Insight

Antibodies are glycoproteins secreted by immune cells and play pivotal roles in innate and adaptive immune responses. Among therapeutic proteins, antibodies rank first in clinical application. Conventionally, mammalian cell lines are adopted for industrial-scale production of monoclonal antibodies (mAbs) and biopharmaceutical antibody products. The quantity and pattern of antibody glycosylation markedly affect antibody stability, safety, immunogenicity and pharmacodynamic properties. Additionally, glycosylation significantly alters antibody pharmacodynamics; deletion of glycan moieties drastically reduces the binding affinity between antibodies and Fc-gamma Receptor I (FcγRI), thereby inhibiting complement component C1 activation.

Correct disulfide bond formation between heavy and light chains is essential for maintaining the domain structure, biological activity and stability of antibodies. Unpaired free sulfhydryl groups derived from aberrant disulfide bonding tend to trigger antibody aggregation and may further induce adverse immune responses in clinical therapy. Escherichia coli and Pichia pastoris are two dominant microbial hosts with favorable fermentation performance for antibody production, yet they still suffer from drawbacks including low production titer and excessive IgG glycosylation.

Escherichia coli, the most widely used prokaryotic host for recombinant protein cloning and expression, lacks native glycosylation systems and possesses inefficient protein folding machinery, thus it is mainly applied to produce aglycosylated IgG and antibody fragments. As a eukaryotic expression system, Pichia pastoris features human-like glycosylation patterns, low-cost culture media, high cell density and convenient large-scale production feasibility. This paper systematically compares the applicability of E. coli and P. pastoris in the biosynthesis of full-length IgG antibodies and antibody fragments, summarizes various strategies for yield improvement in microbial expression systems, and discusses the existing challenges and future prospects of these two hosts in antibody manufacturing.

Among the five major antibody isotypes (IgA, IgD, IgE, IgG and IgM), IgG is the most extensively utilized subtype for therapeutic purposes, which consists of four subclasses. As a typical Y-shaped heterotetramer, IgG1 is composed of two identical heavy chains (50–70 kDa) and two identical light chains (25 kDa), serving as the primary template for the development of human therapeutic mAbs. Functionally, intact IgG is divided into antigen-binding fragment (Fab) and crystallizable fragment (Fc).

Compared with full-length IgG molecules, antibody fragments exhibit superior tissue permeability and shorter retention time in non-target tissues. Common antibody fragments include Fab fragments, single-chain variable fragments (scFv) and single-domain antibodies (sdAbs).

Post-translational modification is an enzyme-driven biological process that facilitates the assembly of proteins into mature tertiary and quaternary structures. Functional IgG undergoes diverse post-translational modifications, mainly including glycosylation, disulfide bond formation, phosphorylation, acetylation and occasional proteolytic cleavage.

Glycosylation is one of the most structurally complex and diversified post-translational modifications that regulate the effector functions of IgG. In this process, oligosaccharides are sequentially linked to specific amino acid residues in a conserved manner under the catalysis of oligosaccharyltransferases. In IgG molecules, N-linked glycosylation predominantly occurs at the asparagine residue at position 297 (Asn297) within the CH2 domain of the Fc region of IgG1. Due to discrepancies in primary sequences, the sites of N-linked glycosylation vary among IgG2, IgG3 and IgG4 subtypes. Moreover, IgG3, IgA1 and IgD can undergo O-linked glycosylation within their hinge regions.

Glycosylation increases protein polarity, thereby enhancing molecular solubility and structural stability. Glycan chains can shield target proteins from proteolytic degradation via steric hindrance that blocks the binding between proteases and substrate proteins, and protect antibody drug structures from oxidative damage during production and storage. Meanwhile, glycosylation improves the conformational stability of antibodies against thermal, pH and chemical-induced denaturation and aggregation.

Non-human expression hosts generate heterogeneous glycan profiles distinct from human-derived glycans, which endow recombinant antibodies with potential immunogenicity, consequently compromising therapeutic efficacy and biosafety, and even triggering anti-drug antibody responses and hypersensitivity reactions. Therefore, it is critical to explore the potential of E. coli and yeast hosts in synthesizing functionally glycosylated IgG, and evaluate the consistency between microbial glycan patterns and human native glycan characteristics to predict antibody effector functions.

Disulfide bonds are formed via post-translational modification. Deficient or incorrectly paired disulfide bonds hinder antibodies from folding into native spatial conformations. The heavy chains and light chains of antibodies are cross-linked by intrachain and interchain disulfide bonds. In mAb development, two core criteria must be satisfied: compliance with ICH quality guidelines regarding the number and positional distribution of disulfide bonds, and strict control over host-derived immunogenicity to guarantee clinical therapeutic potential. Disulfide bonds are indispensable for maintaining antibody spatial structure, physicochemical stability and biological effector functions; interchain disulfide bonds ensure accurate pairing of heavy chains and maintain the monospecificity of antibodies.

Apart from Escherichia coli, multiple bacterial species including Corynebacterium glutamicum, Bacillus subtilis and Pseudomonas putida have been verified for antibody biosynthesis. In comparison with other prokaryotic hosts, E. coli stands out for antibody production owing to easy genetic manipulation, rapid proliferation in low-cost culture media and abundant commercially available genetically engineered strains. Nevertheless, the absence of intrinsic glycosylation machinery restricts its application in producing fully functional therapeutic glycosylated IgG, limiting its mainstream application to the manufacture of antibody fragments.

The prominent strengths of E. coli include rapid growth rate, simple culture conditions, low production cost, higher cell density and volumetric productivity than mammalian cell systems, as well as straightforward genetic manipulation. Exogenous plasmids can be efficiently transformed into E. coli cells with a transformation efficiency exceeding 10¹¹ CFU/μg. Rational combination of plasmid vectors enables predictable regulation of target gene expression, and various commercialized engineered strains are available for customized application scenarios.

Its main limitations cover the lack of native glycosylation pathways, intracellular inclusion body formation, insufficient intracellular folding microenvironment, host codon usage bias and endogenous proteolytic degradation. The solubilization and renaturation of inclusion bodies involve cumbersome procedures and high processing costs. Human-derived antibody genes present obvious codon bias in E. coli, and inhibition of endogenous proteases requires expensive inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and other protective reagents.

To achieve commercially viable cytoplasmic expression of antibodies in E. coli, multiple optimized strategies have been established, including rational promoter selection, co-expression of folding chaperones and fusion tag fusion, construction of oxidizing cytoplasmic microenvironment, construction of cysteine-free scFv variants and preliminary glycosylation modification of antibody fragments. Other effective approaches involve monocistronic expression design, novel expression vector construction, knockout of endogenous membrane components and protease genes, and optimization of fermentation culture parameters.

Mammalian cell lines remain the gold standard for producing clinical-grade full-length therapeutic IgG. Since E. coli lacks inherent glycosylation machinery, it cannot synthesize native glycosylated IgG with complete physiological functions. Relevant studies have compared the effects of antibody-drug conjugation on the thermal stability and metabolic stability of glycosylated and aglycosylated antibodies. In addition, directed molecular evolution technology has been applied to engineer aglycosylated IgG expressed in E. coli. After multiple rounds of fluorescence-activated cell sorting screening, Fc domain variants with higher binding affinity to FcγRIIIa than clinical-grade trastuzumab have been successfully obtained.

Pichia pastoris possesses complete co-translational and post-translational modification systems, and has been successfully applied to express various membrane proteins including histamine receptors, calcium/potassium ion channels, nitrate and phosphate transporters. This expression system effectively reduces host protein contamination and endotoxin residue risks, and has become a mature industrial expression platform capable of synthesizing over 300 types of recombinant proteins including diverse antibody fragments for large-scale therapeutic protein production. In contrast to mammalian cell culture, P. pastoris requires simpler medium components and allows convenient genetic modification, which is highly suitable for industrial mass production of antibodies.

The primary drawbacks of this system include low exogenous gene transformation efficiency and high susceptibility to miscellaneous bacterial and fungal contamination during fermentation. Its powerful secretory expression system also leads to the extracellular secretion of endogenous proteases into culture supernatant, resulting in proteolytic degradation of secreted target antibodies and reduced final product yield. Although yeast owns evolutionarily conserved N-linked glycosylation mechanisms, it predominantly generates high-mannose-type glycan structures distinct from human glycans.

Based on existing research progress, the optimization directions for improving antibody and antibody fragment production in P. pastoris are summarized as follows: rational selection of engineered strains, optimization of fermentation culture conditions, modification of genetic expression elements, enhancement of secretory expression efficiency and optimization of downstream protein renaturation protocols.

In 1999, researchers first achieved the heterologous expression of aglycosylated full-length antibodies in Pichia pastoris. After 96 hours of methanol-induced expression, the secretory expression titer of intact IgG reached 36 mg/L in culture medium. Subsequently, Pichia pastoris has gradually developed into a mainstream eukaryotic host for glycosylated antibody production by virtue of complete endogenous glycosylation pathways.

The clinical applicability of Pichia pastoris-derived glycosylated IgG is determined by multiple biochemical and biophysical properties, including glycan composition profile, aggregation tendency, charge heterogeneity, methionine oxidation level, as well as thermal and storage stability. Intact IgG consists of four polypeptide chains cross-linked by interchain disulfide bonds, and tends to spontaneously form dimers, trimers and high-molecular-weight polymers at high concentrations. Charge heterogeneity of antibodies is mainly derived from diverse post-translational modifications such as deamidation, acetylation, glycation, phosphorylation and sialylation.

Both Escherichia coli and Pichia pastoris are reliable microbial hosts capable of biosynthesizing low-immunogenicity antibodies and related fragments. E. coli exhibits prominent advantages in producing aglycosylated full-length IgG and various antibody fragments including Fab, scFv and nanobodies for biomedical research and industrial preparation. Nevertheless, E. coli inevitably releases endotoxins during cell lysis and antibody separation processes, leading to potential endotoxin contamination in final products.

Pichia pastoris can be engineered to synthesize humanized glycan-modified therapeutic IgG, yet the inherent high-mannose glycosylation and abnormal O-linked glycosylation patterns in yeast-derived IgG may induce immunogenic reactions, severely limiting its direct clinical application.

In summary, E. coli is more suitable for the production of antibody fragments and aglycosylated antibody products, while P. pastoris serves as an ideal platform for the preparation of glycosylated antibodies. Future research priorities include constructing endotoxin-deficient E. coli strains and introducing heterologous eukaryotic glycosylation pathways without compromising expression titer. For Pichia pastoris, the development of vesicle-based cell-free glycoprotein synthesis systems is expected to eliminate glycan heterogeneity, which will greatly improve the production efficiency and clinical applicability of therapeutic antibodies.