Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase‐mediated gene amplification and their stability in the absence of selective pressure

Abstract

Recombinant Chinese hamster ovary (CHO) cells expressing a high-level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, 1.0, and 4.0 microM. The highest producer (HP) subclone was isolated from each MTX level and was characterized with respect to cell growth and antibody production in the corresponding level of MTX. The specific growth rate of the HP subclone was inversely proportional to the MTX level. On the other hand, its specific antibody productivity (qAb) rapidly increased with increasing MTX level up to 0.08 microM, and thereafter, it gradually increased to 20 microg/10(6) cells/day at 4 microM MTX. Southern blot analysis showed that the enhanced qAb at higher MTX level resulted from immunoglobulin (Ig) gene amplification. The stability of the HP subclones isolated at 0.02, 0.08, 0.32, and 1.0 microM MTX in regard to antibody production was investigated during long-term culture in the absence of MTX. The qAb of all subclones significantly decreased during the culture. However, the relative extent of decrease in qAb was variable among the subclones. The HP subclone isolated at 1 microM MTX was most stable and could retain 59% of the initial qAb after 80 days of cultivation. Southern blot analysis showed that this decrease in qAb of the subclones resulted mainly from the loss of Ig gene copies during long-term culture. Despite the decreased qAb, the HP subclone isolated at 1 microM MTX could maintain high volumetric antibody productivity over three months because of improved cell growth rate during long-term culture.

Arginine Residues Are Important in Determining the Binding of Human Monoclonal Antiphospholipid Antibodies to Clinically Relevant Antigens

“…MTX amplification increased the yield of IgG by up to 75-fold in the case of IS4VH/IS4VL. The variable expression from CHO cells is well documented in both our hands (31) and others (38). This phenomenon occurs during subcloning in successive amplification steps when cells can increase the copy number of the dhfr gene without increasing their production rate (38).…”

“…The variable expression from CHO cells is well documented in both our hands (31) and others (38). This phenomenon occurs during subcloning in successive amplification steps when cells can increase the copy number of the dhfr gene without increasing their production rate (38). Thus, cells that do not produce an exogenous protein will usually have a higher growth rate and take over the culture, thereby reducing the chance of selecting a high producer cell.…”

A study of monoclonal antibody‐producing CHO cell lines: What makes a stable high producer?

“…In general the degree of gene amplification is proportional to the level of gene expression (Guarna et al, 1995;Jiang et al, 2006;Pendse et al, 1992). However, high gene copy numbers do not always result in high productivity, probably the result of transcriptional and post-transcriptional limitations in highly amplified subclones (Kim et al, 1998b;Lattenmayer et al, 2007). Gene amplification and high-level expression of a foreign gene product can challenge the cells with different levels of toxicity and metabolic burden.…”

“…In most studies addressing this issue a loss in transgene copy numbers was reported to be the major cause for the decrease in recombinant protein productivity (Fann et al, 2000;Hammill et al, 2000;Kim et al, 1998a,b;Pallavicini et al, 1990). In some cases, no decrease in transgene copy numbers was observed and decreased transcriptional efficiency was discussed to be the reason for the decline in productivity (Kim et al, 1998b). The chromosomal site of integration of a recombinant gene has a major effect on its transcription rate, a phenomenon called the ''position effect'' (Wilson et al, 1990).…”

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“…A detailed understanding of how cells regulate this pathway is a prerequisite for designing genetic strategies for increasing antibody production [1]. Methotrexate (MTX), which is widely used in the creation of high-producing stable cell lines by amplification of gene copy number, provides an effective means to alter Mab production rates for mechanistic studies of the regulation of this pathway [2]. …”