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Successful parallel development and integration of a plasmid-based biologic, container/closure system and electrokinetic delivery device
Abstract
We have developed three major technologies that allow plasmid-based products to be used for large-scale vaccination or therapeutic protein applications. Our team has integrated these components into one complete, cost-effective, easy-to-use system capable of rapid implementation under field conditions. The proprietary manufacturing process uses a lysis method and membrane-based chromatography to rapidly produce large-scale batches of plasmid. Plasmid doses are filled into the Becton-Dickinson Uniject container/closure system. The Uniject adapts to the electrode array of our constant current electrokinetic device, such that the plasmid is delivered in the area of tissue defined by the electrodes. Thus, plasmid uptake and expression levels are dramatically improved. This is the first completely integrated delivery system for plasmid-based products.
... Continuous mixing of the liquids is a common principle in these processes. In several cases, mixing is achieved by static (Urthaler et al., 2007;Wan et al., 1998) or active mixers (Hebel et al., 2006). Simple T-connectors have also proven to be very effective when using sufficient flow rates (Voss, 2007;Voss et al., 2005) which will be outlined in more details in the results section. ...
... With respect to product recovery, the cell disruption processes were compared to a "manual" lysis according to common plasmid purification protocols where mixing was achieved by inverting. Incubation times for alkaline lysis and subsequent neutralization were in good aggreement with optimal values determined by Clemson and Kelly (2003 Although the concept of continuous lysis has been described occassionally (Urthaler et al., 2007;Wan et al., 1998) and even a comparable process to ours has been described (Hebel et al., 2006), the method presented here has not been analyzed in such details yet. The process described by Urthaler et al. (2007) works with specially designed equipment as does the froth flotation in our case. ...
... i.e. chromosomal DNA. The same applies for the work of Hebel et al. (2006). Here, the depletion of chromosomal DNA was only analyzed by agarose gel electrophoresis which is a rather improper analytical technique for this purpose. ...
Plasmid DNA is currently used in gene therapy and genetic vaccination as a vector system for the delivery of therapeutic genes. Clinical trials as well as future therapeutics demand large amounts of high quality plasmid DNA that fulfils the specifications set by regulatory authorities. This thesis describes the development, analysis, and evaluation of pharmaceutical plasmid DNA production processes comprising cultivation, product isolation, and purification as well as stability assessment during storage and application. Cultivations on defined media have been analyzed and compared to state of the art cultivations on semidefined medium. The influence of amino acid supplementation as well as the effect of the physiological conditions of the inoculation culture on growth and product formation have been determined. In this way, batch cultivation processes utilizing glycerol based defined media could be established having yield coefficients Y_x/s and product selectivities S_p/x comparable to or exceeding the values obtained with a semidefined medium. Additional proteome analysis indicated a stringent response that could influence plasmid replication. A semi continuous alkaline lysis was combined with froth flotation for large scale product isolation. The procedure was able to produce a highly clear lysate that could directly be applied to subsequent purification. This process was compared to other modes of operation with respect to product formation, contamination with chromosomal DNA, and plasmid form distribution. For the purification of plasmids, DNA-binding proteins were analyzed as potential affinity ligands. In addition, a recombinant RNase has been produced and its capability for RNA depletion could successfully be demonstrated. The partitioning of nucleic acids in reverse micellar two-phase systems was examined and used to develop an extraction process for plasmid purification which could be integrated into different purification schemes that allowed the complete depletion of all contaminants. Finally, the stability of purified plasmid DNA during long term storage and its implication on the effectivity after gene transfer has been investigated.
... Vaccine Preparation. Plasmids were manufactured to high concentrations and purified using the manufacturing procedure described by Hebel et al [24] in US patent 7238522 with modifications . All plasmid preparations were formulated and prepared with 1% weight/weight with high performance liquid chromatography purified low-molecular-weight poly-L-glutamate in sterile water, as previously described [24]. ...
