Epitope tagging: development of an innovative ionic-strength modulate system for enhaced detection, quantification and purification of recombinant proteíns

LEOPOLD, M. J.; KRATJE, R. B.; OGGERO M.; CEAGLIO N.

Viña del Mar. Valparaíso

Simposio; X Simposio Latinoamericano de Tecnología de Cultivo de Células Animales (X SLATCC); 2024

Pontificia Universidad Católica de Valparaíso. Universidad de Chile. Universidad de Concepción.

Background: Epitope tagging is a well-established and a very useful tool for tracking recombinantproteins. These highly specific systems use peptide tags and anti-peptide monoclonal antibodies(mAbs). In particular, in our lab, we developed a mAb called CC1H7 which recognizes an epitopepresent in a novel O-glycosylated tag named mGMOP and exhibits ionic strength-modulated binding. This characteristic has turned this system into a versatile strategy to set up methods such as ELISA, chromatography, and western blot.Methods: To begin with this study, two new molecules were obtained: one with a single copy andanother with four copies of mGMOP tag fused to human interferon-α2b (hIFN-α2b). These proteinswere produced in suspension CHO-K1 cells. mAb-CC1H7 producing hybridoma was adapted tosuspension in vitro culture. For this, FBS was progressively reduced from 5 to 1.25% in adherenceculture medium, and then cells were directly subcultured into serum-free medium with agitation. The mAb specificity was assessed through a specific indirect ELISA and western blot. Protein-A purified mAb facilitated techniques for detecting, quantifying, and purifying proteins labeled with mGMOP peptide. An experimental design explored the impact of antichaotropic salts and pH on CC1H7-mGMOP interaction. Optimal conditions were applied in an immunoaffinity chromatography. Another experimental design was performed to find optimal conditions for quantifying tagged proteins using a competition ELISA.Results: Interferon variants with different number of mGMOP tags were successfully obtained fordevelopment of detection, quantification, and purification assays. Hybridoma mAb productivityincreased concomitantly with serum reduction in culture media. The mAb specificity after adaptation was demonstrated by indirect ELISA and by western blot assay, showing a detection limit of 10 ng for both proteins. Using an experimental design, a competition ELISA with appropriate sensitivity and detection limit to quantify variants containing different copies of mGMOP peptide could be developed.Also, another experimental design allowed to identify optimal high ionic strength conditions thatenhanced epitope paratope interaction. In this way, incubation of the complex in the presence ofNa2SO4 1 M (pH 8) increased the apparent affinity about 15 times compared to the control (withoutsalt). The use of salts allowed the improvement of the dynamic capacity of the resin. Under theseconditions, purification achieved 80% yield and 96% purity.Conclusion: mAb-CC1H7 was successfully obtained through in vitro, serum-free, suspension culture, and used to develop a mAb-mGMOP system useful for detection, quantification and purification of proteins tagged with the O-glycosylated peptide.