Development of a Pichia pastoris-Based Expression System for High-Activity Anti-SARS-CoV-2 Neutralizing Antibodies

doi:10.21203/rs.3.rs-7070370/v1

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Research Article

Development of a Pichia pastoris-Based Expression System for High-Activity Anti-SARS-CoV-2 Neutralizing Antibodies

https://doi.org/10.21203/rs.3.rs-7070370/v1

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published 11 Dec, 2025

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The growing need for rapid and cost-effective antibody production highlights the potential of Pichia pastoris as an alternative to mammalian systems. This study aimed to develop and optimize engineered single-chain antibody fragments (scFvs) against SARS-CoV-2 using Pichia pastoris. Four scFv formats, scFv-His, scFv-CH3-His, scFv-ZIP-His, and scFv-Fc, were evaluated for binding activity, neutralization potency, expression yield, and in vivo stability. Among them, scFv-Fc demonstrated the best overall performance, with strong antigen binding (EC₅₀ = 0.003 µg/mL), effective neutralizing activity (IC₅₀ = 0.570 µg/mL), and a prolonged serum half-life exceeding 5 days. Optimization of fermentation conditions increased its expression from 23 mg/L to 110 mg/L. These results demonstrate the feasibility of engineering scFv-based therapeutics using yeast systems and establish a framework for the rapid and economical production of next-generation antibody formats.

Pichia pastoris

SARS-CoV-2

Antibody

scFv

Fermentation optimization

The worldwide COVID-19 pandemic has significantly impacted global public health and socioeconomic stability(Shademan et al., 2021). Mechanistically, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection begins when its viral spike (S) glycoprotein binds to the angiotensin-converting enzyme 2 (ACE2) receptor on host cell membranes. Neutralizing antibodies, which recognize and bind to key epitopes on the viral spike protein, block this interaction, effectively neutralizing the virus and preventing its infectivity (Baum et al., 2020). Based on this viral neutralization mechanism, neutralizing antibodies have been established as an indispensable foundation of antiviral therapy. Clinical evidence supports their use for both therapeutic and prophylactic purposes (O'Brien et al., 2021; Pang et al., 2021), with their inclusion in emergency use authorization programs highlighting their critical role in controlling the pandemic(Zhang et al., 2024).

Traditional antibody production platforms, primarily using mammalian cell systems like CHO, HEK293F, are effective in ensuring proper folding and post-translational modifications required for functional immunoglobulin G (IgG) antibodies. However, these systems are limited by high costs and lengthy production timelines, presenting challenges for large-scale clinical application(Zhang et al., 2022; Li et al., 2022). These limitations have spurred the pursuit of more efficient and economical production alternatives in antibody manufacturing.

Pichia pastoris, a methylotrophic yeast, has emerged as a promising alternative in antibody production. It offers remarkable cost-effectiveness and suits high-density fermentation processes. This yeast system also exhibits proficient eukaryotic protein processing capabilities, including disulfide bond formation and glycosylation(Das et al., 2025). Despite these advantages, producing full-length IgG antibodies in Pichia pastoris remains challenging. The structure of IgG requires precise endoplasmic reticulum-mediated folding and assembly, which is often inefficient in yeast, resulting in poor secretion, misfolding, and low yields(Krishna et al., 2024). To overcome these challenges, antibody engineering strategies have focused on simplifying constructs, such as single-chain variable fragments (scFv), Fab fragments, or VHH antibodies, to match the yeast’s secretion capabilities while maintaining antigen-binding function(Gómez-Ramírez et al., 2023; Gagné et al., 2023; Xi et al., 2021; Montoliu-Gaya and Villegas, 2022).

Building on our previous work, in which we isolated the high-affinity anti-SARS-CoV-2 antibody 11-2G from humanized antibody mice, targeting the receptor-binding domain (RBD) of the viral S protein (Yang et al., 2022), this study aims to develop a cost-effective expression pipeline using the Pichia pastoris X33 strain. We designed four antibody constructs with varying structural domains (Fc, CH3, leucine zipper and His-tag) to compare expression efficiency, binding affinity, and neutralizing potency. Through systematic optimization of fermentation conditions, including methanol concentration, pH, and temperature, we enhanced the secretion of our lead candidate, the scFv-Fc construct. By integrating structural design, functional screening, and bioprocess optimization, this work highlights the feasibility of yeast-based systems for producing high-potency neutralizing antibodies. These findings establish a technically robust and economically viable strategy for producing functional neutralizing antibodies in Pichia pastoris, bridging the gap between molecular antibody design and industrial-scale manufacturing, offering a practical solution for rapid, low-cost therapeutic deployment against current and future viral threats.

Strains, plasmids, enzymes, reagents

Heterologous protein expression was performed in Pichia pastoris X33 strain (Invitrogen), using protocols outlined in the manufacturer’s expression manual. Most reagents were sourced from Sigma-Aldrich (USA), except for molecular biology enzymes and antibiotics, which were obtained from Thermo Fisher Scientific (USA).

Vector Construction

As shown in Fig. 1, four recombinant expression vectors were generated (1) scFv-Fc: the Fc fragment (hinge, CH2, and CH3 domains) of antibody 11-2G was fused to the C-terminus of scFv. The scFv consists of the heavy chain variable region (VH) and light chain variable region (VL). A flexible (G4S) ₃ linker connects these two regions. The Fc region included six mutations—L264A, L265A (LALA) to reduce antibody-dependent cellular cytotoxicity (ADCC) (Hessell et al., 2007), and F271T, N327A, D386E, L388M to improve glycosylation and stability, as reported in Eptinezumab (DrugBank: DB14040), the first monoclonal antibody produced in Pichia pastoris (https://go.drugbank.com/drugs/DB14040#reference-L12318). (2) scFv-CH3-his: This construct contains the scFv fused to the hinge and CH3 domains, with D386E and L388M mutations for enhanced stability. (3) scFv-ZIP-His: A leucine zipper dimerization motif was fused via the Human IgG1 upper hinge region at the C-terminus of scFv to promote dimer formation (Klement et al., 2015). (4) scFv-his: The control construct included only scFv with a C-terminal His tag. All sequences were synthesized (Beijing Qingke Biotech Co.), cloned into the pPICZαA vector under the AOX1 promoter, and transformed into Pichia pastoris X33.

Protein Expression and Purification

Recombinant plasmids were linearized with SacI and introduced into Pichia pastoris X33 by electroporation (1.5 kV, 25 µF, and 200 Ω). Transformants were screened on YPD plates. The plates contained 1% yeast extract (w/v), 2% peptone (w/v), 2% agar (w/v) and 100 mg/L Zeocin™, then incubated at 30°C until colonies formed(Piboonpocanun, 2024).

Individual yeast colonies were randomly selected and cultured for small-scale expression screening. Recombinant protein expression was evaluated by ELISA. SARS-CoV-2 RBD proteins were kindly provided by Zhongyuan Huiji Biotechnology Co., Ltd. (China) and used as coating antigens. RBD-his was employed for scFv-Fc constructs, and RBD-mFc for His-tagged constructs (scFv-His, scFv-CH3-His, scFv-ZIP-His), each antigen was diluted to 0.5 µg/mL (pH 9.6). The ELISA procedure was as follows: 50 µL of diluted antigen was added to 96-well plates and incubated overnight at 4°C; Plates were washed three times with 0.05% (v/v) PBST (250 µL Tween-20 in 500 mL PBS) and blocked with 1% (w/v) BSA-PBS at 37℃for 1 h. After washing, 50 µL of recombinant yeast culture supernatant or blank control (1% BSA-PBS) was added to each well and incubated at 37℃for 2 h. Plates were then washed three times with PBST. HRP-conjugated secondary antibodies (anti-human IgG Fc for scFv-Fc, anti-6×His for His-tagged constructs; Abcam, UK) were diluted 1:5000 in blocking buffer, added to plates, and incubated for 2 h. Following washing, TMB substrate (50 µL) was added for 15 min, then reactions were stopped with 50 µL of 2.5 M H₂SO₄. Absorbance at 450 nm (OD₄₅₀) was measured using a microplate reader, and the clone yielding the highest OD value was selected for scale-up expression(Gao et al., 2023).

For scale-up, positive transformants were inoculated into 250 mL Erlenmeyer flasks. Each flask contained 50 mL of BMGY medium (1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM potassium phosphate buffer [pH 6.0], 1.34% (w/v) yeast nitrogen base without amino acids, 0.4 mg/L biotin, 2% (v/v) glycerol and 200 µg/mL ampicillin). Cultures were inoculated into BMGY medium and incubated at 30℃ with shaking at 220 rpm until saturation. The OD₆₀₀ was measured by diluting the supernatant 10-fold, and the culture was considered saturated when the diluted OD₆₀₀ reached 1, which typically took 1–2 days. Cells were pelleted (4,000 × g, 5 min) and transferred to BMMY medium (identical composition to BMGY except glycerol replaced with 0.5% (v/v) methanol). Methanol was added daily to maintain a final concentration of 0.5% (v/v) throughout a 3-day induction period under the same culture conditions(Cahyati et al., 2021).

After induction, cultures were centrifuged at 8,000 × g for 5 min. The supernatants were collected into sterile tubes for purification. Prior to chromatography, all supernatants and buffers were filtered through 0.45 µm polyethersulfone (PES) membranes to remove particulates. His-tagged proteins (scFv-His, scFv-ZIP-His, scFv-CH3-His) were purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, while scFv-Fc constructs were purified using Protein A affinity chromatography, leveraging its high specificity for the IgG Fc domain. The chromatography system was initially flushed with deionized water (5 column volumes, 5 mL/min, 0.3 MPa). Subsequently, the column was equilibrated with binding buffer: for Ni-NTA, this comprised 20 mM Phosphate Buffer, 200 mM NaCl, pH 7.5 (10 column volumes at 2 mL/min); for Protein A, it was 20 mM Phosphate Buffer, 150 mM NaCl, pH 7.0 (10 column volumes at 2 mL/min). After baseline correction, filtered supernatants were loaded onto the column at 0.5 mL/min. Unbound proteins were removed by washing with binding buffer at 2 mL/min. Target antibodies were eluted using elution buffer (20 mM Phosphate Buffer, 200 mM NaCl, 500 mM imidazole, pH 7.2 for Ni-NTA; 0.1 M glycine-HCl, pH 3.0-3.6 for Protein A) at 3 mL/min until UV absorbance (280 nm) returned to baseline. Eluates were immediately neutralized to pH 7.2–7.4 with 1 M Tris-HCl (pH 9.0). Proteins were then dialyzed against 2 L of 1×PBS at 4℃overnight with three buffer changes using a 3 kDa molecular weight cutoff dialysis membrane. Finally, the concentrated proteins were aliquoted into sterile microcentrifuge tubes and stored at -80°C.

SDS - PAGE Analysis

Protein samples (30 µL) were mixed with 10 µL of 4× loading buffer. The buffer contained 5% β-mercaptoethanol for reducing conditions but excluded it for non-reducing electrophoresis. Samples were heated at 100°C for 5 min to denature proteins. After brief cooling, centrifugation proceeded at 12,000 × g for 2 min. We loaded 20 µL of each supernatant onto precast gels containing a 12% (w/v) separating gel layered over a 4% (w/v) stacking gel. Electrophoresis began at 80 V until the dye front migrated into the separating gel. Voltage was then increased to 100 V, continuing until the bromophenol blue dye front reached approximately 1 cm from the gel bottom. Post-electrophoresis, gels were stained with Coomassie Brilliant Blue R-250 for 4 h. Background staining was removed by destaining in methanol:acetic acid:water (4:1:5, v/v/v) until clear. Gel images were captured using an Odyssey CLx Imaging System (LI-COR, USA).

Western Blotting Analysis

For immunoblotting under reducing conditions, samples were prepared and electrophoresed identically to the SDS-PAGE protocol. Post-electrophoresis, proteins were transferred to a 0.45 µm PVDF membrane (Millipore, USA) at 30 V for 60 min. Transfer buffer contained 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol (pH 8.3). Membranes underwent a 10-min wash in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.6). Blocking followed with 5% (w/v) non-fat dry milk in TBST for 1 h at room temperature with 50 rpm shaking.

scFv-Fc detection: Membranes were probed with DyLight™ 680-conjugated Rabbit anti-Human IgG (H + L) secondary antibody (Invitrogen, USA; 1:5000 in blocking buffer). Incubation proceeded for 2 h at room temperature with 30 rpm shaking.

His-tag detection: Membranes were first incubated overnight at 4°C with anti-His Tag antibody (H-3, Santa Cruz Biotechnology, USA; 1:500 in blocking buffer). After three 10 min TBST washes, membranes were incubated with Alexa Fluor 680-conjugated Donkey anti-Rabbit IgG secondary antibody (Invitrogen, USA; 1:5000 in blocking buffer) for 1–2 h at room temperature.

Finally, the membranes received three additional 10-min TBST washes. Fluorescent signals were visualized using the Odyssey CLx Imaging System (LI-COR, USA).

EC₅₀ Assay

The half-maximal effective concentration (EC₅₀) of purified recombinant antibodies was determined by indirect ELISA. ELISA plates were coated with 0.5 µg/mL RBD antigen in carbonate buffer (pH 9.6) and incubated overnight at 4°C. After removing the coating solution, plates were washed three times with 0.05% PBST. Blocking was performed using 1% (w/v) BSA-PBS at 37°C for 1 h, following the same protocol as for the initial screening ELISA. Purified antibodies were serially diluted (2-fold or 4-fold) in 1% BSA/PBS. Following a 2-h incubation at 37°C, plates were washed and incubated with HRP-conjugated secondary antibodies: anti-human IgG Fc for scFv-Fc constructs, and anti-6×His for His-tagged constructs (both from Abcam, UK), each diluted 1: 5000 in blocking buffer. Colorimetric detection was performed using TMB substrate, and absorbance was measured at 450 nm following the addition of 2.5 M H₂SO₄ to stop the reaction. Dose-response curves were analyzed using GraphPad Prism 7.0 software to calculate EC₅₀ values (Yang et al., 2022).

