An innovative platform to improve asymmetric bispecific antibody assembly, purity, and expression level in stable pool and cell line development

Abstract

Asymmetric Immunoglobulin G (IgG)-like antibody is a fast-growing class of therapeutics among various bispecific antibody formats. However, the expression of correctly assembled products with high purity and yield remains challenging. Approaches have been investigated to facilitate antibody heterodimerization, including protein engineering, cell line development (CLD) screening, and vector element customization. In this paper, an innovative CLD platform was developed to express HLX101, an asymmetric IgG antibody consisting of two different heavy chains and two common light chains. We combined Leap-In Transposase® technology, a novel transposon-based integration strategy with two ORF “dual” and four ORF “quad” expression vector designs, and a microcapillary electrophoresis (µM) based high-throughput screening method, which efficiently identified clones expressing heterodimers at early stages of CLD. In this single case study of HLX101, the percentage of single clones expressing desired heterodimer among outgrown clones isolated from the stable pool was increased from 6% to 35% by varying dual vector ratios during co-transfection. This number was further improved to 90 % by a “quad” vector design. The final titer of a single clone fed-batch production reached 6 g/L. The established CLD platform demonstrated a simple and effective way to obtain high-yield and high-quality cell lines for asymmetric antibody production.