Analytical Chemistry, Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, United States.
Analytical Chemistry, Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, United States.
Anal Biochem. 2022 Sep 15;653:114773. doi: 10.1016/j.ab.2022.114773. Epub 2022 Jun 7.
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.
明确确认单克隆抗体 (mAb) 的完整二硫键连接模式是生物制药行业面临的一项挑战。尽管在非还原条件下肽图消化物中适当的天然二硫键连接对于分析目的而言含量很高，但二硫键重排也可能存在，但难以检测，更不用说表征了，尤其是在低水平下。在这里，我们开发了一种用于鉴定 mAb 中明确二硫键连接的超灵敏高置信度方法。通过在非还原 mAb 消化物的 LC 洗脱液中添加三(2-羧乙基)膦盐酸盐 (TCEP)，在初始肽分离后实现二硫肽的部分还原，允许母体二硫键及其还原的子肽共同洗脱，以便同时进行质谱 (MS) 检测。将这一概念与最近发现的在 LC 洗脱液中添加甘氨酸可增强 MS 信号的能力相结合，我们展示了一种用于检测、表征和定量 mAb 中低丰度二硫键重排的方法。
