MedImmune, Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA.
MedImmune, Purification Process Sciences, One MedImmune Way, Gaithersburg, MD 20878, USA.
J Chromatogr A. 2018 Jun 15;1554:45-60. doi: 10.1016/j.chroma.2018.03.060. Epub 2018 Apr 7.
We describe a comprehensive evaluation of 12 Protein A stationary phases for capture of biotherapeutics. We first examine the morphological properties of the stationary phases using a variety of orthogonal techniques including electron microscopy, particle sizing, pressure-flow behavior, and isocratic pulse response. A panel of nine proteins spanning a wide range of structures and biochemical properties was then used to assess equilibrium uptake, mass transport, dynamic binding capacity, and elution pH. Process performance and product quality were also examined under realistic bioprocess conditions using clarified mammalian cell culture broth. Equilibrium isotherms were found to be highly favorable, with equilibrium binding capacity for monoclonal and bispecific antibodies ranging from 47-100 mg/mL packed bed across all stationary phases tested. Effective pore diffusivities, D, were obtained by fitting the chromatography general rate model to breakthrough data. The fitted D values for monoclonal antibodies ranged from 1.1-5.7 × 10 cm/s. The stationary phases had high dynamic binding capacities for the model proteins. The highest dynamic capacities for monoclonal and bispecific antibodies were seen with MabSelect SuRe pcc and MabSelect PrismA, which ranged from 58-74 mg/mL packed bed at 4 min residence times. Product capture using clarified cell culture broth as a feedstock showed high yields and elution pool volumes that ranged from 2-3 column volumes in most cases. Host cell protein, DNA, and aggregate levels in the elution pool were dependent on the specific nature of protein being purified, and levels were consistent between stationary phases. Lastly, we perform an analysis of bivariate correlations and discuss considerations for process design and optimization.
我们描述了对 12 种 Protein A 固定相进行的综合评估，以用于捕获生物疗法。我们首先使用各种正交技术（包括电子显微镜、粒度分析、压力-流量行为和等度脉冲响应）来检查固定相的形态特性。然后，使用涵盖广泛结构和生化特性的九种蛋白质来评估平衡摄取量、质量传递、动态结合容量和洗脱 pH 值。在使用澄清的哺乳动物细胞培养物的实际生物工艺条件下，还检查了工艺性能和产品质量。平衡等温线非常有利，单克隆抗体和双特异性抗体在所有测试的固定相上的填充床平衡结合容量范围为 47-100mg/mL。通过将色谱一般速率模型拟合到突破数据，获得有效孔扩散系数 D。单克隆抗体的拟合 D 值范围为 1.1-5.7×10cm/s。这些固定相对模型蛋白具有高动态结合容量。MabSelect SuRe pcc 和 MabSelect PrismA 对单克隆抗体和双特异性抗体的最高动态容量最高，在 4min 停留时间下，填充床的容量分别为 58-74mg/mL。使用澄清的细胞培养物作为进料进行产物捕获显示出高收率和洗脱池体积，在大多数情况下范围为 2-3 个柱体积。洗脱池中的宿主细胞蛋白、DNA 和聚集体水平取决于要纯化的蛋白质的特定性质，并且在固定相之间保持一致。最后，我们进行了二元相关性分析，并讨论了工艺设计和优化的注意事项。