... Plasmids were manufactured to high concentrations and purified using the manufacturing procedure described by Hebel et al [24] in US patent 7238522 with modifications . All plasmid preparations were formulated and prepared with 1% weight/weight with high performance liquid chromatography purified low-molecular-weight poly-L-glutamate in sterile water, as previously described [24]. All plasmids (pGX4001 to pGX4008) were combined to make a single vaccine preparation consisting of 125 lg of each plasmid in a total volume of 0.1 mL for the ID or 0.5 mL for the IM administration. ...
The threat of a smallpox-based bioterrorist event or a human monkeypox outbreak has heightened the importance of new, safe
vaccine approaches for these pathogens to complement older poxviral vaccine platforms. As poxviruses are large, complex viruses,
they present technological challenges for simple recombinant vaccine development where a multicomponent mixtures of vaccine
antigens are likely important in protection. We report that a synthetic, multivalent, highly concentrated, DNA vaccine delivered
by a minimally invasive, novel skin electroporation microarray can drive polyvalent immunity in macaques, and offers protection
from a highly pathogenic monkeypox challenge. Such a diverse, high-titer antibody response produced against 8 different DNA-encoded
antigens delivered simultaneously in microvolumes has not been previously described. These studies represent a significant
improvement in the efficiency of the DNA vaccine platform, resulting in immune responses that mimic live viral infections,
and would likely have relevance for vaccine design against complex human and animal pathogens.
... Transient changes in plasma membrane permeability contribute to increased intracellular uptake and expression of injected DNA. Proinflammatory cytokine induction and mobilization of monocyte/macrophages to the site of EP are additional factors that result in improved antigen presentation [19][20][21][22][23][24][25][26][27][28]. ...
... It thus appears that the dose of DNA plasmid employed might be one of the critical factors in inducing sufficient antibody titers to mediate transmission blocking effects, especially for larger animals. As discussed earlier, DNA vaccines in general have shown rather poor immunogenicity in larger animals and humans and attempts to enhance the potency of DNA plasmids include various approaches such as codon optimization of antigen coding gene, use of genetic and chemical immunomodulatory adjuvants, administration of plasmids as cationic lipid formulations and alternate methods of delivery, including in vivo EP [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. ...
Pfs25 is a promising target antigen for the development of a malaria transmission-blocking vaccine and prior research has demonstrated induction of high and functionally effective antibodies in mice with IM injection of Pfs25 encoding DNA plasmid. Likewise, Pfs25 DNA vaccine was immunogenic in rhesus macaques but required a protein boost to elicit significant transmission-blocking antibodies. The translation of these encouraging findings to human clinical trials has been impeded largely by the relatively poor immunogenicity of DNA plasmids in larger animals. In vivo electroporation (EP) has revealed significant enhancement of the potency of DNA plasmids. The results reported here compared the immunogenicity and functional transmission-blocking effects of immunization with DNA plasmid (25 microg) by the traditional IM route compared to coupling the IM injection (0.25, 2.5 and 25 microg doses) with in vivo EP. Significantly, a 0.25 microg dose of DNA plasmid, when administered with EP, induced antibody titers (1:160,000) and functional transmission-blocking effects that were equivalent to those achieved by a one hundred fold higher (25 microg) dose of DNA plasmid given without EP. At a 25.0 microg DNA dose with or without EP there was sufficient antigenic stimulation to result in effective antibody titers; however EP method yielded antibody titer of 1:1,280,000 as compared to only 1:160,000 titer without EP. This observed two log reduction in the amount of DNA plasmid required to induce significant transmission-blocking effects makes a compelling argument in favor of further evaluation of DNA vaccines by in vivo EP method in larger animals. Further experiments in non-human primates and eventually in phase I human trials will determine if the use of EP will induce effective and sustained malaria transmission-blocking effects at acceptable doses of plasmid DNA.
... In industrial processes, strategies such as tangential flow filtration (TFF), expanded bed adsorption, depth filtration, ultra/diafiltration, and the use of specialized filtration devices are employed to circumvent these challenges. [37,38,51,[56][57][58][59] Here, we discovered that filtering the supernatant through cheesecloth sterilized with 70% ethanol followed by an ammonium acetate precipitation is sufficient to achieve the desired clarification of the lysate, circumventing the need for additional apparatuses and greatly reducing cost and time associated with 0.2 μm filtration. ...
