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CC BY 4.0 · Pharmaceutical Fronts
DOI: 10.1055/a-2642-2305
Original Article

High-Yield Biosynthesis Process and Characterization of Brolucizumab

Xiaoyue Hu
1   National Key Laboratory of Lead Drugability Research, China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
,
Shuaiyi Huang
1   National Key Laboratory of Lead Drugability Research, China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
,
Tao Luo
1   National Key Laboratory of Lead Drugability Research, China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
,
Yapeng Wang
1   National Key Laboratory of Lead Drugability Research, China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
2   Shanghai Duomirui Biotech Co., Ltd., Shanghai, People's Republic of China
,
Jianguang Lu
1   National Key Laboratory of Lead Drugability Research, China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
2   Shanghai Duomirui Biotech Co., Ltd., Shanghai, People's Republic of China
,
Jun Feng
1   National Key Laboratory of Lead Drugability Research, China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China
› Author Affiliations
Funding None.
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Abstract

Brolucizumab was the first single-chain fragment variable (scFv) antibody approved by the FDA for the treatment of age-related macular degeneration (AMD). However, the manufacturing process of brolucizumab remains rarely reported. This study aimed to explore a bioprocess for the production of brolucizumab, where it is expressed as inclusion bodies (IBs) in Escherichia coli (E. coli) BL21 (DE3) cells. In this work, IBs were initially obtained via high cell density fermentation (HCDF) at a high expression level of 30 g/L, followed by denaturation, refolding, and purification to obtain brolucizumab. The refolding parameters were systematically optimized to ensure a high yield of brolucizumab, with 413 mg of the target protein from a 1-L fermentation broth, and purity exceeding 98%. In addition, the amino acid sequence coverage and disulfide bond pairing of the protein were further verified. The results confirmed that brolucizumab has excellent structural integrity, high purity, and notable biological activity. The biosynthetic process holds significant potential for therapeutic applications of brolucizumab and provides valuable insights for the further development of additional scFv bioprocesses.

Keywords

age-related macular degeneration - brolucizumab - inclusion bodies refolding - recombinant expression

Supporting Information

Effects of denaturant type and concentration on inclusion bodies solubilization ([Supplementary Fig. S1] [available in the online version]); RP-HPLC analysis of refolding yield before and after optimization, HPLC spectrum of brolucizumab ([Supplementary Fig. S2] [available in the online version]); purification process of brolucizumab and optimization, screening of chromatographic resins and mobile phase pH for primary ion-exchange chromatography ([Supplementary Table S1] [available in the online version]); as well as screening chromatography for the secondary purfication experiments ([Supplementary Table S2] [available in the online version]); brolucizumab undergoing tryptic digestion under non-reducing conditions to preserve disulfide bonds, with resultant fragments confirmed by LC-MS/MS analysis ([Supplementary Fig. S3] [available in the online version]), are included in “[Supplementary Material], available in the online version” section of this article's webpage.


Supplementary Material



Publication History

Received: 18 February 2025

Accepted: 24 June 2025

Article published online:
18 July 2025

© 2025. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)

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