正辛酸沉淀在单克隆抗体纯化中的工艺优化与应用

Optimization and application of caprylic acid precipitation in the purification of monoclonal antibody

摘要:

为应对治疗性抗体快速增长的市场需求，抗体上游细胞培养规模和表达量水平已显著提高，而下游纯化工艺的生产效率则相对落后，下游处理能力已成为限制抗体产能的瓶颈。本研究以单克隆抗体mab-X为实验材料，优化了细胞培养液、低pH病毒灭活收集液2种模式的正辛酸(caprylic acid, CA)沉淀工艺条件，并研究了CA处理去除聚体、CA处理灭活病毒等2种应用，在小试的基础上，采用低pH病毒灭活收集液CA沉淀的模式进行了500 L细胞培养规模生产放大研究，对沉淀前后的产品质量和收率进行了检测和对比。结果显示，两种模式的CA沉淀均可显著降低宿主细胞蛋白(host cell protein, HCP)残留和聚体含量，在聚体去除实验中CA沉淀可去除约15%的聚体，病毒灭活研究显示CA对逆转录模型病毒具有完全的病毒灭活能力。在放大生产规模中，下游依次进行了深层过滤收获、亲和层析、低pH病毒灭活、CA沉淀及深层过滤、阳离子交换层析，CA沉淀过程中混合时间和搅拌速度显著影响CA沉淀效果，CA沉淀处理后低pH病毒灭活液中的HCP残留量降低了895倍，沉淀后产品纯度和HCP残留均已控制在单克隆抗体质量要求范围内，CA沉淀可以减少传统纯化工艺中的一个精纯步骤。总之，下游工艺中采用CA沉淀，能够精简传统纯化工艺，并完全满足mab-X的纯化质量要求，而且能提高生产效率、降低生产成本。本研究结果将推动CA沉淀在单克隆抗体下游纯化生产中的应用，为解决目前传统纯化工艺的问题提供参考。

Abstract:

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.