DNA serves as a model system in polymer physics due to its ability to be obtained as a uniform polymer with controllable topology and nonequilibrium behavior. Currently, a major obstacle in the widespread adoption of DNA is obtaining it on a scale and cost basis that accommodates bulk rheology and high‐throughput screening. To address this, recent advancements in bioreactor‐based plasmid DNA production is coupled with anion exchange chromatography producing a unified approach to generating gram‐scale quantities of monodisperse DNA. With this method, 1.1 grams of DNA is obtained per batch to generate solutions with concentrations up to 116 mg mL⁻¹. This solution of uniform supercoiled and relaxed circular plasmid DNA, is roughly 69 times greater than the overlap concentration. The utility of this method is demonstrated by performing bulk rheology measurements at sample volumes up to 1 mL on DNA of different lengths, topologies, and concentrations. The measured elastic moduli are orders of magnitude larger than those previously reported for DNA and allowed for the construction of a time‐concentration superposition curve that spans 12 decades of frequency. Ultimately, these results can provide important insights into the dynamics of ring polymers and the nature of highly condensed DNA dynamics.
... In addition to CMOs, pDNA vaccine developers and innovators have also been active in this field. Researchers at ADViSYS (now part of Inovio) published a production process for DNA vaccines [74]. Key characteristics of their process include a highshear, in-line mixing device for alkaline cell lysis with a residence time of only milliseconds. ...
The demand for plasmid DNA (pDNA) has vastly increased over the past decade in response to significant advances that have been made in its application for gene therapy and vaccine development. Plasmid DNA-based vaccines are experiencing a resurgence due to success with prime-boost immunization strategies. The challenge has always been poor productivity and delivery of pDNA. Plasmid DNA-based vaccines have traditionally required milligram scale of GMP-grade product for vaccination due to the relatively low efficacy and duration of gene expression. However, efforts to increase pDNA vaccine effectiveness are evolving in genetic manipulations of bacterial host, improvements in product recovery and innovative delivery methods. This review summarizes recent advances in large-scale pDNA vaccine manufacturing, ranging from upstream processing, downstream processing and formulation, as such information is usually not available to the scientific community. The article will highlight technology gaps and offer insight on further scope of innovation.
... Devices and Methods for Biomaterial Production, 2007, US Patent 7,238,522) as previously described which was developed on the principle of alkaline lysis. 8,12 The neutralized lysate was clarified with a decreasing pore size nylon monofilament mesh bag filter (Filter Specialist Inc.) followed by a 0.2 μm depth filter (Millipore) before being subjected to chromatography purifications. The anion exchange membrane chromatography assay detection limit (Table2). ...
Two DNA vaccine plasmids encoding Herpes simplex virus type 2 (HSV-2) glycoprotein D, NTC8485-O2-gD2 and NTC8485-O2-UgD2tr, were produced at large scale under current good manufacturing practice (cGMP) for use in a Phase I human clinical trial. These DNA vaccines incorporate the regulatory agency compliant, minimal, antibiotic-free (AF) NTC8485 mammalian expression vector. Plasmid yields of > 1 g/L were achieved using the HyperGRO™ fed-batch fermentation process, with successful scale up from 10L process development scale to 320L culture volume for cGMP production. The DNA vaccines were purified using a low residence time, high shear lysis process and AIRMIX (TM) technology, followed by chromatographic purification. This combination of optimized plasmid vector, high yield upstream production, and efficient downstream purification resulted in purified HSV-2 DNA vaccines with > 99% total supercoiled plasmid, ≤ 0.2% RNA, ≤ 0.1% host cell genomic DNA, and ≤ 0.1 endotoxin units per mg.
... 18,19 The plasmids were produced by ADViSYS, Inc. (The Woodlands, TX, USA), according to previously described methods. 20 In vivo gene delivery Mice were mildly anesthetized using isoflurane (5.0% isoflurane in 100% O 2 until unconscious and anesthesia was maintained with 1.25% isoflurane in 100% O 2 at a flow rate of 1 liter min À 1 ) and 100 mg of plasmid containing either adiponectin or empty control vector in 25 ml sterile saline (control) were injected directly into each gastrocnemius muscle of the mice. Transcutaneous electric pulses were applied to the muscle using two stainless steel needle electrodes placed 1-cm apart on each side of the injection site immediately after DNA injection. ...
Background and Design:
Adiponectin is an adipokine secreted primarily from adipose tissue that can influence circulating plasma glucose and lipid levels through multiple mechanisms involving a variety of organs. In humans, reduced plasma adiponectin levels induced by obesity are associated with insulin resistance and type 2 diabetes, suggesting that low adiponectin levels may contribute the pathogenesis of obesity-related insulin resistance.
Methods and Results:
The objective of the present study was to investigate whether gene therapy designed to elevate circulating adiponectin levels is a viable strategy for ameliorating insulin resistance in mice fed a high-fat, high-sucrose (HFHS) diet. Electroporation-mediated gene transfer of mouse adiponectin plasmid DNA into gastrocnemius muscle resulted in elevated serum levels of globular and high-molecular weight adiponectin compared with control mice treated with empty plasmid. In comparison to HFHS-fed mice receiving empty plasmid, mice receiving adiponectin gene therapy displayed significantly decreased weight gain following 13 weeks of HFHS diet associated with reduced fat accumulation, and exhibited increased oxygen consumption and locomotor activity as measured by indirect calorimetry, suggesting increased energy expenditure in these mice. Consistent with improved whole-body metabolism, mice receiving adiponectin gene therapy also had lower blood glucose and insulin levels, improved glucose tolerance and reduced hepatic gluconeogenesis compared with control mice. Furthermore, immunoblot analysis of livers from mice receiving adiponectin gene therapy showed an increase in insulin-stimulated phosphorylation of insulin signaling proteins.
Conclusion:
Based on these data, we conclude that adiponectin gene therapy ameliorates the metabolic abnormalities caused by feeding mice a HFHS diet and may be a potential therapeutic strategy to improve obesity-mediated impairments in insulin sensitivity.
... 4 A five-electrode array was used in all experiments, with the plasmid administered through a syringe introduced in the muscle area delineated by the electrodes. 72 EP conditions in these very large mammals were generally: electric field intensity of 0.5-0.6 A, 3 pulses, 52 ms/pulse. ...
Type I diabetes mellitus (T1D) is due to a loss of immune tolerance to islet antigen and thus, there is intense interest in developing therapies that can re-establish it. Tolerance is maintained by complex mechanisms that include inhibitory molecules and several types of regulatory T cells (Tr). A major historical question is whether gene therapy can be employed to generate Tr cells. This review shows that gene transfer of immunoregulatory molecules can prevent T1D and other autoimmune diseases. In our studies, non-viral gene transfer is enhanced by in vivo electroporation (EP). This technique can be used to perform DNA vaccination against islet cell antigens and when combined with appropriate immune ligands results in the generation of Tr cells and protection against T1D. In vivo EP can also be applied for non-immune therapy of diabetes. It can be used to deliver protein drugs such as glucagon-like peptide 1 (GLP-1), leptin or transforming growth factor beta (TGF-beta). These act in T1D or type II diabetes (T2D) by restoring glucose homeostasis, promoting islet cell survival and growth or improving wound healing and other complications. Furthermore, we show that in large animals EP can deliver peptide hormones, such as growth hormone releasing hormone (GHRH). We conclude that the non-viral gene therapy and EP represent a safe and efficacious approach with clinical potential.
... While plasmids manufactured under GMP have limited levels of endotoxins, residual genomic DNA or bacterial RNA, as plasmid-dose increases, the absolute levels of these contaminants increase and can negatively impact expression as well as increase the risk of adverse effects, both locally and systemically. 33 Distant site distribution and integration of plasmids at the injection site is thought to be correlated to the plasmid-dose and electroporation conditions, with higher current and voltage settings associated to higher risk of unwanted effect. 22 The ability to lower the dose and frequency of necessary vaccinations may allow this method to be both economically viable and safer by using less vaccine per human patient. ...
