0, while all the other cysteine residues were not affected. The racemization of Cys220 was confirmed by mass spectrometry in conjunction with extracted ion chromatography of the tryptic digest of IgG1 forced to degrade in D₂O and a series of synthetic hinge fragments containing d-amino acid, as well as the detection of d-cysteine in the acid hydrolysate. To rationalize the possible relationship between the racemization of Cys220 and isopeptide formation at the neighboring residue of Asp221, we suggest a new reaction mechanism that assumes a base catalyst to initiate these reactions by activating the amide nitrogen of Lys222. Due to the highly flexible nature of the hinge region, Lys222 can participate in either the formation of a cyclic imidazoline intermediate involving the 伪-carbonyl carbon of Cys220 to facilitate the racemization of Cys220 or that of a succinimide structure leading to the isomerization of Asp221.