Swartz Andrew R, Shieh Yvonne, Gulasarian Amanda, Curtis Erik, Hofmann Carl F, Baker Jack B, Templeton Neil, Olson Jessica W
Process Research and Development, Merck & Co., Inc., Rahway, NJ, United States.
Analytical Research and Development, Merck & Co., Inc., Rahway, NJ, United States.
Front Bioeng Biotechnol. 2023 Jun 15;11:1193454. doi: 10.3389/fbioe.2023.1193454. eCollection 2023.
Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two-three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250 L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5 L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.
治疗性病毒载体是一种新兴技术，在基因治疗、疫苗和免疫治疗中有多种临床应用。需求的增加促使人们重新开发传统的、低通量的细胞培养和纯化生产方法，如静态细胞堆叠和超速离心。在这项工作中，研究了可扩展的方法来生产一种溶瘤病毒免疫疗法产品，该产品由在贴壁MRC-5细胞中产生的柯萨奇病毒A21（CVA21）原型毒株组成。在搅拌罐微载体生物反应器中建立细胞培养，并开发了一种高效的亲和色谱方法，通过病毒衣壳与固定化谷胱甘肽（GSH）配体的结合来纯化收获的CVA21。研究了感染期间生物反应器的温度以最大化滴度，温度从37°C降至34°C可使感染性提高两到三倍。纯化34°C收获物后，GSH亲和色谱洗脱不仅使感染性和病毒基因组增加了两倍以上，而且与37°C收获物相比，空衣壳的比例也增加了。使用从两个感染温度设定点产生的材料在实验室规模研究色谱参数和流动相组成，以最大化感染性颗粒产量和清除细胞培养杂质。从34°C感染温度收获物中与完整衣壳共洗脱的空衣壳在测试的各种条件下分辨率较差，但随后开发了抛光阴离子交换和阳离子交换色谱步骤以清除残留的空衣壳和其他杂质。溶瘤CVA21的生产从实验室规模扩大了75倍，并在250 L一次性微载体生物反应器中进行了七批生产，并用定制的、预装的一次性1.5 L GSH亲和色谱柱进行纯化。感染期间控制在34°C的大规模生物反应器在GSH洗脱中保持了三倍的生产率提高，并且在所有批次中均观察到宿主细胞和培养基杂质的出色清除。本研究提出了一种用于生产溶瘤病毒免疫疗法产品的稳健方法，该方法可用于可扩展生产其他与谷胱甘肽相互作用的病毒和病毒载体。
