Strategies and Applications of Antigen-Binding Fragment (Fab) Production in Escherichia coli

Abstract

With the advancement of genetic engineering, monoclonal antibodies (mAbs) have made far-reaching progress in the treatment of various human diseases. However, due to the high cost of production, the increasing demands for antibody-based therapies have not been fully met. Currently, mAb-derived alternatives, such as antigen-binding fragments (Fab), single-chain variable fragments, bispecifics, nanobodies, and conjugated mAbs have emerged as promising new therapeutic modalities. They can be readily prepared in bacterial systems with well-established fermentation technology and ease of manipulation, leading to the reduction of overall cost. This review aims to shed light on the strategies to improve the expression, purification, and yield of Fab fragments in Escherichia coli expression systems, as well as current advances in the applications of Fab fragments.

The Dawn of a New Era: Targeting the “Undruggables” with Antibody-Based Therapeutics

“…115 The Fab framework has the longest clinical history among all types of antibody fragments, with 4 molecules (i.e., Abciximab, Ranibizumab, Certolizumab pegol, and Idarucizumab) having successfully received FDA approval. 116 They have been used to treat acute indications where fast clearance is not a major concern. When being used to treat chronic inflammatory conditions, the free cysteine located near the heavy chain C-terminus of these drugs was used to be site-specifically conjugated to ∼40-kDa polyethylene glycol (PEG) (e.g., Certolizumab pegol), leading to an increase in serum half-life.…”

“…IgGs can be cleaved by papain into one Fc and two identical antigen-binding fragments (Fabs) consisting of a light chain (V L + C L ) and a heavy chain (V H + C H 1) linked by disulfide bonds with a molecular weight (MW) of ∼55 kDa, , or by pepsin to form F­(ab′) 2 of ∼110 kDa (a bivalent fragment containing two Fab segments) . The Fab framework has the longest clinical history among all types of antibody fragments, with 4 molecules (i.e., Abciximab, Ranibizumab, Certolizumab pegol, and Idarucizumab) having successfully received FDA approval . They have been used to treat acute indications where fast clearance is not a major concern.…”

Design of an alternate antibody fragment format that can be produced in the cytoplasm of Escherichia coli

“…As a potential solution, antibody fragments (Fabs) have emerged as key players in the biopharmaceutical industry. They offer certain advantages such as improved and deep tumor penetration, binding to specific epitopes inaccessible to Mabs, monovalent antigen binding with potentially reduced immunogenicity, higher stability than smaller antibody fragment formats and faster clearance 3 – 5 . A multitude of applications of antibody-based molecules as therapeutic and diagnostic agents have intensified their demand and subsequently the need for their production in high yields.…”

Full-length recombinant antibodies from Escherichia coli : production, characterization, effector function (Fc) engineering, and clinical evaluation

“…Currently, seven approved antibody-fragment-based therapeutics are in the market ( Table 1 ). 5 , 12 Over the years, substantial advances have been made with respect to the optimization of expression plasmids and host engineering that enable the scale of full-length antibody production in E. coli . 13–17 This has led to clinical development of several E. coli -produced therapeutic mAb and bispecific antibodies (BsAb) in recent years.…”

“…Reviews have recently been published on E. coli as a host for production of recombinant proteins in general 10 , 35–40 and for antibody fragment in particular. 5 , 12 , 41–43 This review, however, is focused exclusively on the production of full-length antibodies in E. coli and their characterization. The potential of E. coli remaining a host of choice for production of antibody-based drugs in the future is also discussed.…”