Pharmaceutical Research and Development, Biotherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, Missouri, 63017, USA.
Saha Institute of Nuclear Physics, Biophysics & Structural Genomics, 1/AF Biddhannagar Road, Kolkata, 700064, India.
Pharm Res. 2022 Mar;39(3):529-539. doi: 10.1007/s11095-022-03200-6. Epub 2022 Feb 16.
The viscosity of highly concentrated therapeutic monoclonal antibody (mAb) formulations at concentrations ≥ 100 mg/mL can significantly affect the stability, processing, and drug product development for subcutaneous delivery. An early identification of a viscosity prone mAb during candidate selection stages are often beneficial for downstream processes. Higher order structure of mAbs may often dictate their viscosity behavior at high concentration. Thus it is beneficial to gauge or rank-order their viscosity behavior using noninvasive structural fingerprinting methods and to potentially screen for suitable viscosity lowering excipients.
In this study, Dynamic Light Scattering (DLS) and 2D NMR based methyl fingerprinting were used to correlate viscosity behavior of a set of Pfizer mAbs. The viscosities of mAbs were determined. Respective Fab and Fc domains were generated for studies.
Methyl fingerprinting of intact mAbs allows for differentiation of viscosity prone mAbs from well behaved ones even at 30-40 mg/ml, where bulk viscosity of the solutions are near identical. For viscosity prone mAbs, peak broadening and or distinct chemical shift changes were noted in intact and fragment fingerprints, unlike the well-behaved mAbs, indicative of protein protein interactions (PPI).
Fab-Fab or Fab-Fc interactions may lead to formation of protein networks at high concentration. The early transients to these network formation may be manifested through peak broadening or peak shift in the 2D NMR spectrum of mAb/mAb fragments. Such insights go beyond rank ordering mAbs based on viscosity behavior, which can be obtained by other methods as well..
在浓度≥100mg/mL 的高浓度治疗性单克隆抗体（mAb）制剂的粘度会显著影响皮下给药的稳定性、处理和药物产品开发。在候选物选择阶段早期识别易产生粘度的 mAb 通常对下游工艺有益。mAbs 的高级结构通常决定其在高浓度下的粘度行为。因此，使用非侵入性结构指纹分析方法来评估或对其粘度行为进行排序，并可能筛选合适的降低粘度赋形剂是有益的。
在这项研究中，动态光散射（DLS）和基于 2D NMR 的甲基指纹分析用于关联一组辉瑞 mAb 的粘度行为。测定 mAb 的粘度。分别生成 Fab 和 Fc 结构域以进行研究。
完整 mAb 的甲基指纹分析能够将易产生粘度的 mAb 与表现良好的 mAb 区分开来，即使在 30-40mg/ml 时也是如此，此时溶液的整体粘度非常接近。对于易产生粘度的 mAb，在完整和片段指纹中都观察到峰展宽和/或明显的化学位移变化，与表现良好的 mAb 不同，表明存在蛋白质-蛋白质相互作用（PPI）。
Fab-Fab 或 Fab-Fc 相互作用可能导致在高浓度下形成蛋白质网络。这些网络形成的早期瞬态可能表现为 mAb/mAb 片段的 2D NMR 光谱中的峰展宽或峰位移。这些见解超越了基于粘度行为对 mAb 进行排序的方法，这些方法也可以通过其他方法获得。
