Manufacture of plasmid DNA

The overall generalized approach used to produce plasmid DNA for the purposes of gene therapy trials is presented in Figure 14.11. Prior to its manufacture, researchers would have constructed an appropriate vector housing the therapeutic gene and introduced it into a producer microorganism, usually E. coli. Routine large-scale plasmid manufacture then entails culture of a batch of producer microorganisms by fermentation, followed by plasmid extraction and purification. In this regard, the overall approach used resembles the approaches taken in the large-scale manufacture of recombinant therapeutic proteins, as described in Chapters 5 and 6.

Industrial-scale microbial fermentation (upstream processing) has also been described in Chapter 5, to which the reader is referred. Fermentation promotes microbial cell replication and, thus, the biosynthesis of large quantities of plasmid. Subsequent to fermentation, the microbial cells are harvested (collected) by either centrifugation or microfiltration. Following resuspension in a low volume of buffer, the cells must be disrupted in order to release the plasmids therein. This appears to be most commonly achieved by the addition of a lysis reagent consisting of NaOH and SDS. The combination of high pH and detergent action disrupts the microbial cell wall/membranes with consequent release of the intracellular contents. In addition to the desired plasmid DNA, this crude mixture will also contain various impurities, which must be removed by subsequent downstream processing steps. Notable impurities include:

• 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 and

Figure 14.11 Overview of the manufacturing process for the large-scale production of plasmid DNA. Refer to the text for further details

SDS-protein complexes, which can subsequently be removed by centrifugation or filtration. The plasmids can then themselves be precipitated from the resultant solution by the addition of appropriate solvent (usually either isopropanol or ethanol). Upon resuspension, the plasmid preparation can then be subjected to chromatographic purification. The major contaminants likely still present include RNA, genomic DNA 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 genomic DNA 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, genomic DNA 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. involving plasmids binding to the chromatographic beads) is the low plasmid-binding capacities observed. 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 media are used to purify (much smaller) therapeutic proteins (Chapter 6).

Purified plasmids may then be analysed using various analytical techniques. Freedom from contaminating nucleic acid/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/stabilizing lipoplex-based gene delivery systems. Aqueous suspensions of these (and other) non-viral-based systems tend to aggregate quickly (in a matter of minutes to hours). In order to circumvent this problem, the final delivery systems were often actually formulated at the patient bedside in earlier clinical trials.

Research aimed at identifying appropriate stabilizing excipients/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.

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