Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor.
The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.
Department of Chemical and Biological Engineering, University of Colorado, CB 424, Boulder, Colorado 80309-0424, USA.
Matthew L
ML
Lipscomb
Mark C
MC
Mowry
Dhinakar S
DS
Kompala
D000818:Animals; D019149:Bioreactors / Q000382:microbiology*; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods*; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D010865:Pilot Projects; D011401:Promoter Regions, Genetic / Q000235:genetics; D011991:Receptors, Virus / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012212:Rheology / Q000295:instrumentation / Q000379:methods
C111417:Fam89b protein, mouse; D006023:Glycoproteins; D008565:Membrane Proteins; D011991:Receptors, Virus; D011994:Recombinant Proteins