| Autor: |
Megan Mason, Bernadette Sweeney, Katharine Cain, Paul Stephens, Susan T. Sharfstein |
| Jazyk: |
angličtina |
| Rok vydání: |
2014 |
| Předmět: |
|
| Zdroj: |
Antibodies, Vol 3, Iss 3, Pp 253-271 (2014) |
| Druh dokumentu: |
article |
| ISSN: |
2073-4468 |
| DOI: |
10.3390/antib3030253 |
| Popis: |
Reduced culture temperature is an increasingly popular practice to improve recombinant protein yields in CHO cells. Recent studies have attributed the enhancement of protein titers at sub-physiological temperatures to increased mRNA levels as well as extended stationary phase. We observed that reducing the culture temperature arrested cell growth, prolonged viability, and increased cell size. However, the reduced culture temperature had a differential effect on protein and mRNA expression of closely related antibody mutants from stable cell lines. The highly expressing mutant (Ala) exhibited similar or decreased specific productivity and decreased volumetric productivity over the culture lifetime at 32 °C compared to 37 °C. In contrast, the specific and volumetric productivity of the poorly expressing mutant (Gly) was enhanced at the lower culture temperature. The difference in specific productivity was reflected in the amounts of heavy- and light-chain mRNA. Analysis of the secondary and tertiary configurations of the purified antibodies by circular dichroism revealed fundamental structural differences imposed by the Ala to Gly mutation as well as reduced culture temperature. We propose that the effect of reduced culture temperature on expression is protein-dependent; protein folding fidelity and assembly is improved at lower temperatures, enhancing the expression of proteins that have a propensity to misfold. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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