Predicting the expression of recombinant monoclonal antibodies in Chinese hamster ovary cells based on sequence features of the CDR3 domain

Abstract

Despite the development of high-titer bioprocesses capable of producing >10 g L(-1) of recombinant monoclonal antibody (MAb), some so called "difficult-to-express" (DTE) MAbs only reach much lower process titers. For widely utilized "platform" processes the only discrete variable is the protein coding sequence of the recombinant product. However, there has been little systematic study to identify the sequence parameters that affect expression. This information is vital, as it would allow us to rationally design genetic sequence and engineering strategies for optimal bioprocessing. We have therefore developed a new computational tool that enables prediction of MAb titer in Chinese hamster ovary (CHO) cells based on the recombinant coding sequence of the expressed MAb. Model construction utilized a panel of MAbs, which following a 10-day fed-batch transient production process varied in titer 5.6-fold, allowing analysis of the sequence features that impact expression over a range of high and low MAb productivity. The model identified 18 light chain (LC)-specific sequence features within complementarity determining region 3 (CDR3) capable of predicting MAb titer with a root mean square error of 0.585 relative expression units. Furthermore, we identify that CDR3 variation influences the rate of LC-HC dimerization during MAb synthesis, which could be exploited to improve the production of DTE MAb variants via increasing the transfected LC:HC gene ratio. Taken together these data suggest that engineering intervention strategies to improve the expression of DTE recombinant products can be rationally implemented based on an identification of the sequence motifs that render a recombinant product DTE.

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

“…[6][7][8][9] The ratio of LC:HC expression affects both mAb expression level and quality, such as aggregation and glycosylation. [8][9][10][11][12][13][14][15][16][17] Tricistronic vectors using internal ribosome entry sites (IRES) or 2A peptides to express the LC, HC and a selection marker gene in one transcript provides accurate control of the relative expression of LC over HC. 6 Expressing the LC, HC and selection marker genes on one transcript also minimizes non-expressing clones because none of the 3 genes will be expressed should the vector be fragmented.…”

A protein chimera strategy supports production of a model “difficult‐to‐express” recombinant target

“…Amino acid sequences and secondary structures of specific proteins contain features associated with limitations in recombinant protein production in CHO cells 30, 31, 32. Functionally related proteins TIMP‐2 (well‐secreted) and TIMP‐3 (poorly secreted), with significant sequence and structural identity have been used as models to test sequence‐specific determinants of secretion in a transient CHO system 26.…”

“…Reports surrounding production of monoclonal antibodies (mAbs) suggest that unique sequence features influence the effectiveness of recombinant protein production 30, 31, 32. For bacterial expression systems, computational tools have been implemented to predict of surface properties to allow rational design of recombinant targets with increased protein solubility of otherwise insoluble or poorly soluble target proteins 29, 33, 34, 35, 36.…”

Unraveling what makes a monoclonal antibody difficult‐to‐express: From intracellular accumulation to incomplete folding and degradation via ERAD

“…Since these artificially generated molecules have never undergone an evolutionary optimization, mammalian cellular expression systems often show low performance with cell lines displaying inferior product titer, low specific productivity, or insufficient product quality (Johari, Estes, Alves, Sinacore, & James, ; Lee et al, ). In addition, even some classical mAb candidates can be difficult‐to‐express (DTE), which might result in the termination of the product development (Pybus et al, ). To provide access to sufficient amounts of innovative drugs, there is an urgent need to identify any rate limiting processing step during cellular synthesis and transport, which might represent a secretion bottleneck.…”