Key determinants in the occurrence of clonal variation in humanized antibody expression of CHO cells during dihydrofolate reductase mediated gene amplification

Recombinant Chinese hamster ovary (CHO) parental clones expressing a humani zed antibody against S surface antigen of hepatitis B virus were obtained b y cotransfection of heavy chain (HC) and light chain (LC) cDNA expression v ectors into dihydrofolate reductase (DHFR)-deficient CHO cells. When 23 rep resentative parental clones were subjected to stepwise selection for increa sing methotrexate (MTX) resistance, such as 0.02, 0.08, 0.32, and 1.0 muM, their clonal variations in regard to antibody expression were found to be s ignificant. Among 23 parental clones, only one clone (hu17) showed the sign ificant increment of specific antibody productivity (q(Ab)) With increasing MTX concentration up to 0.32 muM. Compared with the parental clone (hu17), the q(Ab) Of hu17 resistant at 0.32 muM MTX (hu17-0.32) was enhanced appro ximately 12.5-fold. To clarify the reason for the occurrence of clonal vari ations, Southern blot analyses of chromosomal DNAs derived from each amplif ied clone at 0.32 muM MTX were performed. Only the hu17-0.32 clone did not experience severe genetic rearrangement during gene amplification, and it h ad only one 49-kb amplification unit including the LC and HC cDNAs. A fluor escent MTX competition assay showed that the resistance against MTX toxicit y of the other clones without enhanced q(Ab) at 0.32 muM MTX was obtained b y mechanisms such as an impaired MTX transport system. Taken together, the data obtained here show that clonal variations in regard to antibody expres sion are found to be significant because clones can acquire MTX resistance by mechanisms other than DHFR-mediated gene amplification despite the stepw ise selection.