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 μM. 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 μM, and thereafter, it gradually increased to 20 μg/106 cells/day at 4 μM 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 μM 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 μM 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 μM MTX could maintain high volumetric antibody productivity over three months because of improved cell growth rate during long‐term culture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:73–84, 1998.

Osmoprotective effect of glycine betaine on foreign protein production in hyperosmotic recombinant Chinese hamster ovary cell cultures differs among cell lines

“…Cell growth of CS13-1.00* and CS13-0.02* was recovered but at a cost of a significantly reduced q Ab . They were derived from the same parental clone (CS13), but had different Ig gene copynumbers (Kim et al, 1998). Thus, regardless of gene dosage, the effect of glycine betaine inclusion in the hyperosmolar medium on improved foreign protein production is cell-line-specific.…”

“…The gene copynumber of CHO/dhfr-B22-4 was approximately 33, whereas the heavy-and light-chain gene copies of CS13-1.00* were approximately 88 and 285, respectively (Kim et al, 1998). In highly amplified rCHO cells, the pool of cellular machinery may not be sufficient to produce foreign protein and grow simultaneously under osmoprotective conditions.…”

“…CS13-0.02* and CS13-1.00* express a chimeric antibody directed against the S surface antigen of hepatitis B virus. They were derived from the same parental clone but were selected at different MTX levels (0.02 and 1.0 M, respectively) (Kim et al, 1998).…”

Artificial Transcription Factors Increase Production of Recombinant Antibodies in Chinese Hamster Ovary Cells

“…CRL-11268) were grown in Dulbecco's modified Eagle medium (DMEM) (JBI) containing 10% (v/v) heat-inactivated FBS. The SH2-0.32 and SO-0.32 cell lines were constructed as described previously (Kim et al 1998(Kim et al , 2001 and were grown in a-MEM or Iscove's modified Dulbecco's medium (IMDM) (JBI) that contained 10% (v/v) dialyzed FBS and either 320 or 80 nM methotrexate (MTX), respectively.…”

Expression and purification of recombinant human Angiopoietin-1 produced in Chinese hamster ovary cells

“…To determine the specific growth rate (μ) and specific rhAng1 productivity (q Ang1 ) of clones at each MTX level, batch cultures were performed in six-well tissue culture plates containing 3 mL of Isocove's modified Dulbecco's medium (IMDM) with 5% dialyzed fetal bovine serum (dFBS) and the corresponding level of MTX. The μ and q Ang1 were evaluated as described previously (Kim et al 1998;Hwang et al 2005). Briefly, the μ was calculated by plotting the logarithm of viable cell concentration versus culture time during the exponential growth phase.…”