Process development for a recombinant Chinese hamster ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system.
The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 microM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 10(7) cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system.
Bioengineering Centre, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, 2052, Australia. edwin.huang@unsw.edu.au
Edwin P
EP
Huang
Christopher P
CP
Marquis
Peter P
PP
Gray
D000818:Animals; D019149:Bioreactors; D016466:CHO Cells / Q000166:cytology / Q000502:physiology*; D002104:Cadmium / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000502:physiology; D019382:Human Growth Hormone / Q000096:biosynthesis* / Q000235:genetics; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D010865:Pilot Projects; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015032:Zinc / Q000494:pharmacology*
D008670:Metals; D011994:Recombinant Proteins; D002104:Cadmium; D019382:Human Growth Hormone; D008668:Metallothionein; D015032:Zinc