R&D Cell Engineering, Lonza Biologics, Little Chesterford, UK.
Cell Line Development, Biotherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, MO, 63017, USA.
Biotechnol Prog. 2021 Jul;37(4):e3137. doi: 10.1002/btpr.3137. Epub 2021 Mar 23.
There are an ever-increasing number of biopharmaceutical candidates in clinical trials fueling an urgent need to streamline the cell line development process. A critical part of the process is the methodology used to generate and screen candidate cell lines compatible with GMP manufacturing processes. The relatively large amount of clone phenotypic variation observed from conventional "random integration" (RI)-based cell line construction is thought to be the result of a combination of the position variegation effect, genome plasticity and clonal variation. Site-specific integration (SSI) has been used by several groups to temper the influence of the position variegation effect and thus reduce variability in expression of biopharmaceutical candidates. Following on from our previous reports on the application of the Fer1L4 locus for SSI in CHOK1SV (10E9), we have combined this locus and a CHOK1SV glutamine synthetase knockout (GS-KO) host to create an improved expression system. The host, CHOK1SV GS-KO SSI (HD7876), was created by homology directed integration of a targetable landing pad flanked with incompatible Frt sequences in the Fer1L4 gene. The targeting vector contains a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line. SSI clones expressing four antibody candidates, selected in a streamlined cell line development process, have mAb titers which rival RI (1.0-4.5 g/L) and robust expression stability (100% of clones stable through the 50 generation "manufacturing window" which supports commercial manufacturing at 12,000 L bioreactor scale).
临床试验中生物制药候选药物的数量不断增加，这迫切需要简化细胞系开发过程。该过程的一个关键部分是用于生成和筛选与 GMP 制造工艺兼容的候选细胞系的方法。从传统的基于“随机整合”（RI）的细胞系构建中观察到的相对较大数量的克隆表型变异被认为是位置变异效应、基因组可塑性和克隆变异的综合结果。几位研究人员已经使用定点整合（SSI）来缓和位置变异效应的影响，从而降低生物制药候选物表达的可变性。在我们之前关于 Fer1L4 基因座在 CHOK1SV（10E9）中用于 SSI 的应用的报告之后，我们将该基因座与 CHOK1SV 谷氨酰胺合成酶敲除（GS-KO）宿主结合，创建了一个改进的表达系统。该宿主，CHOK1SV GS-KO SSI（HD7876），是通过靶向 Fer1L4 基因侧翼带有不相容 Frt 序列的着陆垫进行同源定向整合而创建的。该靶向载体包含无启动子的 GS 表达盒和单克隆抗体（mAb）表达盒，两侧是 Frt 位点，与宿主细胞系中着陆垫两侧的等效位点兼容。通过简化的细胞系开发过程选择的表达四种抗体候选物的 SSI 克隆，其 mAb 滴度可与 RI（1.0-4.5 g/L）相媲美，并且具有稳健的表达稳定性（在支持 12,000 L 生物反应器规模商业生产的 50 代“制造窗口”中，100%的克隆稳定）。
