Trisulfide Modification Impacts the Reduction Step in Antibody鈥揇rug Conjugation Process

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• 作者：Katherine Cumnock ; Timothy Tully ; Christopher Cornell ; Matthew Hutchinson ; Jeffrey Gorrell ; Ken Skidmore ; Yan Chen ; Fredric Jacobson
• 刊名：Bioconjugate Chemistry
• 出版年：2013
• 出版时间：July 17, 2013
• 年：2013
• 卷：24
• 期：7
• 页码：1154-1160
• 全文大小：273K
• 年卷期：v.24,no.7(July 17, 2013)
• ISSN：1520-4812

文摘

Antibody鈥揹rug conjugates (ADCs) utilizing cysteine-directed linker chemistry have cytotoxic drugs covalently bound to native heavy鈥揾eavy and heavy鈥搇ight interchain disulfide bonds. The manufacture of these ADCs involves a reduction step followed by a conjugation step. When tris(2-carboxyethyl)phosphine (TCEP) is used as the reductant, the reaction stoichiometry predicts that for each molecule of TCEP added, one interchain disulfide should be reduced, generating two free thiols for drug linkage. In practice, the amount of TCEP required to achieve the desired drug-to-antibody ratio often exceeds the predicted, and is variable for different lots of monoclonal antibody starting material. We have identified the cause of this variability to be inconsistent levels of interchain trisulfide bonds in the monoclonal antibody. We propose that TCEP reacts with each trisulfide bond to form a thiophosphine and a disulfide bond, yielding no net antibody free thiols for conjugation. Antibodies with higher levels of trisulfide bonds require a greater TCEP:antibody molar ratio to achieve the targeted drug-to-antibody ratio.