Assessment of IgG stability in a low pH elution buffer using ATR-FTIR spectroscopic imaging and microfluidics - Processed Data

Monoclonal antibodies (mAbs) represent the largest class of biopharmaceuticals, playing an vital role in the treatment of a wide range of diseases. Although the production of high quality mAbs has significantly improved over the last three decades, particularly in terms of scale and yield, the protein’s complex nature poses several challenges during bioprocessing. One of the main challenges in the production of mAbs is the formation of aggregates, which may cause harmful immunogenic responses in patients if not removed from the final drug product. Exposure to a low pH environment during protein A chromatography and viral inactivation is thought to be the major contributor to aggregate formation and has therefore been a topic of study for many years. Here, we investigate the stability of an IgG4 molecule in low pH elution buffer (pH 3.5) under flow using ATR-FTIR spectroscopic imaging. This method, making use of a microfluidic setup, enables non-destructive monitoring of the mAb’s structural stability under bioprocessing-relevant conditions. Samples were (i) prepared through dialysis into elution buffer, and (ii) collected directly after elution from the protein A column, after which their stability was assessed under flow at two different temperatures (30 °C or 45 °C). Spectroscopic images and associated IR absorption spectra revealed that in both cases the protein in low pH buffer underwent small, but measurable, structural changes at 30 °C. However, at 45 °C, the protein rapidly aggregated as indicated by a major shift in the Amide I peak position from 1637 cm-1 to 1625 cm-1, representing the formation of inter-molecular beta sheets. These results confirm the destabilising effect of the low pH environment and demonstrate the applicability of ATR-FTIR spectroscopic imaging in combination with microfluidics as a powerful analytical tool for the analysis of protein structural stability under flow.单克隆抗体（monoclonal antibodies, mAbs）是生物制药（biopharmaceuticals）领域规模最大的一类产品，在多种疾病的治疗中发挥着至关重要的作用。尽管高质量mAbs的生产在过去三十年中已有显著提升，尤其是在生产规模与产率方面，但该蛋白质的复杂特性仍给生物加工过程带来了诸多挑战。mAbs生产过程中的主要挑战之一是聚集体的形成，若最终药品未将其去除，可能会引发患者的有害免疫原性反应。研究表明，蛋白A层析（protein A chromatography）与病毒灭活（viral inactivation）环节中，蛋白质暴露于低pH环境是聚集体形成的主要诱因，因此该方向多年来一直是研究热点。本研究采用衰减全反射傅里叶变换红外光谱成像（ATR-FTIR spectroscopic imaging）技术，探究了免疫球蛋白G4（IgG4）分子在流动状态下，于pH 3.5的低pH洗脱缓冲液中的稳定性。该方法借助微流控装置（microfluidic setup），可在贴合生物加工过程的相关条件下，对mAbs的结构稳定性进行无损监测。实验样本通过两种方式制备：（i）经透析（dialysis）处理后转入洗脱缓冲液；（ii）直接从蛋白A层析柱（protein A column）洗脱后收集。随后在流动状态下，分别于30℃与45℃两种温度下对其稳定性进行评估。光谱成像与配套的红外吸收光谱结果显示，在30℃条件下，两种样本中的低pH缓冲液内蛋白质均发生了微小但可被检测到的结构变化。但在45℃条件下，蛋白质会快速聚集，表现为酰胺I峰（Amide I peak）的位置从1637 cm⁻¹显著偏移至1625 cm⁻¹，这一变化对应分子间β折叠（inter-molecular beta sheets）的形成。上述结果证实了低pH环境的去稳定化效应，同时证明了衰减全反射傅里叶变换红外光谱成像与微流控技术联用，可作为一种高效的分析工具，用于流动状态下蛋白质结构稳定性的检测分析。