| Abstract [eng] |
Few years ago, Sicor Biotech UAB had faced an issue – decrease of E.coli K802/pT7a-GCSF fermentation process parameters and increase of impurity of RRT 1.10 in the final product. These problems appeared after changed lot of yeast extract. In this study, we found out, that these decrease of fermentation parameters and increase of impurity level were caused by the lower iron level than usually in one of culture media components – yeast extract. These issues can be eliminated by adding iron and manganese ions to the culture media. This also reduces level of RRT 1.10, but additional impurities of lower molecular mass forms of target protein appear. After series of experiments required concentration of iron ions in yeast extract was determined. 49±5 µg/g is an optimal iron concentration in yeast extract. This concentration keeps fermentation parameters at optimal level, reduces level of RRT 1.10 and also does not induce production of smaller molecular mass target protein forms. Moreover, in this study, characterization of RRT 1.10 was performed. This impurity consists of two components: deamidated forms of target protein and mutant of target protein (W119→F119). Increased level of RRT 1.10 was caused by higher amount of mutant, which was generated during starve of iron. |