Antibody Affinity Determination

The binding and dissociation kinetics between recombinant antibodies and RBD protein were characterized using a Fortebio Octet RED96 biolayer interferometry system. Sensors were pre- equilibrated in equilibration buffer (PBS containing 0.1% BSA and 0.02% Tween 20) for 10 min. The RBD protein sample was diluted to 20 µg/mL in equilibration buffer for subsequent immobilization. For the analytes (four recombinant antibodies), a stock solution was prepared at 400 nM in equilibration buffer, followed by 2-fold serial dilutions to obtain final concentrations of 400, 200, 100, 50, and 25 nM). Sample loading was conducted in a 96-well plate, where columns 1 to 4 were filled sequentially with equilibration buffer, ligand solution, equilibration buffer, and analyte dilutions. Within the analyte column, rows A to H corresponded to the five antibody concentrations and a blank control (equilibration buffer only). Real-time binding interactions were monitored using the Octet system. Kinetic parameters, including the equilibrium dissociation constant (KD), association rate constant (ka), and dissociation rate constant (kd), were calculated by analyzing the binding and dissociation curves using Fortebio Data Analysis software.

IC₅₀ Assay

The half-maximal inhibitory concentration (IC₅₀) of the four purified recombinant antibodies was determined using a competitive ELISA. RBD protein (2 µg/mL in carbonate coating buffer, pH 9.6) was added to 96-well plates (100 µL/well) and incubated overnight at 4°C. After three washes with PBST, wells were blocked with 2% (w/v) BSA-PBS at 37℃for 1 h. Following washing, serial dilutions of the antibodies (2- or 4-fold in 1% (w/v) BSA-PBS, 100 µL/well) were added, with 1% BSA-PBS serving as the blank. After incubation at 37℃for 2 h, wells were washed and incubated ACE2 protein (1 µg/mL, 100 µL/well) at 37℃for 1 h. Following three additional PBST washes, HRP-conjugated secondary antibodies were applied at a 1:5000 dilution: Anti-6×His Tag Antibody (Abcam, UK) for scFv-Fc (plates coated with RBD-mFc; ACE2-His target) and Goat anti-Mouse IgG Fc Cross-Adsorbed Secondary Antibody (Invitrogen, USA) for His-tagged antibodies (plates coated with RBD-His; ACE2-mFc target).

After a 1h incubation at 37℃and final washes, 50 µL of TMB substrate was added per well and developed for 15 min. Reactions were stopped with 50 µL of 2.5 M H₂SO₄. Absorbance measurements at 450 nm yielded percentage inhibition:

$$\:Inℎibitions\:\left(\%\right)=\left(1-\frac{B}{\:{B}_{0}\:}\right)\%$$

B is the OD₄₅₀ of the test well and B₀ is the OD₄₅₀ of the negative control. Four-parameter logistic (4-PL) modeling in GraphPad Prism 7.0 generated IC₅₀ values from triplicate experiments (Yang et al., 2022).

Pharmacokinetics in Kunming Mice

Pharmacokinetic analysis was conducted in 6–8-week-old female Kunming mice (n = 3 per antibody group, 30–42 g) housed under specific pathogen-free (SPF) conditions. All animal experiments were approved by the Experimental Animal Ethics Committee of Chongqing Academy of Animal Sciences. The four recombinant antibodies (scFv-Fc, scFv-His, scFv-CH3-His, scFv-ZIP-His) were administered via single intravenous injection into the tail vein at a dose of 10 mg/kg. Blood samples were collected at 0 h and 5 min, 0.5 h, 1 h, 3 h, 6 h, 24 h, 48 h, 72 h, 120 h post-injection. Samples were centrifuged at 10,000 × g for 10 min at 4°C to isolate serum, which was then stored at -80°C until analysis(Song et al., 2021).

Serum antibody concentrations were determined by ELISA following the same protocol as for single-colony screening. Plates were coated with RBD (0.5 µg/mL, carbonate buffer, pH 9.6), blocked with 1% BSA-PBS, and incubated with diluted serum (1:100) or antibody standards (4.88–5000 ng/mL). Detection was performed using HRP-conjugated secondary antibodies: Anti-Human IgG Fc for scFv-Fc, and Anti-6×His for His-tagged antibodies (1:5000 dilution). OD₄₅₀ values were used to construct standard curves and determine antibody concentrations using a four-parameter logistic model.

