Download (direct link):
cell wall debris and some intact cells;
low molecular mass metabolites;
After lysis is complete, the next step can entail the addition of a high-salt neutralization solution, such as potassium acetate. This promotes formation of aggregates of genomic DNA (gDNA) and SDS-protein complexes, which can subsequently be removed by centrifugation or filtration. The plasmids can then be themselves precipitated from the resultant solution by the addition of appropriate solvent (usually either isoproponal or ethanol). Upon resuspension, the plasmid preparation can then be subjected to chromatographic purification. The major contaminants likely still present include RNA, gDNA fragments, nicked or other plasmid variants and endotoxins. Gel filtration chromatography can effectively remove contaminants that differ substantially in shape/size from the desired plasmid. These can include most gDNA fragments, RNA and (most) endotoxins. It can also achieve partial removal of plasmid variants, such as open circular plasmids, from the main (supercoiled) plasmid preparation. Ion exchange can remove many protein contaminants, as well as RNA. However, gDNA and endotoxins generally co-purify with the plasmid DNA. Additional chromatographic approaches based upon reverse-phase and affinity systems have been developed at laboratory scale at least.
A significant feature of plasmid purification employing capture chromatography (i.e. involves plasmids binding to the chromatographic beads) is the low plasmid-binding capacities observed.
NUCLEIC ACID THERAPEUTICS 481
Cellular recovery and lysis
Removal of cell debris
Concentration, if required
Formulation and packaging
Figure 11.10. Overview of the manufacturing process for the large-scale production of plasmid DNA. Refer to the text for further details
The pore size of commercially-available capture chromatographic media is insufficiently large to allow entry of plasmids, restricting binding to the bead surface. Binding capacities can, therefore, be 100-fold or more lower than those observed when the same medium is used to purify (much smaller) therapeutic proteins (Chapter 3).
Purified plasmids may then be analysed using various analytical techniques. Freedom from contaminating nucleic acid and/or proteins can be assessed electrophoretically. Endotoxin and sterility tests would also be routinely undertaken. The purified plasmid DNA must next be formulated to yield the final non-viral delivery system. Formulation studies relating to such systems remain an area requiring further investigation. Most work reported to date relates to formulating and stabilizing lipoplex-based gene delivery systems. Aqueous suspensions of these (and other) non-viral based systems tend to quickly aggregate (in a matter of minutes to hours). In order to circumvent this problem, the final delivery systems were often actually formulated at the patients bedside in earlier clinical trials.
Research aimed at identifying appropriate stabilizing excipients and formulation formats is ongoing. Simple freezing is an option, particularly as frozen formulations would be immune to agitation-induced aggregation. However, the process of freezing, particularly slow freezing, in itself induces aggregation. This can be minimized by flash freezing (e.g. by immersion in liquid nitrogen), although this approach may not prove practicable at an industrial scale. The addition of cryoprotectants may help minimize this problem and initial studies indicate that various sugars (e.g. glucose, sucrose and trehalose) show some potential in this regard. Another avenue under investigation relates to the generation of a final freeze-dried product. Again issues, such as the (relatively) slow freezing process characteristic of industrial-scale freeze-driers, complicate attaining this goal in practice.
Gene therapy and genetic disease
Well over 4000 genetic diseases have been characterized to date. Many of these are caused by lack of production of a single gene product or are due to the production of a mutated gene product incapable of carrying out its natural function. Gene therapy represents a seemingly straightforward therapeutic option which could correct such genetic-based diseases. This would be achieved simply by facilitating insertion of a healthy copy of the gene in question into appropriate cells of the sufferer.
Although simple in concept, the application of gene therapy to treat or cure genetic diseases has, thus far, made little impact in practice. The slow progress in this regard is likely due to a number of factors. These include: