The absence of effect of gene copy number and mRNA level on the amount of mAb secretion from mammalian cells

Abstract

Recombinant human antibody production represents a major growing class of biopharmaceuticals based on the technological progress within the last decades especially in CHO cells. The HIV neutralizing human monoclonal antibody 2F5 was developed as hybridoma from human lymphocyte preparations. In order to estimate the potential of recombinant 2F5-expressing CHO cells, we generated different recombinant CHO cell lines by varying regulatory sequences, the codon usage, the signal peptides, and the transfection technique. These 2F5-expressing cell lines were developed by selection of the best producer, clone homogeneity, and clone stability. The gene copy number of the clones differed significantly due to methotrexate amplification. In one cell line, we identified only one copy of heavy chain and two copies of light chain. Neither the gene copy number nor the promoter was found to influence the amount of transcript exclusively emphasizing the positioning effect of the transgene. Messenger RNA levels were highest in 2F5/CO and may have resulted from a combination of the promoter and codon-optimized sequences, but unexpectedly, the amount of secreted product was not elevated in this configuration. In our example, translational and post-translational limitations are responsible for decreased antibody secretion.

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells

“…However they had lower transcription and expression level. The results are consistent with former studies in which GCN did not reflect the content of mRNA [36]. Studies have shown that mammalian cells lines which contain higher copies of the heterologous plasmids were are susceptible to silencing [37, 38], and early methylation coincide with high transgene copy numbers [26].…”

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

“…In both, CHO as well as HEK cells, IgM-012 producing cell line showed several times lower specific productivity and antibody titer levels than IgM-617 producing cell line (sevenfold difference in CHO cells, 22-fold difference in HEK cells). Although CHO and HEK IgM-617 cell lines manifested same or higher gene copy numbers for HC, LC, and JC coding genes than IgM-012 cell lines (with dramatically higher values for CHO IgM-617 cells), this fact is not reflected at the transgene transcript and specific productivity level, similarly as shown previously (Lattenmayer et al 2007b;Reisinger et al 2008). These studies as well as our results indicated that many of coding gene copies are probably incorporated into places, which are not as favorable for transcription initiation or are not active at all.…”

Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastorisan alpha 1-antitrypsin in Pichia pastoris

“…Moreover, there are potential bottlenecks for the optimum expression of every heterologous protein including promoter strength (Kim et al 2009), the gene copy number (Vassileva et al 2001), the codon usage of recombinant gene (Xu et al 2010), and finally processing in ER and Golgi and secretion out of cell in case of secretary protein (Inan et al 2006). Based on previous experiments, each of these factors can be considered for elevating the heterologous protein expression, though there are no reliable means of predicting the precise effect of each factor (Strausberg and Strausberg, 2001;Luo et al 2006;Geymonat et al 2007;Ramachandran et al 2008;Reisinger et al 2008;Pérez-García et al 2010). AAT protein was expressed and partially characterized in the previous work (Arjmand et al 2011).…”