Optimization of scFv-Fc Expression in Pichia pastoris

Cultures were inoculated into BMGY medium at 3% (v/v), shaken at 220 rpm until the OD₆₀₀ reached approximately 1.0 with a 10-fold dilution. Subsequently, cells were harvested by centrifugation and resuspended in BMMY medium. To optimize scFv-Fc expression in Pichia pastoris, parameters including methanol concentration (0.5-3% v/v), pH (5.5-8.0), temperature (20–30°C), and induction duration (0-120 h) were systematically varied. Methanol supplementation was performed every 24 h after transferring to BMMY. For the induction duration experiments, supernatants were collected at 0, 24, 48, 72, 96, and 120 h post-induction. All other parameter groups had supernatants harvested at 72 h post-induction. Following fermentation, supernatants were harvested by centrifugation (4,000 × g, 5 min) and stored at -80°C for subsequent analysis. Protein expression levels were assessed by 12% SDS-PAGE with Coomassie Brilliant Blue staining to identify the optimal conditions for protein expression(Zhan et al., 2021).

Expression and Purification of Antibodies with Different Structures

Four recombinant expression vectors were constructed (Fig. 1A), encoding four antibody variants: scFv-Fc, scFv-CH3-His, scFv-ZIP-His, and scFv-His (Fig. 1B). All constructs were successfully expressed as soluble proteins in Pichia pastoris X33. ScFv-Fc was purified using Protein A affinity chromatography, while the His-tagged variants (scFv-CH3-His, scFv-ZIP-His, and scFv-His) were purified using Ni-NTA agarose resin.

As shown in Fig. 1C, in the reducing SDS-PAGE analysis, scFv-Fc, scFv-CH3-His, and scFv-ZIP-His appeared as monomeric proteins with sizes of approximately 52.3 kDa, 41.2 kDa, and 32.6 kDa, respectively. In non-reducing SDS-PAGE, all of them existed as dimers with sizes of approximately 104.6 kDa, 82.4 kDa, and 65.2 kDa, which was consistent with expectations. For scFv-His, reducing SDS-PAGE exhibited two monomeric bands of comparable molecular weight (≈ 29 kDa), likely resulting from differential glycosylation modifications. Non-reducing analysis revealed that most of scFv-His existed as monomers, with minor multimeric species; upon reduction, multimers nearly vanished, accompanied by a marked increase in the intensity of the larger monomeric band, indicating this form may serve as the primary multimer precursor. (Fig. 1C).

Biological Activity Comparison of Antibody Variants

Indirect ELISA was used to evaluate binding affinity to the SARS-CoV-2 receptor-binding domain (RBD). As shown in Fig. 2A, scFv-Fc exhibited the highest binding activity, with an EC₅₀ of 0.0034 µg/mL, comparable to the full-length IgG1 antibody (11-2G) expressed in 293F suspension cells [12]. In contrast, scFv-His showed a 5-fold lower affinity (EC₅₀ = 0.018 µg/mL), while scFv-CH3-His and scFv-ZIP-His displayed > 100-fold reduced binding (EC₅₀ = 0.360 µg/mL and 0.693 µg/mL, respectively) (Fig. 2A).

Biolayer interferometry (BLI) further characterized binding kinetics. ScFv-Fc, scFv-CH3-His, and scFv-ZIP-His demonstrated exceptionally high affinity for RBD, with equilibrium dissociation constants (KD) < 1.0×10^− 12 M (Fig. 2B). However, in contrast to the EC₅₀ results, scFv-His showed very weak affinity for RBD (KD = 6.62×10^− 10 M).

Competitive ELISA was used to assess the ability of the antibodies to block RBD-ACE2 interactions. ScFv-Fc and scFv-ZIP-His strongly inhibited this interaction, with IC₅₀ values of 0.570 µg/mL and 0.532 µg/mL, respectively. ScFv-CH3-His and scFv-His showed reduced inhibitory activity (IC₅₀ = 1.531 µg/mL and 1.608 µg/mL, respectively) (Fig. 2C).

In Vivo Pharmacokinetics of Antibody Variants

Pharmacokinetic analysis in mice revealed significant differences in serum persistence among the antibody variants (Fig. 3). His tagged constructs (scFv-His, scFv-CH3-His, and scFv-ZIP-His) were rapidly cleared: scFv-His became undetectable within 1 h, while scFv-CH3-His and scFv-ZIP-His fell below the limit of quantification by 24 h. In contrast, scFv-Fc exhibited a prolonged circulation profile, with measurable serum levels persisting for more than 5 days post-injection, highlighting the critical role of the Fc domain in extending the in vivo half-life.

