An experimental and computational fluid dynamics study of the influence of fluid mixing and fluid stress on DNA purification

Interest in the field of pure DNA manufacture has been driven in recent years by the explosion of research into gene therapy. Gene therapy technology offers a new paradigm for treating human diseases where defective cells are transformed with gene vectors capable of expressing therapeutic protein. Administration is often via direct injection of naked or lipid-coated plasmid DNA. Plasmid for gene therapy is usually produced in Escherichia coli. The challenge in manufacturing plasmid is primarily the removal of impurities like proteins, lipids, lipopolysaccharides, RNA, non-supercoiled plasmid variants, and host chromosomal DNA. Long chain polymers, such as DNA, are uniquely prone to chain scission at moderate to high fluid stresses that commonly occur in biotechnology equipment. Stress-induced degradation of both plasmid DNA and host chromosomal DNA must be minimised to optimise plasmid yield and purity. Such degradation plays a critical role during alkaline lysis, a key step in DNA isolation. The effect of lysis reagent on DNA stability, the required level of fluid mixing, the effect of the resultant fluid stresses on DNA degradation, and the effect of DNA fragmentation on subsequent downstream purification performance are all poorly understood. This thesis sets out to characterise the effect of lysis reagent concentration on DNA so as to determine the required level of fluid mixing during alkaline lysis, to characterise the effect of the resultant fluid stress on DNA degradation and to determine the effects of stress-induced degradation on downstream processing. The following paragraphs outline the key finding of the thesis, which together provide a framework for the design of a robust lysis process. Two novel HPLC-based procedures were developed, based on polyethylenimine and quaternary amine anion exchange chromatography resins, capable of simultaneously measuring supercoiled plasmid DNA and chromosomal DNA in process samples, in addition the form of the chromosomal DNA. Experiments using E. coli cells containing 6 kb to 116 kb plasmids showed that cell lysate should be maintained below 0.13 + 0.03 M NaOH to prevent irreversible denaturation of supercoiled plasmids and above 0.08 M NaOH to ensure complete conversion of chromosomal DNA to single-stranded form. Conversion of chromosomal DNA to single-stranded form was shown not to significantly affect its removal during alkaline lysis, but was advantageous for subsequent purification. Complete conversion of chromosomal DNA to single-stranded form enabled complete removal by a variety of inexpensive and scaleable purification methods, significantly reducing the cost of plasmid DNA manufacture. Denaturation-renaturation of DNA, either during alkaline lysis or further downstream, was shown to be an effective method of removing non-supercoiled plasmid variants.The level of mixing required is highly dependent on the sodium hydroxide (NaOH) concentration in the lysis buffer. More highly concentrated lysis buffer reduced the overall lysate volume, but rapid mixing was essential to avoid irreversible supercoiled plasmid degradation. Mixing tanks provided adequate mixing only at low NaOH concentrations. Opposed jets provided excellent mixing characteristics for lysis buffer addition, and concentrated NaOH could be used, significantly reducing the volume increase over alkaline lysis. Opposed jets provided a suitable method for denaturing residual double-stranded chromosomal DNA downstream of alkaline lysis. Hence, inexpensive methods for singlestranded DNA removal could be utilised to remove all residual chromosomal DNA. Computational fluid dynamics (CFD) simulations were used to develop appropriate scaling rules for opposed jets, and the CFD predictions were verified against published experimental data. Capillary shear degradation studies with pure solutions of 6kb to 116 kb plasmids and chromosomal DNA, determined that DNA degraded at capillary entrances, not internally. Large plasmids degraded at significantly lower fluid flow rates than small plasmids. CFD simulations were used to determine fluid flow properties (turbulent energy dissipation rates, shear stresses, elongational stresses and pressure drops) at the entrance to, and within, capillaries and to correlate breakage of chromosomal and plasmid DNA with fluid flow parameters. Results indicated that elongational fluid stresses caused significantly more DNA degradation than shear stresses. An assay to monitor plasmid degradation in dilute solutions was developed using Picogreen dye, enabling different size plasmids to be used as probes for fluid stress-induced degradation in large-scale industrial equipment. Results showed that fluid stresses during alkaline lysis led to chromosomal DNA fragmentation. Despite causing chromosomal fragmentation, it was shown that fluid stresses during lysis did not significantly increase chromosomal contamination in cell lysates; chromosomal DNA removal over alkaline lysis/neutralisation not being a strong function of chromosomal DNA size. High levels of fluid stress during the neutralisation step were also shown not to increase chromosomal DNA contamination. The effects of chromosomal DNA fragment size on its removal in different downstream purification steps demonstrated which steps were sensitive to DNA size, enabling better selection of downstream unit operations based on DNA fragmentation upstream.