Biomedical subjects
Publications and source records attributed to D M Prazeres.
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.
This work presents studies on the interactions of supercoiled plasmid DNA and Escherichia coli genomic DNA (gDNA) and RNA, with an hydrophobic interaction chromatography (HIC) gel, obtained by derivatisation of Sepharose CL-6B with 1,4-butanediol diglycidyl ether. Nucleic acids purified from E. coli were injected separately in the above HIC column and eluted with 1.5 M (NH4)2SO4 in the buffer. The column was able to separate single-stranded from double-stranded nucleic acids. RNA and denatured gDNA were retarded in a different way due to the interactions of the exposed hydrophobic bases with the ligands. Supercoiled plasmid DNA, on the contrary, eluted in the flowthrough.
The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded plasmid DNA. RNA, denatured genomic and plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled plasmid, were retained and separated from nonbinding plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded plasmid was recovered after HIC. RNA and denatured plasmid in the final plasmid preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/microg pDNA and 0.048 EU/microg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns.
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.
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.
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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.
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.
Large scale manufacturing of gene vectors such as plasmid DNA is an important issue in gene therapy. Anion-exchange chromatography is fundamental in the downstream processing of plasmids both as a process and analytical technique. This work reports the use of Q-Sepharose columns (1, 10 and 40 ml) for the preparative purification of plasmid pUC18. NaCl gradient elution enabled the isolation of supercoiled plasmid from low-M(r) RNA, cDNA and plasmid variants. A compact covalently closed, supercoiled form of denatured plasmid carrying large stretches of single-stranded DNA was identified as one of the major contaminants. Anion-exchange HPLC on a Poros QE 20 column was used to quantify plasmid yield. Supercoiled plasmid was recovered in a single fraction with a 62 +/- 8% yield. Loadings higher than 40 micrograms/ml gel could be used but at the expense of a loss of resolution between open circular and supercoiled forms. Plasmid quality was evaluated by gel electrophoresis, restriction analysis, transformation experiments and protein assays.
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.
Biotransformation of poorly water soluble compounds in organic media by immobilized enzyme and whole cells is illustrated in this paper taking the following examples from the author's laboratory: (1) controlled hydrolysis of triglycerides and synthesis reactions by a recombinant lipolytic enzyme (cutinase); (2) enzymatic synthesis of dipeptides; (3) continuous production of isovaleraldehyde by Gluconobacter oxydans in isooctane; and (4) sitosterol side chain cleavage by Mycobacterium sp. The role of water and organic solvent are evaluated, namely the increase in the volumetric productivity of the reaction system and the shift of the reaction equilibrium in favour of product synthesis. High product yields have been obtained due to the reduction of substrate/product inhibition. Biocatalyst stability in the presence of the organic phase was also performed.
A commercially available super absorbent polymer from Hoechst (Sanwet IM-5000-SG) was tested for the concentration of dilute solutions of bovine serum albumin (BSA). A systematic study was undertaken in order to evaluate the possibility of scaling-up the process. The polymer was first characterized by determining the swelling ratio (or mass increase) in aqueous solution as a function of time, temperature, pH, salt and polymer concentration. The swelling ratio was found to be independent of the polymer concentration, temperature (range 15-50 degree C), and pH (range 4-10), but decreased significantly with an increase in NaCL concentration. The polymer was capable of absorbing as much as 300-times its own weight in water, when using the most favorable conditions (0 mM NaCL). BSA was concentrated up to 3.5-times when using the appropriate polymer concentration. The recovery of protein was around 100% for concentration factors below 2.0, but decreased for higher concentration factors. As expected from the characterization results, higher amounts of polymer were needed to concentrate BSA solutions with higher salt concentrations. The performance of the process improved when using lower concentration BSA solutions (0.15 to 0.5 mg ml-1). The initial volume (10 to 500 ml) had a slight effect on the process due to a decrease in the rate of the absorption process. The concentration factor was predicted from the NaCL and polymer concentrations through a semi empirical model.
The synthesis of dipeptide AcPheLeuNH2 catalyzed by alpha-chymotrypsin encapsulated in TTAB/octanol/heptane reversed micelles was investigated in a tubular ceramic membrane reactor, operated in a batch mode. The reaction medium conditions (TTAB concentration, buffer molarity, and pH) were optimized using a factorial design in order to achieve maximum synthesis rates. Hydrated reversed micelles permeated through the membrane together with the substrate ester, dipeptide, and by-products. However, as a result of the low solubility of the peptide in the reaction medium, selective precipitation occurred, thus enabling the complete retention of the solid product by the ultrafiltration membrane and therefore an integration of a separation step in the biotransformation process. In spite of the continuous accumulation of solids inside the reactor, constant permeation flow rates could be maintained throughout the operation. The influence of alpha-chymotrypsin, TTAB, and water concentration on the kinetics and mass transfer of the system was also investigated. The behavior of the system during a continuous experiment was also evaluated.
