Sangeetha L. Sagar

Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification

Abstract The past several years have witnessed a rapidly increasing number of reports on utilizing plasmid DNA as a vector for the introduction of genes into mammalian cells for use in both gene therapy and vaccine applications. “Naked DNA vaccines” allow the foreign genes to be transiently expressed in transfected cells, mimicking intracellular pathogenic infection and triggering both the humoral and cellular immune responses. While considerable attention has been paid to the potential of such vaccines to mitigate a number of infections, substantially less consideration has been given to the practical challenges of producing large amounts of plasmid DNA for therapeutic use in humans, for both clinical studies and, ultimately, full-scale manufacturing. Doses of naked DNA vaccines are on the order of milligrams, while typical small-scale Escherichia coli fermentations may routinely yield only a few mg/l of plasmid DNA. There have been many investigations towards optimizing production of heterologous proteins over the past three decades, but in these cases, the plasmid DNA was not the final product of interest. This review addresses the current state-of-the-art means for the production of plasmid DNA at large scale in compliance with existing regulatory guidelines. The impact of the nature of the plasmid vector on the choice of fermentation protocols is presented, along with the effect of varying cultivation conditions on final plasmid content. Practical considerations for the large-scale purification of plasmid DNA are also discussed.

The Development of Recombinant Subunit Envelope-Based Vaccines to Protect Against Dengue Virus Induced Disease

Challenges associated with the interference observed between the dengue virus components within early tetravalent live-attenuated vaccines led many groups to explore the development of recombinant subunit based vaccines. Initial efforts in the field were hampered by low yields and/or improper folding, but the use of the Drosophila S2 cell expression system provided a mechanism to overcome these limitations. The truncated dengue envelope proteins (DEN-80E) for all four dengue virus types are expressed in the S2 system at high levels and have been shown to maintain native-like conformation. The DEN-80E proteins are potent immunogens when formulated with a variety of adjuvants, inducing high titer virus neutralizing antibody responses and demonstrating protection in both mouse and non-human primate models. Tetravalent vaccine formulations have shown no evidence of immune interference between the four DEN-80E antigens in preclinical models. Based on the promising preclinical data, the recombinant DEN-80E proteins have now advanced into clinical studies. An overview of the relevant preclinical data for these recombinant proteins is presented in this review.

Plasmid DNA purification by selective calcium silicate adsorption of closely related impurities.

The selective adsorption of supercoiled plasmid, open‐circular plasmid, and genomic DNA to gyrolite, a compound from the class of crystalline calcium silicate hydrates, is investigated and exploited for purification purposes. Genomic DNA and open‐circular plasmid bind to gyrolite adsorbents with greater affinity than the more conformationally constrained supercoiled plasmid. As such, the gyrolite adsorbents are an economical and scaleable alternative to chromatographic purification for the removal of DNA impurities from solutions containing supercoiled plasmid. The advantage of gyrolite adsorbents is their lower unit price and ability to selectively adsorb DNA impurities without binding supercoiled plasmid under certain conditions. The effects of ionic strength, temperature, chelating agent, divalent cation, and lyotropic salts on adsorption of highly purified plasmid are studied to understand the forces that bind DNA to gyrolite, a structure with hydrophilic and hydrophobic characteristics. The results indicate that DNA binding is governed by hydrogen bonding, electrostatic bridging with divalent cations, shielding of electrostatic repulsion, hydrophobic adsorption, and disruption of integral surface water layer on gyrolite. On the basis of results from a range of Hofmeister series salts, strongly hydrated anions may enhance DNA adsorption by promoting hydrophobic interactions between DNA and gyrolite. Conversely, the very weakly hydrated chaotrope I− may enhance adsorption by strongly associating with hydrophobic siloxanes of gyrolite, thereby disrupting an integral water layer, which competes for hydrogen bonding sites.

Large-Scale, Nonchromatographic Purification of Plasmid DNA

A large-scale approach to the purification of plasmid DNA has been developed that overcomes many of the limitations of current chromatography-based processes. The process consists of a scaleable lysis using recombinant lysozyme and a rapid heating and cooling step followed by a selective precipitation with cetyltrimethylammonium bromide (CTAB). Calcium silicate batch adsorption is then utilized to remove residual genomic DNA, linear plasmid, open circular plasmid, endotoxin, detergents, and proteins. Finally, a concentration and diafiltration step utilizing ultrafiltration and a terminal sterile filtration complete the process. The final product exceeds the requirements for clinical-grade plasmid DNA, and the process has been scaled up to yield an average of 18 +/- 4 g (over five lots) of pharmaceutically pure plasmid DNA per 140 L of lysate (from approx 1.3 kg Escherichia coli dry cell weight).

Scaleable purification of adenovirus vectors.

Adenovirus vectors currently are being evaluated in gene delivery studies ranging from prophylactic vaccination to therapeutic gene therapy. The quantity of purified virus required for these studies necessitate that purification methods must shift from classical density gradient ultracentrifugation to scaleable approaches. A methodology is described herein using batch centrifugation, tangential flow ultrafiltration, and chromatography to purify adenovirus particles at a scale of approximately 10(13) viral particles. This method has been demonstrated to easily scale an additional 40-fold. While purification of human adenovirus type 5 is exemplified, modifications are suggested for the purification of other serotypes.

Monitoring of RNA Clearance in a Novel Plasmid DNA Purification Process

As the field of plasmid DNA‐based vaccines and therapeutics matures, improved methods for impurity clearance monitoring are increasingly valuable for process development and scale‐up. Residual host‐cell RNA is a major impurity in current large‐scale separation processes for the production of clinical‐grade plasmid DNA. Current RNA detection technologies include quantitative rtPCR, HPLC, and fluorescent dye‐based assays. However, these methodologies are difficult to employ as in‐process tests primarily as a result of impurity and buffer interferences. To address the need for a method of measuring RNA levels in various process intermediates, a sample pretreatment strategy has been developed that utilizes spermidine affinity precipitation to eliminate a majority of solution impurities, followed by a quantitative precipitation with alcohol to concentrate RNA and allow detection at lower concentrations. RNA concentrations as low as 80 ng/mL have been measured using detection with gel electrophoresis and 20 ng/mL if microplate‐based detection with Ribogreen fluorescent dye is used. The assay procedure has been utilized to troubleshoot RNA clearance issues encountered during scale‐up of a novel, non‐chromatographic purification process for plasmid DNA. Assay results identified residual liquor removal inadequacies as the source of elevated RNA levels, enabling process modifications in a timely fashion.