Pharmaceutical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, MO, 63017, USA.
Analytical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Andover, MA, 01810, USA.
Pharm Res. 2019 Jul 1;36(9):130. doi: 10.1007/s11095-019-2652-1.
An understanding of higher order structure (HOS) of monoclonal antibodies (mAbs) could be critical to predicting its function. Amongst the various factors that can potentially affect HOS of mAbs, chemical modifications that are routinely encountered during production and long-term storage are of significant interest.
To this end, two Pfizer mAbs were subjected to forced deamidation stress for a period of eight weeks. Samples were aliquoted at various time points and high resolution accurate mass liquid chromatography-mass spectrometry (LC-MS/MS) was performed using low-artifact trypsin digestion (LATD) peptide mapping to identify and quantify chemical modifications. 2D backbone amide and sidechain methyl NMR spectra were acquired to gauge the effect of HOS changes upon chemical modification. Differential scanning calorimetry was also performed to assess the effect of thermal stability of mAbs upon modification. Finally, functional studies via target-binding based ELISA were performed to connect HOS changes to any loss of potency.
The extent of deamidation in the mAb domains were quantified by LC-MS/MS. The HOS changes as obtained from 2D NMR were mostly localized around the affected sites leaving the overall structure relatively unchanged. The antigen-antibody binding of the mAbs, in spite of deamidation in the Fab region, remains unchanged.
This case study provides an integrated approach of relating chemical modifications in mAb domains with possible changes in HOS. This can be potentially used to assess a possible loss of potency within the structure-function paradigm of proteins in an orthogonal manner.
了解单克隆抗体 (mAb) 的高级结构 (HOS) 对于预测其功能可能至关重要。在可能影响 mAb 的 HOS 的各种因素中，在生产和长期储存过程中经常遇到的化学修饰是非常重要的。
为此，对辉瑞的两种 mAb 进行了为期八周的强制脱酰胺应激处理。在各个时间点等分样品，并使用低伪影胰蛋白酶消化 (LATD) 肽图进行高分辨率精确质量液相色谱-质谱 (LC-MS/MS) 分析，以鉴定和定量化学修饰。获得二维骨架酰胺和侧链甲基 NMR 谱，以评估 HOS 变化对化学修饰的影响。还进行了差示扫描量热法 (DSC) 以评估 mAb 修饰后的热稳定性变化。最后，通过基于靶标结合的 ELISA 进行功能研究，将 HOS 变化与任何效力损失联系起来。
通过 LC-MS/MS 定量了 mAb 结构域中的脱酰胺程度。从 2D NMR 获得的 HOS 变化主要集中在受影响的部位，使整体结构相对不变。尽管 Fab 区域发生了脱酰胺，但 mAb 的抗原-抗体结合保持不变。
该案例研究提供了一种将 mAb 结构域中的化学修饰与 HOS 可能发生的变化联系起来的综合方法。这可以潜在地用于以正交方式评估蛋白质结构-功能范式中可能发生的效力损失。
