Antibodies | Structural and Functional Characterization of Anti-SARS-CoV-2 Spike Monoclonal Antibodies

This study systematically compares the impact of light chain and heavy chain gene positioning in bicistronic expression vectors on monoclonal antibody expression efficiency, and validates antibody structural and functional characteristics through multiple analytical techniques, providing critical references for optimizing recombinant antibody production strategies.

 

Literature Overview
This article titled 'Structural and Functional Characterization of Anti-SARS-CoV-2 Spike Monoclonal Antibodies Produced via Bicistronic Expression in CHO Cells' published in the journal Antibodies reviews and summarizes expression and quality attribute analyses of anti-SARS-CoV-2 spike protein monoclonal antibodies. The study systematically evaluates expression levels of two distinct bicistronic vector configurations in CHO cells, employing SDS-PAGE, Western blot, circular dichroism, endogenous tryptophan fluorescence, SEC-HPLC, dot blot, surface plasmon resonance, N-glycosylation analysis, and transmission electron microscopy for comprehensive structural and functional characterization.

Background Knowledge
Monoclonal antibodies (mAbs) represent one of the fastest-growing product categories in biopharmaceuticals, where efficient expression remains critical for scalable production. Proper light chain (LC) and heavy chain (HC) expression ratios are essential for correct folding, assembly, and secretion during expression. Current mainstream expression strategies include co-transfection of two independent plasmids, multi-promoter vectors, F2A peptide-based multicistronic systems, and IRES (internal ribosome entry site)-dependent expression systems. The IRES element, derived from viral mRNA's 5' untranslated region, enables cap-independent translation of downstream genes in bicistronic vectors. However, translation efficiency and expression ratio modulation through different IRES elements require further optimization. Additionally, critical quality attributes of mAbs include secondary/tertiary/quaternary structural integrity, glycosylation modifications, and antigen-binding capacity. Contemporary anti-SARS-CoV-2 antibody research emphasizes both expression yields and broad neutralization capabilities against viral variants. This study provides experimental evidence by constructing bicistronic vectors using native EMCV IRES elements to systematically evaluate expression efficiency and quality attributes of two neutralizing antibodies in CHO cells.

 

 

Research Methods and Experiments
The research team constructed two bicistronic expression vectors with either light chain (LC) or heavy chain (HC) genes positioned upstream of the IRES element, comparing expression levels using transient transfection in CHO cells (ExpiCHO™ system). Structural and functional analyses were conducted through protein A affinity purification, quantitative analysis, SDS-PAGE, Western blot, circular dichroism spectroscopy, endogenous tryptophan fluorescence, SEC-HPLC, dot blot, surface plasmon resonance (SPR), N-glycosylation analysis (HILIC-HPLC and LC-MS), and transmission electron microscopy (TEM). Statistical analysis employed one-way ANOVA with Sidak multiple comparisons tests to evaluate expression differences between constructs.

Key Conclusions and Perspectives

• Expression levels significantly increase when light chain genes are positioned upstream of the IRES element compared to heavy chain upstream configurations
• Structural analyses confirm both constructs maintain proper immunoglobulin G1 (IgG1) molecular characteristics, including β-sheet-dominated secondary structures, tryptophan-environment tertiary structures, and single elution peaks indicating correct quaternary assembly
• Both antibodies bind spike proteins from D614G, Delta, Gamma, and Beta variants but show no binding capacity toward Omicron BA.2
• LC-MS analysis identifies core-fucosylated G0-F as predominant N-glycans, consistent with CHO cell expression features
• TEM confirms typical Y-shaped structure of mAb LBL-01 but achieved only low-resolution 3D reconstruction due to particle limitations and antibody flexibility, necessitating cryo-EM for antigen-binding conformation analysis

Research Significance and Prospects
This study provides experimental evidence for optimizing bicistronic antibody expression strategies, demonstrating that prioritized light chain expression enhances mAb yield while reducing aggregation. The differential variant-binding patterns of the two antibodies suggest antigen recognition diversity potentially linked to light chain variable gene usage. Future research should focus on IRES element sequence optimization, stable CHO cell line screening, and high-resolution structural analysis techniques like cryo-EM to resolve antigen-binding conformational dynamics. Broadly neutralizing antibodies against immune-evasive variants such as Omicron remain a critical development priority.

 

 

Conclusion
This study systematically evaluated the impact of light chain and heavy chain gene positioning in bicistronic vectors on anti-SARS-CoV-2 spike protein antibody expression efficiency and quality attributes. Results demonstrate that positioning the light chain upstream of the IRES element significantly improves expression levels while reducing aggregation. Structural analyses confirm both antibodies maintain correct secondary, tertiary, and quaternary structures with variant-binding capabilities against multiple SARS-CoV-2 strains, except Omicron BA.2. The findings emphasize gene positioning's critical role in expression optimization and provide experimental evidence for improving monoclonal antibody design in CHO cell expression systems, with broad applications in biopharmaceutical development.

 

Federico Francisco Marsili, Fernanda Bittencourt de Aquino, Hiam Rodrigo da Silva Arruda, Renato Sampaio Carvalho, and Leda dos Reis Castilho. Structural and Functional Characterization of Anti-SARS-CoV-2 Spike Monoclonal Antibodies Produced via Bicistronic Expression in CHO Cells. Antibodies.