Biologics Process Development, Biologics Process Research and Development, Merck & Co., Inc., Rahway, New Jersey, USA.
Biologics Analytical Research and Development, Merck & Co., Inc., Rahway, New Jersey, USA.
Biotechnol Bioeng. 2023 Jul;120(7):1902-1913. doi: 10.1002/bit.28416. Epub 2023 May 6.
Precipitation can be used for the removal of impurities early in the downstream purification process of biologics, with the soluble product remaining in the filtrate through microfiltration. The objective of this study was to examine the use of polyallylamine (PAA) precipitation to increase the purity of product via higher host cell protein removal to enhance polysorbate excipient stability to enable a longer shelf life. Experiments were performed using three monoclonal antibodies (mAbs) with different properties of isoelectric point and IgG subclass. High throughput workflows were established to quickly screen precipitation conditions as a function of pH, conductivity and PAA concentrations. Process analytical tools (PATs) were used to evaluate the size distribution of particles and inform the optimal precipitation condition. Minimal pressure increase was observed during depth filtration of the precipitates. The precipitation was scaled up to 20L size and the extensive characterization of precipitated samples after protein A chromatography showed >75% reduction of host cell protein (HCP) concentrations (by ELISA), >90% reduction of number of HCP species (by mass spectrometry), and >99.8% reduction of DNA. The stability of polysorbate containing formulation buffers for all three mAbs in the protein A purified intermediates was improved at least 25% after PAA precipitation. Mass spectrometry was used to obtain additional understanding of the interaction between PAA and HCPs with different properties. Minimal impact on product quality and <5% yield loss after precipitation were observed while the residual PAA was <9 ppm. These results expand the toolbox in downstream purification to solve HCP clearance issues for programs with purification challenges, while also providing important insights into the integration of precipitation-depth filtration and the current platform process for the purification of biologics.
沉淀可以在生物制品下游纯化过程的早期用于去除杂质，可溶性产物通过微滤留在滤液中。本研究的目的是研究聚烯丙胺（PAA）沉淀的使用，通过去除更高的宿主细胞蛋白来提高产物的纯度，以增强聚山梨酯赋形剂的稳定性，从而延长保质期。使用具有不同等电点和 IgG 亚类特性的三种单克隆抗体（mAb）进行实验。建立了高通量工作流程，以快速筛选 pH、电导率和 PAA 浓度作为函数的沉淀条件。过程分析工具（PAT）用于评估颗粒的粒径分布，并为最佳沉淀条件提供信息。在沉淀的深度过滤过程中观察到最小的压力增加。沉淀放大到 20L 规模，在经过蛋白 A 层析后对沉淀样品进行广泛的表征表明，宿主细胞蛋白（HCP）浓度降低了>75%（通过 ELISA），HCP 种类减少了>90%（通过质谱法），DNA 减少了>99.8%。在 PAA 沉淀后，所有三种 mAb 的蛋白 A 纯化中间体中含有聚山梨酯的制剂缓冲液的稳定性至少提高了 25%。质谱法用于获得对具有不同特性的 PAA 和 HCP 之间相互作用的更多了解。沉淀后观察到对产品质量的影响最小，<5%的产率损失，而残留的 PAA<9ppm。这些结果扩展了下游纯化工具包，以解决具有纯化挑战的项目中的 HCP 清除问题，同时还为沉淀-深度过滤的集成和生物制品纯化的当前平台工艺提供了重要的见解。