Optimization of Expression Conditions in Pichia pastoris

Single-factor optimization was performed to enhance scFv-Fc production in Pichia pastoris, evaluating methanol induction concentration, medium pH, incubation temperature, and induction time. Densitometric analysis of SDS-PAGE revealed that 0.5% (v/v) methanol, which was the lowest concentration tested within the range of 0.5–3% (v/v), yielded the highest target protein expression (Fig. 4A). This effect is likely attributed to reduced methanol-induced cytotoxicity and enhanced stability of the secretion pathway. A slightly acidic pH of 5.5 significantly improved expression compared to neutral or alkaline conditions (pH 5.5–8.0), aligning with the native acidic environment optimal for Pichia pastoris secretory pathways (Fig. 4B).

Incubation at 28℃ achieved the highest protein yield, outperforming both lower (20–25°C) and higher (30°C) temperatures (Fig. 4C). Induction duration experiments showed that 72 h post-induction yielded significantly higher target protein expression than other induction time points (Fig. 4D). Based on the results above, optimal conditions (0.5% methanol, pH 5.5, 28°C, 72h induction) for protein expression were selected. SDS-PAGE analysis confirmed a notable increase in antibody concentration within the optimized culture supernatant (Fig. 4E). The optimized culture supernatant was purified using a Protein A column. Purification results demonstrated that scFv-Fc expression increased from 23 mg/L to 110 mg/L following the optimization of induction parameters.

The Pichia pastoris expression system has demonstrated significant advantages over traditional mammalian cell systems such as 293F and CHO in the production of recombinant antibodies. Its core competitiveness lies in its low cost, high scalability, and simplified culture process (Karbalaei et al., 2020; Hebbi et al., 2025). Unlike mammalian cells, Pichia pastoris does not require serum supplementation, and its defined medium is cost-effective, enabling high-density fermentation, which can reach hundreds of grams per liter of cell biomass. This significantly reduces the cost burden associated with large-scale production. As a eukaryote, Pichia pastoris possesses an endoplasmic reticulum-Golgi protein processing pathway, enabling disulfide bond formation and glycosylation. Although the glycosylation pattern differs from that of mammalian cells (high-mannose type versus complex type), product homogeneity can be effectively optimized through in vitro enzymatic modification or genetic engineering (Jacobs et al., 2009; Nett et al., 2011). Recent studies have also explored antibody fragment production in Pichia pastoris. For example, Mokrushina et al. (Mokrushina et al., 2016) optimized the gene constructs of antibody heavy and light chains by identifying proteolytic degradation sites and relevant endogenous proteases, leading to stable production of antibody Fab fragments. Wan et al. (Wan et al., 2013) successfully produced a CD25-targeting antibody in Pichia pastoris expression system, demonstrating its significant functional activity in antibody-dependent cellular cytotoxicity and immunosuppression. Adame produced a functional scFv-6009FV in Pichia pastoris that can inhibit both pure Cn2 neurotoxin and whole venom from N. katiensis, achieving a yield of 31.6 ± 2 mg/L in shake flasks (Adame et al., 2024). Compared to 293F and CHO systems, yeast cultures do not require adherent growth and offer a shorter production cycle (only 7–10 days from cloning to protein purification), making them ideal for rapid-response biomanufacturing, particularly during outbreaks of emerging infectious diseases.

Despite the promising results for antibody fragments (e.g., scFv, Fab), challenges persist in producing full-length IgG antibodies in Pichia pastoris. IgG, a tetramer consisting of two heavy and two light chains, requires precise assembly in the endoplasmic reticulum (ER). The protein folding and secretion machinery in yeast is less efficient for high-molecular-weight proteins, often leading to low secretion efficiency, misfolding, and aggregation (Kuroda et al., 2008; Nylen and Chen, 2018). Our team also attempted to express the full-length IgG1-type 11-2G antibody in yeast; however, the target protein was not detected in the yeast culture supernatant (data was not shown). Therefore, this study shifted focus to engineering single-chain antibody fragments (scFv) by introducing functional domains to construct fusion proteins. This strategy retained antibody activity while circumventing the challenges associated with expressing large, complex IgG structures, thus providing a novel approach for antibody engineering in yeast systems.

