化学
TCEP
色谱法
二硫键
肽
争先恐后
洗脱
柱色谱法
甘氨酸
二硫键
组合化学
半胱氨酸
氨基酸
磷化氢
生物化学
计算机科学
酶
催化作用
算法
作者
Andrew Kleinberg,Rachel Joseph,Yuan Mao,Ning Li
标识
DOI:10.1016/j.ab.2022.114773
摘要
Explicitly confirming the complete disulfide bond linkage pattern of a monoclonal antibody (mAb) presents a challenge in the biopharmaceutical industry. Although proper native disulfide connections are in high abundance for analytical purposes within a peptide mapping digest under non-reducing conditions, disulfide scrambling can also exist but be difficult to detect, let alone characterize, particularly at low levels. Here, we developed an ultrasensitive high-confidence method for identifying explicit disulfide connectivity in mAbs. By applying a post-column addition of tris (2-carboxyethyl)phosphine hydrochloride (TCEP) to the liquid chromatography (LC) eluent of a non-reduced mAb digest, partial reduction of disulfide peptides is achieved after the initial peptide separation, allowing both the parent disulfide and its reduced daughter peptides to co-elute for simultaneous mass spectrometry (MS) detection. Combining this concept with the recently discovered ability of glycine to enhance MS signal when added to the LC eluent, we demonstrate a method for detecting, characterizing and quantifying low-abundance disulfide scrambling in mAbs.
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