Guilherme N.M. Ferreira

Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks

Gene therapy is a promising process for the prevention, treatment and cure of diseases such as cancer, acquired immunodeficiency syndrome (AIDS) and cystic fibrosis. One of the methods used to administer therapeutic genes is the direct injection of naked or lipid-coated plasmid DNA, but this requires considerable amounts of plasmid DNA. There are several problems and bottlenecks associated with the design and operation of large-scale processes for the production of pharmaceutical-grade plasmid DNA for gene therapy.

Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications

Interest in producing large quantities of supercoiled plasmid DNA has recently increased as a result of the rapid evolution of gene therapy and DNA vaccines. Owing to the commercial interest in these approaches, the development of production and purification strategies for gene-therapy vectors has been performed in pharmaceutical companies within a confidential environment. Consequently, the information on large-scale plasmid purification is scarce and usually not available to the scientific community. This article reviews downstream operations for the large-scale purification of plasmid DNA, describing their principles and the strategy used to attain a final product that meets specifications.

Development of Process Flow Sheets for the Purification of Supercoiled Plasmids for Gene Therapy Applications

Human clinical trial of gene therapy with nonviral vectors demands large amounts of pharmaceutical-grade plasmid DNA. Since standard molecular biology methods cannot be used for this purpose, there is a need for the development of processing methodologies for the large-scale production and purification of plasmids. This work describes several studies that were undertaken during the development of process flow-sheets for the downstream processing of supercoiled plasmids. Anion-exchange HPLC was used as a routine technique for monitoring plasmid purity in process streams. The use of RNase or high temperatures during alkaline lysis was proved unnecessary. Instead, RNA could be completely removed by performing sequentially clarification with a chaotropic salt, concentration with PEG, and ion-exchange and size-exclusion chromatography. Also, clarification of streams by precipitation was independent of the chaotropic salt used. Furthermore, by proceeding directly from cell lysis to chromatography it was possible to obtain plasmid with purity/quality identical to that of the one obtained when clarification and concentration were included in the process. This strategy has the advantage of increasing the overall process yield to 38%. The plasmid thus purified was depleted of RNA, chromosomal DNA, and proteins. Additionally, no animal-derived enzymes, alcohols, or toxic solvents were used, rendering validation potentially easier. The results described in this report also indicate that downstream processing times and costs can be considerably reduced without affecting plasmid purity.

Anion exchange purification of plasmid DNA using expanded bed adsorption.

Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH=8.0) buffer at an upward flow of 300 cmh-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1. Purification factors of 36±1 fold, 26±0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed–values of 35±2 and 5±0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.

Studies on the batch adsorption of plasmid DNA onto anion-exchange chromatographic supports.

The adsorption of a supercoiled 4.8 kbp plasmid onto quaternary ammonium anion‐exchangers was studied in a finite bath. Equilibrium experiments were performed with pure plasmid, at 25 °C, using commercial Q‐Sepharose matrices differing in particle diameter (High Performance, 34 μm; Fast Flow, 90 μm; and Big Beads, 200 μm) and a recently commercialized ion‐exchanger, Streamline QXL (dp = 200 μm) at different salt concentrations (0.5, 0.7, and 1 M NaCl). Plasmid adsorption was found to follow second‐order kinetics (Langmuir isotherm) with average association constants KA = 0.32 ± 0.12 mL μg−1 and KA = 0.25 ± 0.15 mL μg−1 at 0.5 and 0.7 M NaCl, respectively. The maximum binding capacities were not dependent on the ionic strength in the range 0.5−0.7 M but decreased with increasing particle diameter, suggesting that adsorption mainly occurs at the surface of the particles. No adsorption was found at 1 M NaCl. A nonporous model was applied to describe the uptake rate of plasmid onto Streamline QXL at 0.5 M NaCl. The overall process rate was controlled by mass transfer in the regions of low relative amounts of adsorbent (initial stages) and kinetically controlled in the later stages of the process for high relative amounts of adsorbent. The forward reaction rate constant (k1 = 0.09 ± 0.01 mL mg−1 s−1) and film mass transfer coefficient (Kf = (6 ± 2) × 10−4 cm s−1) were calculated. Simulations were performed to study the effect of the relative amount of adsorbent on the overall process rate, yield, and media capacity utilization.

A comparison of gel filtration chromatographic supports for plasmid purification

Two gel filtration chromatographic supports, Superose 6 and Sephacryl S1000 SF, were used to purify supercoiled plasmids using a 4.8 kb plasmid as a model. Both supports purified the plasmid from RNA and other small molecular weight contaminants, as shown by agarose electrophoresis and ion exchange HPLC, with an overall yield of 70%. Sephacryl S1000 SF was the better support as it resolved supercoiled, relaxed, linear and concatamer plasmid forms, and chromosomal DNA.

Purification of plasmids for gene therapy and DNA vaccination

This chapter covers the different aspects of the production and purification of plasmids for gene therapy and DNA vaccination. Process issues are extensively covered and complemented with information related to plasmid DNA structure, vector construction, product specifications and quality assurance and control.

Analysis and use of endogenous nuclease activities in Escherichia coli lysates during the primary isolation of plasmids for gene therapy.

Two important issues in the downstream processing of plasmids for gene therapy are the stability of plasmids in the process streams, and the presence of contaminating host RNA. Results with a 4.8-kb plasmid harbored in a non-nuclease-deficient strain of Escherichia coli show that, in spite of the harsh conditions during alkaline lysis, a fraction of endogenous nucleases remains active, degrading both RNA and genomic and plasmid DNA. Although it is possible to minimize plasmid degradation by decreasing temperature and reducing processing times, the presence of endogenous nucleases can be used advantageously to purify the plasmid streams. The kinetics of nucleic acid degradation showed that, by controlling the incubation at 37 degrees C, it was possible to degrade RNA selectively, while maintaining plasmid integrity. A reduction of 40% in RNA content was obtained, corresponding to a 1.5-fold increase in plasmid purity using high-performance liquid chromatography (HPLC). This strategy is simple and straightforward, and the increase in processing time and the associated plasmid loss (9%) are fully justified by the purity increase. Furthermore, the use of endogenous RNase activity is clearly advantageous over alternative procedures, such as the addition of external RNase, in terms of cost, validation, and compliance with guidelines from regulatory agencies.

Purification of supercoiled plasmid DNA using chromatographic processes

The interest in purifying injectable‐grade plasmid DNA has increased with the development of gene therapy and DNA vaccination technologies. In this paper we develop a method for purifying a 4.8 kb plasmid based on chromatographic processes. An NaCl gradient was optimized on a Q Sepharose® column and plasmid was eluted at 800– 820 mM NaCl in a broad peak. Supercoiled plasmid was isolated after a final Sepharcryl S1000 SF® gel filtration step. Final plasmid preparation was depleted of proteins and RNA, as revealed by the BCA assay and 1% agarose gel electrophoresis. Copyright