Development of a pilot-scale bacterial fermentation for plasmid-based biopharmaceutical production using a stoichiometric medium
Development of a pilot-scale bacterial fermentation for plasmid-based biopharmaceutical production using a stoichiometric medium
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DOI：
10.1007/s12257-007-0080-2
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发表时间：
2008-03-01
影响因子：
3.2
通讯作者：
Forde, Gareth M.
中科院分区：
文献类型：
--
作者：
Danquah, Michael K.;Forde, Gareth M.
认识到质粒DNA（pDNA）分子作为载体在治疗和预防新出现的疾病方面的潜在功效，使人们对抗击全球流行病产生了信心。这是由于与细胞转染和靶向中的病毒载体相比，与pDNA载体相关的安全性问题接近于零。人们对pDNA载体的潜力给予了相当大的关注，但相对较少考虑大量生产以满足当前不断增长的需求的实际挑战。通过采用化学计量设计的生长培养基开发了中试规模发酵方案，该生长培养基在摇瓶环境中证实了pUC 19和pEGFP-N1转化到E. coliDH 5 α和E. coliJM 109。E. coliDH 5 α-pUC 19在化学计量培养基中的最大质粒体积产量和比产量分别为62.6 mg/L和17.1 mg/g（mg质粒/g细胞干重）。流加发酵E.在甘油底物上的coliDH 5 α-pUC 19表现出有史以来报道的最高中试规模质粒比产率之一，为48.98 mg/g，体积产率为0.53 g/L。高质粒比产量的获得降低了质粒生产成本，并通过降低细胞内杂质的比例提高了下游工艺的有效性。还认为逐步升温诱导的效果使ColE 1起始质粒复制最大化。(C)KSBB
The recognition of the potential efficacy of plasmid DNA (pDNA) molecules as vectors in the treatment and prevention of emerging diseases has birthed the confidence to combat global pandemics. This is due to the close-to-zero safety concern associated with pDNA vectors compared to viral vectors in cell transfection and targeting. Considerable attention has been paid to the potential of pDNA vectors but comparatively less thought has been given to the practical challenges in producing large quantities to meet current rising demands. A pilot-scale fermentation scheme was developed by employing a stoichiometrically-designed growth medium whose exceptional plasmid yield performance was attested in a shake flask environment for pUC19 and pEGFP-N1 transformed into E. coliDH5 alpha and E. coliJM109, respectively. Batch fermentation of E. coliDH5 alpha-pUC19 employing the stoichiometric medium displayed a maximum plasmid volumetric and specific yield of 62.6 mg/L and 17.1 mg/g (mg plasmid/g dry cell weight), respectively. Fed-batch fermentation of E. coliDH5 alpha-pUC19 on a glycerol substrate demonstrated one of the highest ever reported pilot-scale plasmid specific yield of 48.98 mg/g and a volumetric yield of 0.53 g/L. The attainment of high plasmid specific yields constitutes a decrease in plasmid manufacturing cost and enhances the effectiveness of downstream processes by reducing the proportion of intracellular impurities. The effect of step-rise temperature induction was also considered to maximize ColE1-origin plasmid replication. (C) KSBB.