Modified monoclonal antibody production kinetics kappa/gamma mRNA levels, and metabolic activities in a murine hybridoma selected by continuous Culture

Abstract

During long-term continuous culture of the hybridoma cell line 11317, a better-producing subclone (I1317-SF11), giving improved productivity, has been selected. The comparison of the original cell line (I1317-DC) with this subclone revealed that although the growth patterns of both clones were similar, both in continuous and in batch cultures, considerable differences could be seen between the clones with respect to monoclonal antibody (MAB) accumulation, MAB production rate, the levels of mRNA coding for heavy and light chains of IgG, and some metabolic activities. In continuous culture as well as in batch culture, I1317-SF11 showed increased levels of mRNA coding for kappa and gamma chains compared with I1317-DC and/or a modified ratio of the mRNA species when compared to that in I1317-DC. Using pulse experiments, it could be established that the biosynthesis of both chains was augmented in I1317-SF11. Although the kappa and gamma mRNA levels were modified or inversed for I1317-SF11, the cells always synthesized more kappa than gamma chains. The overall increase in the synthetic activity of I1317-SF11 is suggested as one reason for the considerable increase of IgG productivity and product accumulation in continuous culture as well as in repeated batch cultures. Tests concerning metabolic activity revealed that I1317-SF11 had a predominantly glycolytic metabolism independent of growth requirements, whereas for I1317-DC the metabolism became increasingly glycolytic with increased growth. The antibody yield coefficient of I1317-SF11 on glutamine was significantly higher than that of I1317-DC for the continuous culture, whereas the antibody coefficients on glucose were almost similar for both clones under the different culture conditions used. Both antibody coefficients were considerablly influenced by the specific growth rate.All these facts together lead to the conclusion that subclone I1317-SF11 uses more of the energy available, or it was the energy and/or precursors available for the synthesis and production of MAB more efficiently than the thesis and production of MAB more efficiently than the original cell line. Although the levels of mRNA coding for heavy and light chains of IgG were modified, it could be confirmed that the overall regulation of MAB-synthesis and -production occurs post-translationally and that at higher growth rates, more biosynthetic activity is diverted to biomass production.

Comparative proteomic analysis of GS‐NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate

“…In addition, a number of previous studies have shown no correlation between qMab and the cellular availability of HC or LC mRNA using clonally derived mammalian cell lines or hybridomas (Flickinger et al, 1992;Kim et al 1998a;Leno et al 1992aLeno et al , 1992b. However, qMab has been shown to correlate more strongly with cellular LC mRNA content than with HC mRNA content in other studies Merten et al, 1994;Strutzenberger et al 1999). Therefore, while intracellular Mab mRNA content may be a poor indicator of qMab, these reports and others (Park and Ryu, 1994) suggest that intracellular Mab protein content is positively correlated with qMab.…”

Cell line‐specific control of recombinant monoclonal antibody production by CHO cells

“…Indeed, our parameter sensitivity analysis demonstrates that the importance of HC mRNA abundance as a productivity constraint decreased with increasing qMab as cells with high qMab are limited more by HC translation. Interestingly, some reports have shown either no correlation between mRNA levels and qMab (Reisinger et al, 2008;Smales et al, 2004), or a stronger relationship between LC mRNA and qMab indicating that these productivity correlations can be specific to the cell line and/or gene expression system Merten et al, 1994;Strutzenberger et al, 1999).…”

Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature?

“…In the case of Mabs, which require the coordinated expression, folding and assembly of both heavy (HCs) and light chains (LCs), a variety of studies using clonally-derived mammalian cell lines or hybridomas all demonstrate that at higher rates of Mab production, there is no correlation between qMab and the corresponding cellular availability of HC or LC mRNA (Barnes et al, 2004;Flickinger et al, 1992;Kim et al, 1998;Leno et al, 1992). Although in some studies, qMab correlates more strongly with LC mRNA content than HC mRNA content Merten et al, 1994;Strutzenberger et al, 1999). Accordingly, the process of Mab folding and assembly in the endoplasmic reticulum (ER) (Fig.…”