Next‐Generation Glutamine Synthetase Expression Vector for Therapeutic Protein Manufacturing Using CHO Cells

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology and Bioengineering Pub Date : 2026-10-06 DOI:10.1002/bit.70405

Yusuf B. Johari, Sophie J. Wensley, Malak A. Ammar, Avinash Srivastava, Changhavi Ragurajan, Jenish Patel, Natalie C. Elliott, Alice C. Hodgson‐Casson, Mariano Olivera Fedi, Michael Anbar, Peter M. O'Callaghan, Bernie Sweeney

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引用次数: 0

Abstract

The glutamine synthetase expression system is widely used for therapeutic protein biomanufacturing in Chinese hamster ovary (CHO) cells. Here, we present the design of an enhanced glutamine synthetase vector for generating CHOK1SV GS‐KO stable pools that produce recombinant proteins at multigram‐per‐liter levels. We first developed a synthetic promoter, LHP‐1, for high‐level constitutive recombinant gene transcription, achieving up to 123% increase in titer across monoclonal and bispecific antibody formats compared to the viral CMV promoter. We then demonstrate, for the first time, that the bacterial origin of replication (Ori) can function as a core promoter to drive glutamine synthetase selection in CHO cells. In this novel glutamine synthetase vector, Ori and LHP‐1 each play dual roles: Ori acts as both the plasmid replication origin and a minimal glutamine synthetase promoter, while LHP‐1 drives product expression and optimal enhancement of glutamine synthetase activity without the use of methionine sulfoximine (MSX) supplementation. Together, these two elements further increased product titers by up to 73% compared to the vector harboring the SV40 promoter to drive glutamine synthetase gene expression with 50 µM MSX. Notably, the LHP‐1 Ori‐GS system effectively maintained high bulk pool titers of up to 5.7 g/L with no decline after 30 days—supporting consistent product yield and quality during culture scale‐up for toxicology material supply. Further cell line development showed that clones derived from the LHP‐1 Ori‐GS pool achieved a 23% higher median titer while maintaining long‐term expression stability compared to those generated with the SV40‐GS vector, with the top‐performing Ori‐GS clone reaching 8.3 g/L. Overall, the MSX‐free LHP‐1 Ori‐GS system offers a robust platform for both rapid, high‐yield material generation from bulk pools and efficient progression to high‐producing clonal cell lines.

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下一代谷氨酰胺合成酶表达载体用于CHO细胞的治疗性蛋白制造

谷氨酰胺合成酶表达系统被广泛用于中国仓鼠卵巢（CHO）细胞的治疗性蛋白生物制造。在这里，我们提出了一种增强型谷氨酰胺合成酶载体的设计，用于生成CHOK1SV GS‐KO稳定池，以每升数克的水平产生重组蛋白。我们首先开发了一种合成启动子LHP‐1，用于高水平组成重组基因转录，与病毒CMV启动子相比，在单克隆和双特异性抗体格式中，其滴度提高了123%。然后，我们首次证明，细菌复制起源（Ori）可以作为核心启动子，在CHO细胞中驱动谷氨酰胺合成酶选择。在这种新的谷氨酰胺合成酶载体中，Ori和LHP‐1各自发挥双重作用：Ori既是质粒复制起点，也是最低限度的谷氨酰胺合成酶启动子，而LHP‐1在不使用蛋氨酸亚砜胺（MSX）补充的情况下驱动产物表达并优化谷氨酰胺合成酶活性。与含有SV40启动子驱动谷氨酰胺合成酶基因表达的载体相比，这两个元件共同进一步提高了产品滴度，最高可达73%。值得注意的是，LHP‐1 Ori‐GS系统有效地保持了高达5.7 g/L的大量池滴度，30天后没有下降，这在毒理学材料的培养规模扩大期间支持了一致的产品产量和质量。进一步的细胞系发育表明，与SV40‐GS载体产生的克隆相比，LHP‐1 Ori‐GS克隆的中位滴度提高了23%，同时保持了长期的表达稳定性，表现最好的Ori‐GS克隆达到8.3 g/L。总的来说，不含MSX的LHP - 1 Ori - GS系统提供了一个强大的平台，既可以快速、高产地从大量池中生成材料，又可以高效地向高产克隆细胞系过渡。

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来源期刊

Biotechnology and Bioengineering 工程技术-生物工程与应用微生物

CiteScore

7.90

自引率

5.30%

发文量

280

审稿时长

2.1 months

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