To enable successful mass production of the 11-2G antibody in yeast, we further modified the antibody structure to reduce its molecular weight. ScFv, composed of variable regions of heavy and light chains linked by a short peptide, retains the antigen-binding ability of intact antibodies, but its stability and affinity may be affected by structural changes (Bemani et al., 2018). In this study, we first constructed a scFv-his fusion construct for expression in Pichia pastoris. Compared to full-length IgG1, scFv-His exhibited a slightly reduced EC₅₀, but a significant decrease in affinity and IC₅₀ were observed (Fig. 2). Pharmacokinetic data indicated that after injection into mice, scFv-His was nearly undetectable in serum just 1 h post-administration (Fig. 3). This result is consistent with previous studies(Tadokoro et al., 2024), suggesting that further optimization of this structure is required to improve its binding affinity, stability and in vivo half-life.

To address these limitations, multiple engineering approaches have been explored to enhance scFv functionality. Fusion of scFv with the CH3 domain is another strategic approach. The CH3 region plays a crucial role in the stability and antigen-binding capacity of antibodies (Bertz et al., 2013; Traxlmayr et al., 2012) Studies have shown that structural changes in the CH3 domain during antigen binding can affect antibody function, and mutating specific amino acids in this domain can improve antibody stability and function (Rose et al., 2013).Adding the CH3 domain may alter the spatial conformation of scFv, thereby influencing antigen-binding specificity or affinity. Additionally, CH3 domain modifications may influence antibody aggregation state, which in turn affects vivo stability and function (Saito et al., 2019). In this study, introduction of the CH3 domain into the scFv notably enhanced the antibody’s affinity for the antigen and significantly extended its circulation time in mice. However, the IC₅₀ remained at a relatively low level, suggesting that despite improvements in binding strength and in vivo stability, the neutralizing potency was not proportionally increased.

Leucine zippers are commonly used in antibody engineering to enhance the stability and function of antibody fragments. Dimerizing antibody variable fragments using leucine zippers can improve neutralization potency and stability (Li et al., 2015). Introducing leucine zippers into antibody fragments can also enhance serum stability and prolong in vivo half-life (Long et al., 2018). Nevertheless, in antibody fragment design, leucine zippers have been shown to significantly improve biophysical properties and in vivo stability. For example, a zipFv construct targeting rabies virus exhibited higher binding capacity, affinity, and comparable protective efficacy to rabies immunoglobulin (RIG) (Xi et al., 2018). Leucine zippers have also been used to extend the half-life of other biomolecules, such as small molecules or peptide drugs conjugated to human serum albumin (Vantourout et al., 2021). In this study, scFv-ZIP-His exhibited a substantial reduction in EC₅₀, with binding potency approximately 38 times weaker compared to scFv-His. This decrease may be attributed to conformational changes in the antigen-binding region introduced by the leucine zipper modification, which can negatively affect binding orientation and efficiency (Koenig et al., 2017; Ciaccio et al., 2012). Notably, its IC₅₀ remained within an effective range, indicating that inhibitory activity was largely preserved despite the decrease in binding affinity. Moreover, the serum half-life of scFv-ZIP-His in mice was significantly longer than that of scFv-His, suggesting that the inclusion of the leucine zipper domain effectively improved in vivo stability.

Incorporating the Fc region emerged as a pivotal strategy to address the limitations of scFv. The Fc fragment not only enhances antibody stability but also maintains a more favorable spatial conformation for antigen binding, thereby improving affinity and neutralization activity. For example, fusing scFv with the Fc fragment of human IgG1 to generate a novel monospecific tetravalent IgG1-(scFv)₂ molecule resulted in significantly higher binding activity against spike protein variants and enhanced neutralization of pseudoviruses from variants of concern (VOCs) (Gao et al., 2023). Additionally, the Fc fragment mediates effector functions and prolongs in vivo half-life through engineered interactions with natural ligands, thereby improving therapeutic efficacy (Knopp et al., 2016). Among the tested antibodies, scFv-Fc demonstrated the most superior performance across multiple assays. By adding the Fc region of the 11-2G antibody to scFv, the Fc fragment provides additional structural support, enhancing antibody stability in both in vitro and in vivo environments. Furthermore, the Fc fragment interacts with Fc receptors on immune cells, potentiating effector functions such as ADCC and complement-dependent cytotoxicity (CDC). Additionally, the Fc region maintains an optimal spatial conformation for antigen binding, improving the interaction with the SARS-CoV-2 receptor-binding domain and achieving a lower IC₅₀ value, effectively blocking the binding between the RBD protein and the ACE2 receptor (Gao et al., 2023; Knopp et al., 2016).These properties make scFv-Fc a promising molecular tool for combating COVID-19.

