Characterization of the effects of Bcl-2 and Bcl-xl deletion mutant expression in cell lines used for antibody production.
Strategies to maximize monoclonal antibody (MAb) yields by in vitro production methods entail that hybridoma cells be maintained at high density. Approaches to increase culture density and antibody yields from hybridomas by inhibiting apoptosis through over-expression of exogenous Bcl-2 family genes have produced variable results. In order to determine if expression of mutant forms of Bcl-2 and Bcl-xl could increase viable culture densities and batch MAb yields when compared to parental cell lines, recombinant delta loop deletion mutant of these apoptotic inhibitory genes were expressed in a myeloma and two hybridoma cell lines. Expression of either Bcl-2-delta or Bcl-xl-delta in P3x63Ag8.653 myeloma cells did not significantly increase viable cell densities in cultures over time. However, the rapid post-peak decline in viable cell density was significantly reduced in Bcl-xl-delta-expressing hybridoma cell lines 552 and 7.16.4 and in Bcl-2-expressing hybridoma 7.16.4. Significant increases in MAb yield were only observed in cultures of Bcl-xl-delta-expressing hybridoma 7.16.4. Annexin staining in hybridoma 7.16.4 confirmed that apoptosis was the primary means of cell death in this cell line, and expression of Bcl-2-delta and Bcl-xl-delta inhibited programmed cell death. These results suggest that cell viability in cultures can be improved by transfection and selection of hybridomas that express delta loop deletion mutant forms of Bcl-2 family genes; however, improvements in MAb yields are dependent upon the genetic background of each manipulated cell line.
Peterson
Norman C
NC
Department of Comparative Medicine, Johns Hopkins University, Baltimore, Maryland, USA. petersonn@medimmune.com
Servinsky
Matthew
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D017209:Apoptosis / Q000235:genetics*; D002452:Cell Count; D002460:Cell Line; D002470:Cell Survival; D019254:Genes, bcl-2; D006801:Humans; D006825:Hybridomas; D051379:Mice; D050505:Mutant Proteins / Q000096:biosynthesis; D014162:Transfection; D051020:bcl-X Protein / Q000096:biosynthesis / Q000235:genetics
D000911:Antibodies, Monoclonal; D050505:Mutant Proteins; D051020:bcl-X Protein