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

Abstract

In this study we compare the cellular control of recombinant human IgG 4 monoclonal antibody (Mab) synthesis in different CHO cell lines. Based on comprehensive empirical analyses of mRNA and polypeptide synthetic intermediates we constructed cell line-specific mathematical models of recombinant Mab manufacture in seven GS-CHO cell lines varying in specific production rate (qMab) over 350-fold. This comparative analysis revealed that control of qMab involved both genetic construct and cell line-specific factors. With respect to the former, all cell lines exhibited excess production of light chain (LC) mRNA and polypeptide relative to heavy chain (HC) mediated by more rapid LC transcription and enhanced LC mRNA stability. Downstream of this, cell lines differed markedly in their relative rates of recombinant mRNA translation, Mab assembly and secretion although HC mRNA abundance and the rate of HC translation generally exerted most control over qMabthe latter being directly proportional to qMab. This study shows that (i) cell lines capable of high qMab exceed a threshold functional competency in all synthetic processes, (ii) the majority of cells in parental and transfected cell populations are functionally limited and (iii) cell engineering strategies to increase Mab production should be cell line specific. Biotechnol. Bioeng. 2010;106: 938-951.

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

“…In fact, CHO cells are known for being able to adapt to changes in process conditions, which has been exploited in the industry to generate clonally derived cell lines with enhanced manufacturing capabilities (Frye et al, 2016). Moreover, the majority of cells in host cell lines seem to be intrinsically incapable of high production of MAb, and universally competent cells are likely relatively rare cases (O'Callaghan et al, 2010). Functional heterogeneity residing in the host cell population is typically referred to as clonal variation (Fig.…”

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

“…Several studies have investigated the underlying bottlenecks that limit cell specific productivities with, in part, contradictory results. This is not surprising in view of the phenotypic and genomic diversity observed in CHO cells [4][5][6]. Although the precise genes involved vary from case to case, certain cellular pathways and functions are frequently found to influence cellular productivity (Table 1).…”

“…These studies were initially driven by the hope of finding the rate limiting step for protein production and identify the one gene or the few genes that would either increase cellular productivity if appropriately engineered, or that would allow the efficient identification of high producing cell lines. However, by now it has been acknowledged that it is not a single step or even pathway that controls productivity but the coordinated interplay and cooperation of multiple steps, none of which must be allowed to become limiting, and all of which have to be expressed and active in a balanced way [4,7,8]. In fact, Clarke et al 2011 found a set of 287 genes for the prediction of cellular productivity in the model they established.…”

“…Overall, the focus on fold changes distracts attention from changes in gene expression that are regulated at a more physiologic scale such as for instance transcription factors that have an effect on the cell upon slight changes in their expression [30]: if within a pathway each individual gene is upregulated 1.4-fold, none of these will show up in the list of differentially expressed genes when using a threshold of 1.5, however, the overall flux through the pathway will increase significantly. [7,66,69,70] Down regulation of growth [7,32,66,68] In case of antibodies: enhanced [4,69,71,[76][77][78]] assembly and secretion due to a surplus of light chain over heavy chain These challenges in identifying relevant genes and understanding their biological roles are further compounded by the fact that many genes have functional assignments in the GO or KEGG annotation databases that reflect the context in which they were first discovered, which may be completely irrelevant to cells grown in suspension in a protein free medium (such as organism development, tissue specific functions and similar). Moreover, pleiotropy, the phenomenon where a single gene may have multiple functions, which may not all be known [31], can make the interpretation of results difficult.…”

Improved antibody production in Chinese hamster ovary cells by ATF4 overexpression

“…The heavy and light chains are connected by several inter-and intra-disulfide bonds. The assembly of heavy and light chains inside the ER is strictly controlled (O'Callaghan et al 2010). The heavy chains cannot be secreted without being assembled with the light chains because the CH1 domain controls the assembly and secretion of IgG (Feige et al 2009).…”