Global Product Development and Supply, Bristol-Myers Squibb Company , Devens , MA , USA.
Department of Chemical & Biological Engineering, Polytechnic Institute , Troy , NY , USA.
MAbs. 2019 Nov-Dec;11(8):1502-1514. doi: 10.1080/19420862.2019.1652075. Epub 2019 Aug 19.
Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 10 cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2-10 × 10 cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22-34 × 10 cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3-6 × 10 cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.
尽管通过连续操作进行的过程强化已成功应用于化学工业，但生物制药行业主要使用分批补料，而不是连续或灌注方法，从中国仓鼠卵巢（CHO）细胞生产稳定的单克隆抗体（mAb）。传统的分批补料生物反应器的接种活细胞密度（VCD）可能约为 0.5×10^6 个细胞/ml。通过在生产生物反应器之前的种子步骤（N-1 步骤）中引入灌注操作或过程强化，将接种 VCD 提高到 2-10×10^6 个细胞/ml，最近已被用于增加制造产量，缩短细胞培养生产时间。在这项研究中，我们报告说，在相同的 14 天内，通过 3 种 CHO GS 细胞系生产的 3 种 mAb 的分批补料生产中，增加接种 VCD 可显著提高最终滴度。我们还报告说，在 N-1 步骤中使用分批或补料方式的其他非灌注方法，同时使用富含培养基，可以同样实现高达 22-34×10^6 个细胞/ml 的高 N-1 最终 VCD。这些非灌注 N-1 种子支持以 3-6×10^6 个细胞/ml 的增加接种 VCD 接种随后的生产补料分批生产生物反应器，在这两种情况下，在 5-L 生物反应器中，以及在放大到 500-L 和 1000-L N 阶段生物反应器中，达到的滴度和产品质量属性与使用灌注 N-1 种子接种的结果相当。为了使用分批模式操作 N-1 步骤，在 N-1 和随后的强化分批生产步骤中，基础培养基的富集是至关重要的。与需要灌注设备以及用于容纳大量灌注培养基的准备和储存容器的灌注 N-1 种子相比，这里报道的非灌注 N-1 方法在操作上是用于工艺开发、工艺表征和大规模商业生产的更简单替代方法。虽然本研究仅使用了 3 种由 CHO 细胞培养物生产的稳定 mAb，但缩短种子培养和生产培养时间或提高滴度的非灌注 N-1 种子策略的基本原则应适用于任何规模的生物制品设施中由不同哺乳动物细胞和其他宿主生产的其他蛋白质。
