An Optimized Process for Expression, Scale-Up and Purification of Recombinant Erythropoietin Produced in Chinese Hamster Ovary Cell Culture

The DHFR mediated gene amplification employed for selection of recombinantChinese hamster ovary (rCHO) clones was evaluated by single and multiple step selection ofmethotrexate (MTX). Multiple step selection of MTX resulted in cells with high amplifiedcopies of erythropoietin (EPO) gene. Expression of EPO rapidly increased with increasingMTX concentration up to 1000 nM, further increased to 2000 nM does not affect theexpression. After the MTX selection, cells grown in the presence of MTX were more stableand retained similar amounts of EPO expression & gene copies until 50 doublings. Whereas,cells grown in the absence of MTX were unstable and retained only 50% of initial EPOexpression & gene copies at the 50th doubling. Scale-up from culture flask to wave bioreactorincreases EPO yield, the novel wave bioreactor with a working volume of 1 liter couldproduce more than 0.56 g of EPO in 3 weeks, with a volumetric productivity of 24 mg/l/dayand specific productivity of 5.93 μg/106cells/day. Simple two step chromatographicpurification process was developed with relatively high yield and purity of EPO using bluesepharose affinity chromatography combined with Q-sepharose ion exchangechromatography. A single protein zone with molecular mass of 32-38kDa was appeared inSDS-PAGE analysis of the purified rHuEPO. Densitometric scanning of gels demonstrated,>90% purity of final EPO, giving a 42% recovery.