Freitas S S, Santos J A L, Prazeres D M F
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Lisbon, Portugal.
Biotechnol Prog. 2006 Jul-Aug;22(4):1179-86. doi: 10.1021/bp060052d.
Large-scale processes used to manufacture grams of plasmid DNA (pDNA) should be cGMP compliant, economically feasible, and environmentally friendly. Alcohol and salt precipitation techniques are frequently used in plasmid DNA (pDNA) downstream processing, as concentration and prepurification steps, respectively. This work describes a study of a standard 2-propanol (IsopOH; 0.7 v/v) and ammonium sulfate (AS; 2.5 M) precipitation. When inserted in a full process, this tandem precipitation scheme represents a high economic and environmental impact due to the large amounts of the two precipitant agents and their environmental relevance. Thus, major goals of the study were the minimization of precipitants and the selection of the best operating conditions for high pDNA recovery and purity. The pDNA concentration in the starting Escherichia coli alkaline lysate strongly affected the efficiency of IsopOH precipitation as a concentration step. The results showed that although an IsopOH concentration of at least 0.6 (v/v) was required to maximize recovery when using lysates with less than 80 microg pDNA/mL, concentrations as low as 0.4 v/v could be used with more concentrated lysates (170 microg pDNA/mL). Following resuspension of pDNA pellets generated by 0.6 v/v IsopOH, precipitation at 4 degrees C with 2.4 M AS consistently resulted in recoveries higher than 80% and in removal of more than 90% of the impurities (essentially RNA). An experimental design further indicated that AS concentrations could be reduced down to 2.0 M, resulting in an acceptable purity (21-23%) without compromising recovery (84-86%). Plasmid recovery and purity after the sequential IsopOH/AS precipitation could be further improved by increasing the concentration factor (CF) upon IsopOH precipitation from 2 up to 25. Under these conditions, IsopOH and AS concentrations of 0.60 v/v and 1.6 M resulted in high recovery (approximately 100%) and purity (32%). In conclusion, it is possible to reduce substantially the mass of precipitation agents used without affecting recovery, if a small concession is made regarding purity. This directly translates into an improvement of the process economics and in a reduction of the environmental impact of the process.
用于生产克级质粒DNA（pDNA）的大规模工艺应符合cGMP规范，在经济上可行且对环境友好。乙醇和盐沉淀技术在质粒DNA（pDNA）下游加工中经常分别用作浓缩和预纯化步骤。本研究描述了对标准异丙醇（IsopOH；0.7 v/v）和硫酸铵（AS；2.5 M）沉淀的研究。当应用于整个工艺时，由于这两种沉淀剂用量大及其环境影响，这种串联沉淀方案对经济和环境有很大影响。因此，本研究的主要目标是尽量减少沉淀剂用量，并选择最佳操作条件以实现高pDNA回收率和纯度。起始大肠杆菌碱性裂解液中的pDNA浓度对作为浓缩步骤的IsopOH沉淀效率有很大影响。结果表明，当使用pDNA含量低于80 μg/mL的裂解液时，需要至少0.6（v/v）的IsopOH浓度才能实现回收率最大化，而对于浓度更高的裂解液（170 μg pDNA/mL），可使用低至0.4 v/v的浓度。用0.6 v/v IsopOH产生的pDNA沉淀重悬后，在4℃下用2.4 M AS沉淀，回收率始终高于80%，杂质（主要是RNA）去除率超过90%。一项实验设计进一步表明，AS浓度可降至2.0 M，在不影响回收率（84 - 86%）的情况下，可获得可接受的纯度（21 - 23%）。通过将IsopOH沉淀时的浓缩因子（CF）从2提高到25，可进一步提高连续IsopOH/AS沉淀后的质粒回收率和纯度。在这些条件下，0.60 v/v的IsopOH和1.6 M的AS浓度可实现高回收率（约100%）和纯度（32%）。总之，如果在纯度上稍有妥协，就有可能在不影响回收率的情况下大幅减少沉淀剂用量。这直接转化为工艺经济性的提高和工艺对环境影响的降低。
