Efficient production of recombinant mAbs mediated by a MAR-enhanced transposon vector combined with blasticidin selection in CHO cells

Abstract

Recombinant antibodies, primarily produced in Chinese hamster ovary (CHO) cells, are widely used to treat various diseases. For industrial production, a rapid and efficient method to screen stable, high-expressing clones is essential. However, conventional screening based on random integration is often cumbersome and labor intensive. This study establishes a novel strategy for generating stable, high-yielding clones by combining a MAR-based piggyBac (PB) transposon semitargeted integration system with blasticidin (BSD) selection. Compared to the random integrated vector pMAR-mAb, the MAR-PB system increases the titers (3.95- to 5.61-fold) and specific protein productivity (Qp; 4.28- to 6.07-fold) of four monoclonal antibodies in stable cell pools. When compared to PB-only vectors, the MAR-PB transposon system enhances the titers (by up to 2.50-fold) and Qp (1.96- to 2.77-fold), respectively. The increased antibody production correlates with elevated mRNA expression. Furthermore, this approach increases the proportion of high-expressing clones by more than 10-fold and significantly improves volumetric yield. Importantly, this approach promotes the long-term stability of recombinant mAb expression for over 60 generations. Transcriptome analysis reveals that the system modulates genes involved in DNA binding, transcriptional regulation, and protein binding. In conclusion, the MAR-based PB transposon system combined with BSD selection presents a significant improvement for efficiently generating high-yielding and stable CHO cell clones, offering a valuable tool for recombinant antibody production.

1. Introduction

The global biopharmaceutical industry is growing rapidly, with monoclonal antibodies (mAbs) as a key class of therapeutic proteins used to treat tumors, inflammation, autoimmune disorders and other diseases. The market is projected to reach $856.1 billion by 2030 [1]. Mammalian cells, particularly Chinese hamster ovary (CHO) cells, are the primary expression system for their human-like post-translational modifications (PTMs), compatibility of suspension and adherent culture, and ability to facilitate correct protein folding [2–4]. However, traditional expression vectors lack homology to host DNA, and their random integration into host chromosomes causes cell clone heterogeneity. Additionally, integration into heterochromatic regions may silence the gene of interest (GOI), requiring extensive and labor-intensive screening to obtain stable, high-yielding cell clones [5].

Strategies to enhance protein of interest (POI) expression include genetic editing of cell lines [6,7], optimization of expression vectors [8–12], culture media [13–16] and cultivation processes [17,18]. Among these, selecting efficient expression vectors is an effective approach to increase POI expression in CHO cells [19,20]. Transposon semitargeted integration systems have been proven effective for the production of recombinant proteins [21,22]. As natural mobile genetic elements, transposons comprise a donor vector and a helper vector (transposase) [23]. Transposase recognizes the inverted terminal repeats (ITRs) of the donor vector and uses a “cut-and-paste” mechanism to integrate the GOI into a highly transcriptionally active site, enhancing POI expression [24,25]. Commonly used transposons include Tol2 from the hAT gene family [26], Sleeping Beauty (SB) from the Tc1/mariner superfamily [27], and the insect-derived piggyBac (PB) [28]. Among these, the PB transposon system has broad activity and efficiently mediates transposition of DNA fragments up to 14 kb [29]. Co-transfection of donor and helper vectors enables efficient integration of GOIs in mammalian cells [30–33].

Additionally, chromatin regulatory elements (CREs) mitigate transgene silencing, with matrix attachment regions (MARs) known to prevent heterochromatin-mediated gene silencing. Ley et al. [34] evaluated a piggyBac transposon system containing human MARs and observed that MARX-29 increased protein expression by over 2-fold in CHO cells. Although combining regulatory elements with transposon vectors improves protein expression, an efficient, rapid method for screening stable, high-expressing cell clones remains crucial. In our previous study, we identified an efficient MAR consensus sequence that enhances transgene expression [35]. However, whether these favorable properties can be combined with the transposon system remains largely untested. Moreover, antibiotic selection is widely used to generate stable cell pools for protein expression [36,37]. Blasticidin S (BSD), a nucleoside antibiotic isolated from Streptomyces coelicolor, inhibits translation by binding to the peptidyl transferase center of the large ribosomal subunit [38,39]. By utilizing the PB transposon system to mediate integration into transcriptiona