Biosimilars offer an avenue for potential cost savings and enhanced patient access to various emerging therapies in a budget neutral way. Biosimilars of the granulocyte colony stimulating factor (GCSF) are an excellent example in this regard with as many as 18 versions of the drug being currently approved across globe for treatment of neutropenia. Here, we identified oxidation of the various methionine residues in GCSF as a key heterogeneity that adversely impact its efficacy. In agreement with earlier studies, it was found that oxidation of Met 122 and Met 127 significantly contributes toward reduction of GCSF efficacy, measured using binding affinity to the GCSF receptor. The combination of molecular dynamics simulation along with structural characterization studies established that oxidation of Met 127 and Met 122 brings about a small local conformational change around the B‐C loop in GCSF structure due to slight displacement of Asp113 and Thr117 residues. The simulation studies were validated using fluorescence quenching experiments using acrylamide as quencher and site‐directed mutagenesis by replacing Met 122 and Met 127 residues with alanine. The results of this study lead to an enhanced mechanistic understanding of the oxidation in GCSF and should be useful in protein engineering efforts to design stable, safe, and efficacious GCSF product. In addition, the structure‐function information can provide targets for protein engineering during early drug development and setting specifications of allowable limits of product variants in biosimilar products.

中文翻译：

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蛋氨酸氧化调节粒细胞集落刺激因子构象和受体结合。

生物仿制药为节省潜在成本和增加患者以预算中立方式获得各种新兴疗法的途径提供了途径。在这方面，粒细胞集落刺激因子（GCSF）的生物仿制药就是一个很好的例子，目前全球已批准多达18种药物用于中性粒细胞减少症的治疗。在这里，我们确定了GCSF中各种蛋氨酸残基的氧化是关键的异质性，不利于其功效。与较早的研究一致，发现使用与GCSF受体的结合亲和力测量，Met 122和Met 127的氧化显着有助于降低GCSF功效。分子动力学模拟与结构表征研究相结合，确定了Met 127和Met 122的氧化会由于Asp113和Thr117残基的轻微位移而在GCSF结构中的B-C环周围引起小的局部构象变化。通过使用丙烯酰胺作为猝灭剂和通过用丙氨酸替代Met 122和Met 127残基进行定点诱变的荧光猝灭实验，对模拟研究进行了验证。这项研究的结果使人们对GCSF中的氧化机理有了更深入的了解，应该对蛋白质工程设计稳定，安全和有效的GCSF产品有用。此外，