Research and Early Development, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States.
Anal Chem. 2020 Jun 16;92(12):8584-8590. doi: 10.1021/acs.analchem.0c01567. Epub 2020 Jun 7.
A current trend in drug development involves the use of high molecular weight, branched, and functionalized polymers for protein conjugation and drug delivery. Accurately characterizing these polymers is critical to control the product quality, to monitor the stability, and ultimately to ensure the drug efficacy and patient safety. However, due to the heterogeneity in size, the multiplicity of functional groups, and the highly convoluted charge-distribution profile in mass spectra, the characterization of these polymers is highly challenging from both chromatography and mass spectrometry perspectives. To overcome these challenges, we developed a strategy utilizing charge-reduction mass spectrometry (CRMS) coupled with two-dimensional HPLC (2D-LC). We then applied the workflow to characterize a 40 kDa 8-arm polyethylene glycol (PEG) functionalized with a maleimide terminal group for protein conjugation. The development was carried out in stages, where first we focused on the development of a CRMS method to simplify the charge profile of the polymers and then coupled it to HPLC to obtain discernible mass spectra of key impurities and degradants. Second, the CRMS method was applied to an investigation of the size-variant impurity resolved by reversed-phase size-exclusion 2D-LC. Finally, a separate size-exclusion reversed-phase 2D-LC-CRMS method was developed to capture a wider range of process-related impurities and reaction intermediates from the PEG-maleimide polymers throughout the conjugation process. The combination of these experiments using the 2D-LC-CRMS strategy enables the sensitive characterization of the entire impurity profile of the high molecular weight multifunctionalized PEG-maleimide conjugation intermediate.
目前药物开发的一个趋势是使用高分子量、支化和官能化聚合物进行蛋白质偶联和药物递送。准确地表征这些聚合物对于控制产品质量、监测稳定性以及最终确保药物疗效和患者安全至关重要。然而，由于这些聚合物在尺寸上存在异质性、官能团的多样性以及在质谱中高度卷曲的电荷分布轮廓，从色谱和质谱的角度来看，其表征极具挑战性。为了克服这些挑战，我们开发了一种利用电荷还原质谱（CRMS）与二维高效液相色谱（2D-LC）相结合的策略。然后，我们将该工作流程应用于表征用于蛋白质偶联的 40 kDa 8 臂聚乙二醇（PEG），其末端带有马来酰亚胺基团。该开发分阶段进行，首先我们专注于开发 CRMS 方法以简化聚合物的电荷分布，然后将其与 HPLC 结合以获得关键杂质和降解物的可分辨质谱。其次，将 CRMS 方法应用于反相尺寸排阻 2D-LC 解析的尺寸变异杂质的研究。最后，开发了单独的尺寸排阻反相 2D-LC-CRMS 方法，以捕获整个偶联过程中 PEG-马来酰亚胺聚合物中更广泛的与过程相关的杂质和反应中间体。使用 2D-LC-CRMS 策略进行这些实验的组合，能够对高分子量多功能化 PEG-马来酰亚胺偶联中间体的整个杂质分布进行敏感表征。
