# Structural and Functional Characterization of Anti-SARS-CoV-2 Spike Monoclonal Antibodies Produced via Bicistronic Expression in CHO Cells

## TL;DR

This study compares bicistronic vectors for producing anti-SARS-CoV-2 antibodies in CHO cells, finding that gene order affects expression efficiency and product quality.

## Contribution

The study demonstrates that positioning the light chain first in bicistronic vectors improves mAb expression and quality in CHO cells.

## Key findings

- Vectors with the light chain as the first cistron yielded higher expression levels than those with the heavy chain upstream.
- Comprehensive analytical methods confirmed the quality attributes of the produced monoclonal antibodies.
- The study supports optimization strategies for scalable recombinant antibody production.

## Abstract

Background: Recombinant monoclonal antibodies (mAbs) represent the fastest-growing sector of the biopharmaceutical industry, with their efficient expression being a key technological factor for scalability. Objectives: In this study we compared the performance of two bicistronic vectors, which alternate the positions of the light and heavy chain coding genes, employing a wild-type Encephalomyocarditis virus (EMCV) IRES functional element to drive expression of the second gene. Methods: Using two neutralizing anti-SARS-CoV-2 IgG1 antibodies as model molecules, we conducted transient transfections in the commercially available ExpiCHOTM platform. Following protein A affinity purification and quantification, vectors positioning the light chain as the first cistron consistently yielded higher expression levels than those with the heavy chain upstream. To confirm the quality attributes of the mAbs, we applied a comprehensive analytical workflow, including SDS-PAGE and Western blot for molecular mass and purity, circular dichroism for secondary structure, intrinsic tryptophan fluorescence for tertiary structure, and SEC-HPLC for quaternary structure and aggregate detection. Additionally, we assessed binding affinity to the target using spot blot and surface plasmon resonance, analyzed N-glycosylation profiles by HILIC-HPLC and mass spectrometry, and examined molecular structure by transmission electron microscopy. Results and Conclusions: Together, these results provide insight into the impact of gene positioning within bicistronic vectors on mAb expression efficiency and quality, supporting optimization strategies for scalable recombinant antibody production.