Department of BioMolecular Sciences, University of Mississippi, P.O. Box 1848, University, Oxford, Mississippi, 38677-1848, USA.
GenNext Technologies, Inc., Montara, California, 94037, USA.
AAPS J. 2019 Jul 11;21(5):87. doi: 10.1208/s12248-019-0358-2.
Unlike small molecule drugs, therapeutic proteins must maintain the proper higher-order structure (HOS) in order to maintain safety and efficacy. Due to the sensitivity of many protein systems, even small changes due to differences in protein expression or formulation can alter HOS. Previous work has demonstrated how hydroxyl radical protein footprinting (HRPF) can sensitively detect changes in protein HOS by measuring the average topography of the protein monomers, as well as identify specific regions of the therapeutic protein impacted by the conformational changes. However, HRPF is very sensitive to the radical scavenging capacity of the buffer; addition of organic buffers and/or excipients can dramatically alter the HRPF footprint without affecting protein HOS. By compensating for the radical scavenging effects of different adalimumab biosimilar formulations using real-time adenine dosimetry, we identify that sodium citrate buffer causes a modest decrease in average solvent accessibility compared to sodium phosphate buffer at the same pH. We find that the addition of polysorbate 80 does not alter the conformation of the biosimilar in either buffer, but it does provide substantial protection from protein conformational perturbation during short periods of exposure to high temperature. Compensated HRPF measurements are validated and contextualized by dynamic light scattering (DLS), which suggests that changes in adalimumab biosimilar aggregation are major drivers in measured changes in protein topography. Overall, compensated HRPF accurately measured conformational changes in adalimumab biosimilar that occurred during formulation changes and identified the effect of formulation changes on protection of HOS from temperature extremes.
与小分子药物不同，治疗性蛋白必须保持适当的高级结构（HOS），以保持安全性和有效性。由于许多蛋白质系统的敏感性，即使是由于蛋白质表达或配方的差异而导致的微小变化，也会改变 HOS。以前的工作已经证明了羟基自由基蛋白足迹法（HRPF）如何通过测量蛋白质单体的平均形貌来灵敏地检测蛋白质 HOS 的变化，以及识别治疗性蛋白质受构象变化影响的特定区域。然而，HRPF 对缓冲液的清除能力非常敏感；添加有机缓冲液和/或赋形剂会极大地改变 HRPF 足迹，而不会影响蛋白质 HOS。通过使用实时腺嘌呤剂量测定法补偿阿达木单抗生物类似药制剂的自由基清除效应，我们发现与相同 pH 值的磷酸盐缓冲液相比，柠檬酸钠缓冲液会导致平均溶剂可及性适度降低。我们发现聚山梨醇酯 80 的添加不会改变两种缓冲液中生物类似物的构象，但它确实在短时间暴露于高温下提供了对蛋白质构象扰动的实质性保护。补偿后的 HRPF 测量结果通过动态光散射（DLS）得到验证和背景化，这表明阿达木单抗生物类似物聚集的变化是测量蛋白质形貌变化的主要驱动因素。总的来说，补偿后的 HRPF 准确地测量了在配方变化过程中阿达木单抗生物类似物发生的构象变化，并确定了配方变化对 HOS 免受极端温度影响的保护作用。
