琥珀酰亚胺(Asu)是治疗性蛋白质中天冬酰胺脱酰胺的中间体,并且它可以容易地水解形成天冬氨酸和异天冬氨酸残基。此外,Asu在蛋白质降解途径中起着重要作用,天冬酰胺脱酰胺,和天冬氨酸异构化。这里,首先报道了戈利木单抗框架区(FR)中高丰度的Asu修饰,研究了变性缓冲液pH值对Asu修饰稳态的影响,结果表明,它在6.0-7.0的pH范围内相对稳定,而在pH8.0时迅速下降。然后,基于肽的多属性方法(MAM)分析显示Asu形成在重链FR中的Asn43处。同时,功效[亲和力,结合和生物活性,补体依赖性细胞毒性(CDC)活性,和抗体依赖性细胞介导的细胞毒性(ADCC)活性]评估了戈利木单抗的Asu修饰的稳定性,目前的结果表明,Asu低丰度和高丰度组之间的疗效和稳定性相当。我们的发现为Asu修饰及其对功效和稳定性的影响提供了有价值的见解,这项研究还表明,有必要开发一种广谱的,快速,和准确的平台来识别和表征治疗性蛋白质开发中的新峰,特别是抗体药物。
Succinimide (Asu) is the intermediate for asparagine deamidation in therapeutic proteins, and it can be readily hydrolyzed to form aspartate and iso-aspartate residues. Moreover, Asu plays an important role in the protein degradation pathways, asparagine deamidation, and aspartic acid isomerization. Here, Asu modification with a high abundance in the framework region (FR) of golimumab was first reported, the effect of denaturing buffer pH on the Asu modification homeostasis was studied, and the results revealed that it was relatively stable over a pH range of 6.0-7.0 whereas a rapid decrease at pH 8.0. Then, the peptide-based multi-attribute method (MAM) analyses showed that the Asu formation was at Asn 43 in the FR of the heavy chain. Meanwhile, the efficacy [affinity, binding and bioactivity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated cytotoxicity (ADCC) activity] and stability of the Asu modification of golimumab were evaluated, and the current results demonstrated comparable efficacy and stability between the Asu low- and high-abundance groups. Our findings provide valuable insights into Asu modification and its effect on efficacy and stability, and this study also demonstrates that there is a need to develop a broad-spectrum, rapid, and accurate platform to identify and characterize new peaks in the development of therapeutic proteins, particularly for antibody drugs.