创新的下一代单克隆抗体使用活性炭纯化:对流过滤和无柱工艺的挑战。
Innovative next-generation monoclonal antibody purification using activated carbon: A challenge for flow-through and column-free processes.
机构信息
Bioprocess Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., Takasaki, Gunma 370-0013, Japan.
Bioprocess Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., Takasaki, Gunma 370-0013, Japan.
出版信息
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Jul 15;1121:72-81. doi: 10.1016/j.jchromb.2019.05.009. Epub 2019 May 10.
Activated carbon (AC) is a porous solid with a larger surface area and lower cost than chromatography resins. AC has been used in the field of biopharmaceutical manufacturing for plasma-derived products and recombinant monoclonal antibodies (mAb). In our previous study, AC was employed in the purification process of therapeutic mAb as a replacement for Protein A affinity chromatography (PrA). In addition, we designed an all flow-through purification process using AC. In these investigations, greater effective clearance of high-molecular-weight species (HMW), low-molecular-weight species (LMW), host cell proteins (HCP), and DNA was observed compared to that of the conventional Protein A platform purification process. However, it was revealed that mAb recovery from the AC step was lower than that from the PrA step. In this work, to improve mAb recovery from the AC step while maintaining the effective removal of impurities, a pretreatment procedure conducted prior to the AC treatment was investigated. We found that both an ultrafiltration/dilution and reduction in the conductivity of the filtered cell culture supernatant after acid precipitation could improve both the impurity clearance and mAb recovery from the AC treatment. From the obtained results, we designed a two-step purification process in which AC treatment is followed by either cation exchange column chromatography or anion exchange column chromatography, and we compared this against the Protein A platform purification process. Excellent impurity clearance was achieved, even in the one-column process. Furthermore, we designed an innovative column-free flow-through purification process based on acid precipitation, clarification, ultrafiltration/dilution, and the implementation of an AC filter membrane and an anion exchange chromatography membrane. With this process in the pilot-scale, HCP level can be reduced to below 10 ng/mg, and HMW and LMW can be removed to below 1% while improving mAb recovery. From these results, it is strongly expected that AC is a promising candidate for the next generation of mAb purification processes to improve the economy and efficiency of the process.
活性炭(AC)是一种多孔固体,比层析树脂具有更大的表面积和更低的成本。AC 已在生物制药领域用于血浆衍生产品和重组单克隆抗体(mAb)。在我们之前的研究中,AC 被用于治疗性 mAb 的纯化过程中,以替代蛋白 A 亲和层析(PrA)。此外,我们设计了一种全流通过滤纯化工艺,使用 AC。在这些研究中,与传统的蛋白 A 平台纯化工艺相比,观察到对高分子量物质(HMW)、低分子量物质(LMW)、宿主细胞蛋白(HCP)和 DNA 的有效清除率更高。然而,发现从 AC 步骤回收 mAb 的效率低于从 PrA 步骤回收的效率。在这项工作中,为了在保持有效去除杂质的同时提高从 AC 步骤回收 mAb 的效率,研究了在 AC 处理之前进行预处理的程序。我们发现,超滤/稀释和降低酸沉淀后过滤的细胞培养上清液的电导率都可以提高 AC 处理的杂质清除率和 mAb 回收率。从获得的结果中,我们设计了两步纯化工艺,其中 AC 处理后依次进行阳离子交换柱层析或阴离子交换柱层析,并将其与蛋白 A 平台纯化工艺进行了比较。即使在单柱工艺中,也实现了出色的杂质清除率。此外,我们设计了一种基于酸沉淀、澄清、超滤/稀释以及实施 AC 过滤膜和阴离子交换层析膜的创新无柱流通过滤纯化工艺。在中试规模下,该工艺可将 HCP 水平降低至 10ng/mg 以下,并可去除 HMW 和 LMW 至 1%以下,同时提高 mAb 回收率。从这些结果可以强烈预期,AC 是下一代 mAb 纯化工艺的有前途的候选者,可以提高工艺的经济性和效率。