Insight

Monoclonal antibodies (mAbs) are soluble glycoproteins with a molecular weight of approximately 150 kDa. They adopt a tetrameric structure composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds, comprising two antigen-binding fragments (Fab) and one crystallizable fragment (Fc). In recent years, therapeutic mAbs and their derivatives have been widely applied in the treatment of cancers, autoimmune diseases and other disorders.

Most therapeutic mAbs are manufactured using mammalian cell lines such as Chinese hamster ovary (CHO) cells, while certain antibody fragments have achieved large-scale production in Escherichia coli (E. coli). Despite the absence of post-translational modifications including glycosylation in E. coli, aglycosylated antibodies produced by this strain share analogous biochemical and physical properties as well as in vivo stability with glycosylated counterparts, except for Fc-mediated immune effector functions. Accordingly, aglycosylated antibodies serve as viable alternatives for therapeutic applications independent of immune effector responses. Moreover, Fc domain engineering can restore immune effector activities and endow antibodies with novel functional mechanisms.

Since the 1980s, E. coli has emerged as a preferred host for recombinant protein manufacturing, with over 85 pharmaceutical drugs currently produced via this microbial system. Compared with other expression hosts, E. coli features rapid proliferation, low production cost, convenient manipulation and high productivity. It enables recombinant protein expression in the cytoplasm, periplasm or extracellular culture medium, offering remarkable production flexibility. To date, seven approved antibody fragments and derivatives have been successfully produced in distinct subcellular compartments of E. coli.

Early studies in 1984 revealed that full-length immunoglobulin G (FL-IgG) expressed in the reducing cytoplasmic environment of E. coli tended to form inclusion bodies. Subsequent solubilization and refolding treatments yielded extremely low antigen-binding activity, driving research focus toward the oxidizing periplasmic space. In 2002, Simmons et al. first reported the production of functionally active FL-IgG in the periplasm. Rational regulation of the expression and secretion balance between heavy and light chains, combined with optimized promoters and signal sequences, facilitated FL-IgG biosynthesis in 10-liter fermenters. Reilly and Yansura further achieved high-titer IgG1 production in high-density fermentation via overexpression of oxidative folding and isomerization chaperones DsbA and DsbC.

Genentech established a bispecific antibody expression platform in E. coli periplasm adopting the knobs-into-holes (KiH) technology. Multiple antibody candidates were successfully generated, including an IgG4 bispecific antibody targeting IL-4 and IL-13 for asthma therapy. The company also produced 27 types of bispecific antibodies (BsAbs) through co-culture strategies and accomplished the biosynthesis of anti-CD3 bispecific antibodies.

The periplasmic compartment is constrained by limited volumetric capacity and protein translocation bottlenecks, making the cytoplasm an alternative venue for FL-IgG production. In 1984, Genentech and Celltech independently reported cytoplasmic expression of FL-IgG and FL-IgM in E. coli. Nevertheless, the highly reducing cytoplasmic environment triggers massive accumulation of antibodies into inclusion bodies, and refolding procedures are mandatory to recover native antigen-binding competence.

Excessively reducing cytoplasmic conditions disrupt correct disulfide bond pairing and hinder functional FL-IgG assembly. To address this limitation, genetically modified E. coli strains were constructed to establish a semi-oxidized cytoplasmic microenvironment compatible with disulfide bond formation.

Two intrinsic reductive pathways, namely the thioredoxin and glutathione reductase pathways, mediate disulfide bond reduction in native E. coli cytoplasm. Deletion of reductase encoding genes gor and trxB eliminates these reductive pathways, and mutation of the peroxidase gene ahpC contributes to the construction of the SHuffle® strain. This engineered strain supports robust cytoplasmic synthesis of soluble and functionally intact FL-IgGs. In 2015, Robinson et al. first obtained correctly folded anti-HER2 and anti-VEGF FL-IgGs with authentic disulfide bond configurations using the SHuffle® strain.

Beyond conventional whole-cell expression systems, FL-IgG can also be synthesized via cell-free protein synthesis (CFPS) derived from E. coli extracts. The first functionally active antibody generated by E. coli-based CFPS was documented in 2008. Sutro Biopharma optimized CFPS protocols by modifying translation initiation regions of heavy and light chain genes and supplementing chaperone proteins DsbC and FkpA, achieving an IgG production titer of 1 g/L.

BsAbs carrying KiH mutations were also successfully biosynthesized using this platform. The research team integrated dsbC and fkpA into the chromosome of host strains for cell extract preparation and developed a continuous fermentation-based extraction process to support the manufacturing of antibody-drug conjugates (ADCs).

Biochemical characterization lays the foundation for antibody quality assessment. Analytical techniques including high-performance liquid chromatography, mass spectrometry and SDS-PAGE electrophoresis are applied to identify molecular conformation, amino acid sequence, glycosylation profile and molecular weight. Antigen-binding capacity, affinity and specificity are quantitatively determined simultaneously.

The anti-tissue factor (TF) FL-IgG produced in E. coli periplasm by Genentech was the first fully biochemically characterized E. coli-derived full-length antibody, exhibiting equivalent antigen-binding activity to mammalian cell-produced counterparts. In 2008, characterization data of creatine kinase-targeting antibodies synthesized via CFPS demonstrated nearly identical binding affinity compared with products from mammalian cell lines.

Numerous mAbs, BsAbs and ADCs have been successfully produced, purified and characterized utilizing E. coli cytoplasmic, periplasmic and cell-free expression systems, marking substantial advancements in the application of E. coli-based antibody manufacturing.

Biophysical characterization evaluates antibody homogeneity, solubility and aggregation propensity. Stability profiles are assessed under variable temperature, storage duration, pH value and salinity conditions. Aglycosylated antibodies derived from E. coli were previously presumed to possess inferior stability, while experimental evidence confirms their stability is comparable to glycosylated antibodies. Genentech verified that periplasmic E. coli-produced anti-TF IgG shared similar quaternary structure and stability characteristics with CHO cell-derived antibodies. CFPS-generated anti-MAK33 antibodies also displayed thermal stability equivalent to mammalian cell products. Current biophysical data validates that aglycosylated E. coli-produced antibodies maintain favorable stability under routine detection conditions.

Biological characterization evaluates in vitro and in vivo biological activities via cell-based assays, flow cytometry, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). These assays verify immunological potency, target binding efficiency and subsequent immune response activation. The therapeutic efficacy of IgG drugs relies on two core mechanisms: Fab-mediated specific antigen recognition, and Fc-triggered immune responses against pathogens and malignant cells. Aglycosylated antibodies manifest comparable functional performance in both mechanisms relative to glycosylated equivalents.

Early studies indicated that the lack of glycosylation impairs the binding capacity of E. coli-produced antibodies to FcγRI, FcγRIIIa and C1q, resulting in abolished ADCC and CDC activities. Aglycosylated trastuzumab exhibited reduced affinity toward FcγRI and completely lost ADCC and CDC potency. Nevertheless, such aglycosylated antibodies retain valid therapeutic effects independent of Fc-mediated immune responses.

Aglycosylated IgG maintains prolonged serum half-life attributed to pH-dependent binding interaction with neonatal Fc receptor (FcRn). Hence, E. coli-produced aglycosylated antibodies sustain favorable circulatory retention even without intact Fc effector functions.

Pharmacokinetic assays in chimpanzees revealed consistent half-life between E. coli-derived anti-TF IgG1 and CHO-produced IgG2/IgG4. Similar pharmacokinetic profiles were also observed in rhesus monkey models. Pharmacological evaluation of six KiH-engineered BsAbs manufactured in E. coli further confirmed that aglycosylated antibodies possess pharmacokinetic behaviors matching glycosylated mammalian cell-derived products.

Owing to the absence of N-linked glycans, E. coli-derived aglycosylated antibodies fail to efficiently engage Fcγ receptors and C1q, leading to compromised ADCC and CDC activities. Fc domain genetic engineering serves as an effective strategy to restore or enhance immune effector functions. Jung et al. screened Fc mutant libraries and identified the Fc5 variant capable of selectively binding FcγRI and inducing potent ADCC against HER2-overexpressing tumor cells. Rational Fc structural optimization improves selective binding to diverse Fcγ receptors and C1q, thereby reinforcing ADCC potency.

Engineered aglycosylated antibodies designed to interact with FcγRIIIa elicit robust antibody-dependent cellular phagocytosis predominantly mediated by macrophages. These modified antibodies exert prominent in vitro bioactivity and favorable in vivo immunotherapeutic potential. Fc engineering effectively recovers impaired effector functions of aglycosylated antibodies and broadens their application prospects in tumor immunotherapy.

Remarkable breakthroughs have been achieved in FL-IgG production utilizing E. coli cytoplasmic, periplasmic and cell-free expression platforms over the past decade, contributing to elevated antibody yield, improved product quality and reduced manufacturing costs. These advances expand the clinical application potential of aglycosylated E. coli-produced antibodies in allergy treatment, immunotherapy and oncology. Although native aglycosylated antibodies lack Fc-mediated ADCC and CDC activities, genetic engineering enables functional restoration, potency enhancement and discovery of unprecedented effector mechanisms.

E. coli-synthesized aglycosylated antibodies possess therapeutic potential comparable to glycosylated antibodies manufactured by mammalian cell systems. It is anticipated that an increasing number of E. coli-derived aglycosylated antibody candidates will advance into clinical research stages in the future.