Process development for production of human granulocyte-colony stimulating factor by high cell density cultivation of recombinant Escherichia coli.
The fed-batch process using glucose as the sole source of carbon and energy with exponential feeding rate was carried out for high cell density cultivation of recombinant Escherichia coli BL21 (DE3) expressing human granulocyte-colony stimulating factor (hG-CSF). IPTG was used to induce the expression of hG-CSF at 48 g dry cell wt l(-1) during high cell density culture of recombinant E. coli BL21 (DE3) [pET23a-g-csf]. The final cell density, specific yield and overall productivity of hG-CSF were obtained as approximately 64 g dry cell wt l(-1), 223 mg hG-CSF g(-1) dry cell wt and 775 mg hG-CSF l(-1) h(-1), respectively. The resulting purification process used cell lysis, inclusion body (IB) preparation, refolding, DEAE and Butyl-Sepharose. Effects of different process conditions such as cell lysis and washing of IB were evaluated. The results reveal that the cells lyzed at 1,200 bar, 99.9% and Triton removed about 64% of the LPS but sarcosyl had no effect on removal of nucleic acids and LPS. Further analysis show that DEAE column removes DNA about 84%. Cupper concentration was identified as parameter that could have a significant impact on aggregation, as an unacceptable pharmaceutical form that decrease process yields. The purity of purified hG-CSF was more than 99%. Also the comparison of activity between purified hG-CSF and commercial form do not show valuable decrease in activity in purified form.
Khalilzadeh
Rasoul
R
Faculty of Engineering, Tarbiat Modares University, Tehran, Iran.
Mohammadian-Mosaabadi
Jafar
J
Bahrami
Ali
A
Nazak-Tabbar
Ahmad
A
Nasiri-Khalili
Mohammad Ali
MA
Amouheidari
Alireza
A
D019149:Bioreactors; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis* / Q000302:isolation & purification*; D006801:Humans; D017510:Protein Folding; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor