Signal Peptide Optimization to Prevent N -terminal Truncation of Glucagon Like Peptide-1/IgG-Fc Fusion Protein
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Signal Peptide Optimization to Prevent N-terminal Truncation of Glucagon Like Peptide-1/IgG-Fc Fusion Protein Chunlai Cao1,2 · Suzhen Wei2 · Xukun Xu2 · Suqin Song2 · Yongjie Lai3 · Jing Li2 Received: 26 May 2020 / Revised: 11 August 2020 / Accepted: 22 August 2020 © Springer Nature B.V. 2020
Abstract Dulaglutide (glucagon like peptide-1/IgG-Fc fusion protein, GLP-1-Fc) is a long lasting GLP-1 agonist, which consists of two arms of GLP-1 moieties fused to IgG Fc fragment. Dulaglutide is a safe and effective medication for type 2 diabetes. In an attempt to develop a biosimilar version of dulaglutide, we found that up to 75% of GLP-1-Fc displayed N-terminal truncations in one or both GLP-1 arms. We proposed that the N-terminal heterogeneity was caused by mis-cleavage of signal peptide and solved this problem through signal peptide optimization. Murine immunoglobulin kappa light chain signal peptide (KASP) significantly improves GLP-1-Fc N-terminal integrity and homogeneity. 92.8–95.7% of GLP-1-Fc molecules directed by KASP contain intact N-terminus. The productivity of GLP-1-Fc could reach 2.2 g/L in shaking flask fed batch culture. KASP is an optimal signal peptide for GLP-1-Fc expression in Chinese hamster ovary (CHO) cells. Keywords Biosimilar · Cleavage · Glucagon like peptide · Signal peptide · Truncation
Introduction Glucagon-like peptide-1 (GLP-1) is an incretin hormone which plays an important role in glucose-dependent insulin secretion and β-cell growth. The initial in vivo product of GLP-1 (1–37) (amino acid sequence: HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG) is susceptible to proteolytic cleavage which yields two truncated and equipotent biologically active forms, GLP-1 (7–36) amide (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR) and GLP-1 (7–37) (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG) Chunlai Cao and Suzhen Wei have contributed equally. * Yongjie Lai [email protected] * Jing Li [email protected] 1
Faculty of Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau SAR, China
2
Zhuhai United Laboratories Co., Ltd, 2428 Anji Road, Zhuhai, Guangdong, China
3
Department of Microbiology and Immunology, Zunyi Medical University (Zhuhai Campus), 368 Golden Coast Avenue, Zhuhai, Guangdong, China
(Campbell and Drucker 2013). GLP-1 (7–36) and GLP-1 (7–37) molecules are susceptible to dipeptidyl peptidase IV (DPP-IV), which cleaves specifically between Ala8-Glu9 (Lambeir et al. 2003). This specific cleavage results in the abundant GLP-1 (9–36) amide constituting 60% to 80% of total GLP-1 in circulation. The half-lives of GLP-1 (7–36) amide and GLP-1 (7–37) are approximately 1–2 min in blood circulation. The two amino acids in the N-terminus are critical for GLP-1 biological activity. GLP-1 (9–36) acts as an antagonist at the human GLP-1 receptor (Elahi et al. 2008; Knudsen and Pridal 1996). Amino acid mutations were made to extend biological half-life of GLP-1. Substitution of Ala8 with Gly, Ser, Thr or Aib confers DPPIV resistance to GLP-1. As for receptor binding activity, only Gl
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