The lipolytic activity of Chromobacterium viscosum lipase B (EC 3.1.1.3.; triacylglycerol hydrolase) solubilized both in water and AOT/isooctane reversed micelles has been investigated using triolein as a substrate. The influence of relevant parameters in the catalytic activity such as temperature, pH, surfactant and substrate concentrations, and water content was tested and compared in both media. A study of stability of the lipase was carried out, with particular reference to the influence of pH. Three major effects of the encapsulation of the lipase in the micelles were observed: increased activity (up to 7 times higher than in water), greater stability, specially at pH 7, and higher resistance to thermal deactivation.
BACKGROUND: The basic and applied research efforts devoted to the development of DNA vaccines must be accompanied by manufacturing processes capable of being scaled up and delivering a clinical-grade product. This work describes a rapid process of this kind, based on hydrophobic interaction chromatography (HIC) for the production of milligram quantities of an experimental DNA rabies vaccine. Its properties and protective activity are tested in comparison with the same plasmid DNA purified with a commercial kit. METHODS: The experimental DNA vaccine encoding the rabies virus glycoprotein was amplified in vivo in Escherichia coli. The plasmid was isolated by alkaline lysis, pre-purified and concentrated by isopropanol and (NH4)2SO4 precipitation, and purified by HIC and dialysis. Product quality was controlled by using high-performance liquid chromatography (HPLC), Southern slot blotting, agarose gel electrophoresis, the kinetic-QCL Limulus amoebocyte lysate assay, and protein assays. The expression of the rabies virus glycoprotein was tested in vitro in neuroblastoma cells. The production of rabies-virus-neutralising antibodies and the protection against an intracerebral virus challenge were tested in mice. RESULTS: One hundred and forty-two milligrams of the plasmid, with an HPLC purity greater than 99% were obtained from 4.5 l medium. Control analysis showed that the vaccine conforms to specifications in terms of impurities (endotoxins, genomic DNA, RNA, proteins). Furthermore, the final experimental vaccine induces rabies-virus-neutralising antibodies and protects mice against a rabies virus challenge. CONCLUSIONS: This study demonstrates that the method developed for the purification of milligram amounts of plasmid delivers an endotoxin-free, experimental rabies DNA vaccine, with protective activity similar to that obtained with the vaccine purified using a commercial kit.
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 degrees C, using commercial Q-Sepharose matrices differing in particle diameter (High Performance, 34 microm; Fast Flow, 90 microm; and Big Beads, 200 microm) and a recently commercialized ion-exchanger, Streamline QXL (d(p) = 200 microm) 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 K(A) = 0.32+/-0.12 mL microg(-)(1) and K(A) = 0.25+/-0.15 mL microg(-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 (k(1) = 0.09+/-0.01 mL mg(-1) s(-1)) and film mass transfer coefficient (K(f) = (6 +/- 2) x 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.
This paper describes the bioconversion of 2-methyl-1,3-propanediol to (R)-beta-hydoxyisobutyric acid (HIBA) by Acetobacter ALEI in a hollow fiber membrane bioreaction system arrangement that allows the integration of three liquid phases: the aqueous bioconversion phase, the organic phase consisting of a solution of trioctyl phosphine oxide (TOPO) in isooctane, and the third phase consisting of a basic stripping solution that allows reextraction of HIBA from the organic phase. A comparison of HIBA mass transfer experiments was carried out in the membrane reactor with two and three phases for different pH and TOPO concentrations. The use of the three-phase arrangement allows the extraction of high quantities of HIBA from the aqueous medium (higher than 85%) independently of the pH, whereas in the two-phase system the percentage of HIBA extracted from the aqueous medium was lower, 42% in the best case, and strongly influenced by the pH. The percentage of the extractive agent TOPO in the organic phase influenced on the mass transfer rate in both bi- and triphasic arrangements. By simply integrating the re-extraction phase in the system it was possible to increase the extraction yield by 2-fold, reduce the amount of TOPO by 4-fold, and operate at the more favorable pH 4. A bioconversion experiment was done in these conditions (pH = 4, TOPO = 5%) to confirm the advantages of including the third stripping solution. Fed-batch operation of the triphasic membrane reactor was maintained for more than 20 h, reaching an HIBA concentration in the stripping solution of 29 g L(-)(1).
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.