ScFv-CH3-His and scFv-ZIP-His exhibited differential performance in EC₅₀, KD, and IC₅₀ parameters. The structural modifications in these constructions influenced antibody functional properties in distinct ways. Although their initial binding efficiency was somewhat compromised, enhancements in binding stability and inhibitory capacity resulted in advantages in KD and IC₅₀ values. These findings suggest that antibody engineering must consider multiple functional requirements and balance trade-offs between different properties to design ideal antibody molecules for specific applications (e.g., therapy, diagnostics).

In vivo pharmacokinetic monitoring further highlighted the critical impact of structural design on antibody properties. ScFv-His, scFv-CH3-His, and scFv-ZIP-His were rapidly cleared from mouse circulation, with scFv-His being undetectable shortly after administration due to its small molecular weight (< 30 kDa) and lack of Fc-mediated protection, leading to rapid glomerular filtration. ScFv-CH3-His and scFv-ZIP-His, with slightly larger molecular weights due to additional domains, were cleared more slowly than scFv-His but still faster than scFv-Fc. In contrast, scFv-Fc exhibited a half-life exceeding 5 days, owing to its interaction with the neonatal Fc receptor (FcRn), which protects it from lysosomal degradation through a recycling mechanism (Jaramillo et al., 2017; Larsen et al., 2018) This prolonged circulation enables sustained therapeutic concentrations in vivo, potentially reducing dosing frequency and improving therapeutic efficacy.

Optimization of scFv-Fc expression in Pichia pastoris was achieved by systematically evaluating key parameters including methanol concentration, medium pH, culture temperature, and induction time using a single-factor experimental design. The results showed that 0.5% methanol effectively activated the AOX1 promoter. A slightly acidic pH of 5.5 optimized the secretion pathway, reducing protein degradation and enhancing secretion efficiency. A culture temperature of 28℃balanced cellular metabolic activity and protein secretion, avoiding stress from high temperatures or slow growth at low temperatures. An induction time of 72 h maximized protein production while preventing yield decline due to nutrient depletion or cell aging. These optimizations increased the shake-flask expression yield of scFv-Fc from 23 mg/L to 110 mg/L, a nearly 4.8-fold improvement, confirming the suitability of the Pichia pastoris secretion system for scFv-Fc production and establishing a robust framework for subsequent scale-up and industrial manufacturing.

In summary, our side-by-side comparison of four scFv-based constructs, scFv-His, scFv-CH3-His, scFv-ZIP-His, and scFv-Fc, demonstrates that fusing the Fc domain to scFv confers the greatest gains in antigen affinity, neutralization potency, and in vivo half-life, effectively recapitulating full-length IgG properties. Although CH3 and leucine zipper fusions improved serum stability compared to scFv alone, each exhibited distinct trade-offs between binding efficiency and pharmacokinetics. Systematic optimization of methanol induction, pH, temperature, and induction time in P. pastoris increased scFv-Fc yield nearly fivefold (from 23 mg/L to 110 mg/L), underscoring yeast’s suitability for cost-effective, high-density antibody production. These results offer actionable design principles and fermentation parameters for rapid, scalable manufacturing of therapeutic antibody fragments against SARS-CoV-2 and future viral threats, highlighting the critical balance between structural modifications, functional performance, and production efficiency.

Ethics approval and consent to participate

Ethical approval for experimental procedures involving animal studies was obtained from the Institutional Review Board of Chongqing Academy of Animal Sciences (Ethics Code: [XKY-20220515]). All methods were performed in compliance with international guidelines for the care and use of laboratory animals and recombinant DNA research.

Consent for publication

All authors consent to the submission and publication of this article in the designated journal.

Competing interests

The authors declare no competing financial or non-financial interests.

Funding

This work was supported by the Chongqing Research Institution Performance Incentive and Guidance Special Project, [Grant No: cstc2021jxj180009].

Author Contribution

Xueqin Liu, Xi Yang designed the study. Xueqin Liu, Qiaoli Lang and Nan Huang performed experiments. Xueqin Liu analyzed data and drafted the initial version. Xiaoyan You, Yuchun Ding，Liangpeng Ge and Meng Wu contributed to manuscript review and revision. All authors have read and consented to the final version of the manuscript.

Acknowledgement

The authors sincerely thank the laboratory members at Chongqing Academy of Animal Sciences for their technical support and valuable discussions during this study. The experiments were conducted in strict accordance with the guidelines and regulations for scientific research integrity.

Data Availability

The datasets generated or analyzed in this study are available from the corresponding author upon reasonable request.

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Development of a Pichia pastoris-Based Expression System for High-Activity Anti-SARS-CoV-2 Neutralizing Antibodies

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