A recently developed, adaptive constant-current electroporation technique was used to immunize mice with an intramuscular injection of plasmid coding for the extracellular and transmembrane domains of the product of the rat neu(664V-E) oncogene protein. In wild-type BALB/c mice, plasmid electroporation at lower current settings elicits higher antibody titers, a strong cytotoxic response and completely protects all mice vaccinated with 10, 25 and 50 microg of plasmid against a lethal challenge of rat neu+ carcinoma cells. BALB/c mice transgenic for the transforming rat neu(664V-E) (ErbB-2, Her-2/neu) oncogene (BALB-neuT(664V-E)) develop an invasive mammary gland carcinoma by 20 weeks of age. Remarkably, when transgenic BALB-neuT(664V-E) mice were vaccinated at a 10- week interval with 50 microg of plasmid with 0.2 A electroporation, mice remained tumor free for more than a year. A single administration of plasmid associated with electroporation was enough to markedly delay carcinogenesis progression in mice with multiple microscopic invasive carcinomas, and keep about 50% of mice tumor free at one year of age. Thus, vaccination using a clinically relevant dose of plasmid encoding the extracellular and transmembrane domains of the neu oncogene delivered by electroporation prevents long-term tumor formation. These improvements in the efficacy of this cancer vaccine regimen vastly increase its chances for clinical success.
... The endotoxin-free plasmid (VGX Pharmaceuticals, Immune Therapeutics Division, The Woodlands, Texas) preparation of pSPc5-12-HV-GHRH was diluted in sterile water for injection + 1% poly-l-glutamate sodium salt to 5 mg/ml. The test doses and placebo (sterile water + 1% poly-l-glutamate sodium salt, the vehicle used for the plasmid preparation) were filled individually in Uniject (Becton-Dickenson, Franklin Lakes, NJ) 47 and randomized. IM injection of 0.35 mg of test article (in a total volume of 500 µl) or placebo was followed after 2 minutes by EP using a five-electrode array and a constant-current electroporator (CELLECTRA device, VGX Pharmaceuticals, The Woodlands, TX) under the following conditions: five pulses, 1 Amp, 50 ms/pulse. ...
The use of growth hormone releasing hormone (GHRH) plasmid-based therapy to treat companion dogs with spontaneous malignancies and anemia receiving a cancer-specific treatment was examined in a double-blinded, placebo-controlled trial. The dogs (age 10.5 +/- 2.5 years, weight 24.9 +/- 12.9 kg) received a single 0.35 mg dose of plasmid or placebo intramuscularly (i.m.), followed by electroporation (EP), and were analyzed for up to 120 days. The response rate was defined as > or = 5% increase above the nadir in the red blood cell (RBC), hemoglobin (Hb), and hematocrit (Ht) levels. Plasmid-treated dogs had at least a 7% increase in all three parameters. The initial response rates for the plasmid-treated dogs were 40.6 and 35.5%, respectively on days 40 and 60, which increased to 54.2% on day 90. Although the response rate reduced to 47.1% by day 120, it was still 22.1% higher than in the control dogs. Post-hoc analysis of the GHRH-treated group showed that responder dogs survived 84% longer, 178 +/- 26 days post-treatment, while nonresponders and controls survived for 95 +/- 16 and 97 +/- 31 days post-treatment, respectively. The quality of life, defined by 10 different parameters, dramatically improved with treatment. Overall, the possibility of a GHRH plasmid-based therapy for anemia in cancer-afflicted subjects is important enough to deserve further investigation.
... Male ferrets (Triple F Farms, Sayre, PA), 4–6 month old, weight average 1.260.2 kg at the start of the study were immunized three times in alternate biceps femoris muscles, each one month apart, with 200 mg of DNA per antigen (and/or vector control) and electroporated as previously described [16]. Briefly, electroporation was delivered through a pentagonal 5- electrode array consisting of 21-gauge solid stainless steel electrodes. ...
Background:
The persistent evolution of highly pathogenic avian influenza (HPAI) highlights the need for novel vaccination techniques that can quickly and effectively respond to emerging viral threats. We evaluated the use of optimized consensus influenza antigens to provide broad protection against divergent strains of H5N1 influenza in three animal models of mice, ferrets, and non-human primates. We also evaluated the use of in vivo electroporation to deliver these vaccines to overcome the immunogenicity barrier encountered in larger animal models of vaccination.
Methods and findings:
Mice, ferrets and non-human primates were immunized with consensus plasmids expressing H5 hemagglutinin (pH5HA), N1 neuraminidase (pN1NA), and nucleoprotein antigen (pNP). Dramatic IFN-gamma-based cellular immune responses to both H5 and NP, largely dependent upon CD8+ T cells were seen in mice. Hemaggutination inhibition titers classically associated with protection (>1:40) were seen in all species. Responses in both ferrets and macaques demonstrate the ability of synthetic consensus antigens to induce antibodies capable of inhibiting divergent strains of the H5N1 subtype, and studies in the mouse and ferret demonstrate the ability of synthetic consensus vaccines to induce protection even in the absence of such neutralizing antibodies. After challenge, protection from morbidity and mortality was seen in mice and ferrets, with significant reductions in viral shedding and disease progression seen in vaccinated animals.
Conclusions:
By combining several consensus influenza antigens with in vivo electroporation, we demonstrate that these antigens induce both protective cellular and humoral immune responses in mice, ferrets and non-human primates. We also demonstrate the ability of these antigens to protect from both morbidity and mortality in a ferret model of HPAI, in both the presence and absence of neutralizing antibody, which will be critical in responding to the antigenic drift that will likely occur before these viruses cross the species barrier to humans.
Purification of high-quality plasmid DNA in large quantities is a crucial step in its production for therapeutic use and is usually conducted by different chromatographic techniques. Large-scale preparations require the optimization of yield and homogeneity, while maximizing removal of contaminants and preserving molecular integrity. The advantages of Convective Interaction Media® (CIM®) monolith stationary phases, including low backpressure, fast separation of macromolecules, and flow-rate-independent resolution qualified them to be used effectively in separation of plasmid DNA on laboratory as well as on large scale. A development and scale-up of plasmid DNA downstream process based on chromatographic monoliths is described and discussed below. Special emphasis is put on the introduction of process analytical technology principles and tools for optimization and control of a downstream process.
IntroductionCell HarvestingCell DisruptionConclusions
References
IntroductionAlkaline Versus Nonalkaline Lysis-Based ProcessesChoosing the Intermediate RecoveryThe Use of ChromatographyThe Ubiquitous Role of TFFConclusions
References
IntroductionThe Dynamics of InnovationDNA Versus Conventional VaccinesConclusions
References
The book addresses the basics, applications, and manufacturing of plasmid biopharmaceuticals. The survey of the most relevant characteristics of plasmids provides the basics for designing plasmid products (applications) and processes (manufacturing). Key features that the authors include in the book are: i) consistency and clear line of direction, ii) an extensive use of cross-referencing between the individual chapters, iii) a rational integration of chapters, iv) appellative figures, tables and schemes, and v) an updated, but selected choice of references, with a focus on key papers.
Human cytomegalovirus (CMV) establishes a lifelong persistent infection characterized by periods of latency and sporadic viral replication and is a major infectious cause of birth defects following congenital infection. Currently, no licensed vaccine is available that would prevent CMV infection. In an effort to develop a prophylactic CMV vaccine, the effects of different formulations, immunization routes and delivery devices on the immunogenicity of plasmid DNA (pDNA)-based vaccines were evaluated in rabbits and mice. Compared with PBS- and poloxamer-based formulations, significantly higher antibody responses were obtained with pDNA formulated with Vaxfectin®, a cationic lipid-based adjuvant. With low vaccine doses, the intradermal (ID) route resulted in higher antibody responses than obtained when the same dose was administered intramuscularly (IM). Since the IM route allowed injection of larger volumes and higher doses than could be administered at a single ID site, better antibody responses were obtained using the IM route. The needle-free injection system Biojector® 2000 and electroporation devices enhanced antibody responses only marginally compared with responses obtained with Vaxfectin®-formulated pDNA injected IM with a needle. A single-vial Vaxfectin® formulation was developed in a dosage form ready for use after thawing at room temperature. Finally, in a GLP-compliant repeat-dose toxicology study conducted in rabbits, single-vial Vaxfectin®-formulated vaccines, containing pDNA and Vaxfectin® up to 4.5 mg and 2 mg/injection, respectively, showed a favorable safety profile and were judged as well-tolerated. The results support further development of a Vaxfectin®-formulated pDNA vaccine to target congenital CMV infection.
Plasmid-DNA wird als Klonierungsvektor zum Einbringen rekombinanter Gene in Mikroorganismen genutzt. Seit den 1990er Jahren wird dieses Prinzip auch zur Übertragung therapeutischer Gene in der Gentherapie und der genetischen Impfung verwendet. Diese Form der nicht viralen Gentherapie stellt eine deutlich risikoärmere Variante im Vergleich zur viralen Gentherapie dar. Zur Deckung des Bedarfs an Plasmid-DNA in klinischen Studien und für in Zukunft zugelassene Therapeutika sind Produktionsverfahren erforderlich, die eine Herstellung im Multigramm- bis Kilogramm-Maßstab möglich machen. Besondere Hersausforderungen werden hier im Bereich des Zellaufschlusses, der Abtrennung strukturell verwandter Verbindungen und der Prozess- und Produktanalytik gestellt. Dieser Übersichtsbeitrag fasst die Entwicklungen der letzten Jahre zusammen.
Plasmid DNA is used as a cloning vector to deliver recombinant genetic information into microorganisms. Since the 1990s, this principle has also been applied for the delivery of therapeutic genes in gene therapy and genetic vaccination. This non-viral gene delivery is afflicted with fewer safety concerns in comparison to viral systems. Processes for the production of high-quality plasmid DNA at multi- and kilogram scale are necessary to meet the needs of clinical trials as well as future therapeutics. Cell disruption, the separation of structurally-related impurities and analytical techniques for process and quality control are the main challenges for bioengineering. This review summarizes the development in these fields over the past recent years.
Non-viral gene transfer using plasmid DNA (pDNA) is generally acknowledged as safe and non-immunogenic compared with the use of viral vectors. However, pre-clinical and clinical studies have shown that non-viral (lipoplex) gene transfer to the lung can provoke a mild, acute inflammatory response, which is thought to be, partly, due to unmethylated CG dinucleotides (CpGs) present in the pDNA sequence. Using a murine model of lung gene transfer, bronchoalveolar lavage fluid was collected following plasmid delivery and a range of inflammatory markers was analysed. The results showed that a Th1-related inflammatory cytokine response was present that was substantially reduced, though not abolished, by using CpG-free pDNA. The remaining minor level of inflammation was dependent on the quality of the pDNA preparation, specifically the quantity of contaminating bacterial genomic DNA, also a source of CpGs. Successful modification of a scalable plasmid manufacturing process, suitable for the production of clinical grade pDNA, produced highly pure plasmid preparations with reduced genomic DNA contamination. These studies help define the acceptable limit of genomic DNA contamination that will impact FDA/EMEA regulatory guidelines defining clinical grade purity of plasmid DNA for human use in gene therapy and vaccination studies.
Plasmid DNA for biopharmaceutical applications is produced easily in Escherichia coli bacteria. The cell lysis is the most crucial step for purification of plasmid DNA. In this paper, we describe a continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF using hollow fiber ultrafiltration column as a lysis chamber and compare the plasmid DNA yield and homogeneity with the T-connector and manual processes, respectively. The results show that the plasmid pUDK-HGF yield of the combination process is 13% higher than manual lysis, twice higher than using T-connector. When the proportion of lysed cells and neutralization solution is 3:1, the plasmid pUDK-HGF yield can improve by 70%. This process could be easily scaled up to meet the industrial scale for cell lysis.
Successful liver gene therapy depends on efficient gene transfer techniques and long-lasting gene expression after successful transfer. Over the last decades, important progress has been made with the introduction of viral vectors using animal models, although their use is hampered by a complex and costly preparation compared to the simple and cost-effective preparation of plasmid DNA. These problems become even more critical when considering the application of viral vectors in human gene therapy and gene therapy trials. In a previous study, we were able to show that the hydrodynamics-based gene transfer of plasmid-DNA, containing the adeno-associated-virus specific inverted terminal repeats (AAV-ITR), prolongs gene expression in the liver, although it remained unclear whether plasmid gene transfer could achieve similar expression levels compared to viral-vector gene transfer.
Rat livers were transfected in-vivo with AAV-ITR-containing plasmid-DNA using a modified hydrodynamics-based procedure. Expression levels were monitored thereafter and compared with expression levels after viral-vector gene transfer.
A high and stable long-term expression was achieved after in vivo transfection of rat livers with AAV-ITR-containing plasmids. The expression course resembled that after AAV-mediated gene transfer, and the expression was at least as high, and lasted as long, compared to recombinant AAV-mediated gene transfer.
We consider AAV-ITR-containing plasmids as a simple and cost-effective alternative to recombinant viral vectors, especially for liver-directed gene therapy in rodents. With ongoing progress in gene transfer methods for naked DNA, these plasmids may also become a successful alternative to recombinant viral vectors in human gene therapy.
The increased use of plasmid-based vaccines to replace their more challenging viral counterparts has increased the demand for high purity and high concentration plasmids. Here we report the production of plasmids encoding different transgenes for DNA vaccine candidates at gram scale with an integrated process consisting of batch fermentation and limited steps of purification. Plasmid products encoding for eight smallpox antigens that were combined into a bioterrorism DNA vaccine exhibited high purity with undetectable RNA, protein and endotoxin, concentration of up to 13.6mg/mL and supercoiled percentage of 94.5+/-1.1% after storage at -80 degrees C for over 1 year. The process has been scaled up for the cGMP manufacture of pharmaceutical-grade human papillomavirus and influenza DNA vaccines up to a 50g scale, also demonstrating high purity and high concentration.
Drug and nucleic acids can be delivered in vivo by an injection of the product followed by the application of a train of electric pulses.
The success of the method is linked to the proper distribution of the electric field in the target tissue. This is under the control of the design of the electrodes.
The field distribution can be obtained by computer simulation mainly by using numerical methods and simplifying hypothesis. The conclusions are validated by comparing the computed current and its experimental values on phantoms. A good agreement is obtained.
Targeting the delivery to the skin can be obtained by using an array of very short needle electrodes, by pinching the skin between two parallel plate electrodes, or by using contact wire electrodes.
Die fortschreitende Entwicklung in der Gentherapie führt zu einem immer höheren Bedarf an therapeutisch einsetzbarer Plasmid DNA (pDNA). Zunächst war das Hauptziel der Gentherapie die Behandlung monogenetischer Erkrankungen und Infektionskrankheiten, bis Herz-Kreislauf-Erkrankungen und vor allem Krebs in den Fokus der Forschung rückten. Plasmid-DNA ist als nicht-viraler Vektor für den Transfer therapeutischer Gene geeignet und ist vor allem auch auf dem Gebiet der genetischen Impfung von Interesse. Gegenüber herkömmlichen Vakzinen bietet Plasmid-DNA den Vorteil, dass der Erreger nicht direkt in den Körper gelangt, sondern lediglich die genetische Information eines Antigens, deren Expression im Körper eine zelluläre oder humorale Immunantwort auslöst. Plasmide kommen in Bakterienzellen und anderen Mikroorganismen vor. Die Replikation findet unabhängig von der Wirts-DNA statt. Die kodierten Gene bieten beispielsweise durch Antibiotikaresistenz einen Selektionsvorteil für den Wirtsorganismus. Modifizierte Plasmide sind als Klonierungsvektoren für die molekulare Biologie und Biotechnologie von Bedeutung. Um Plasmid-DNA in ausreichenden Mengen herstellen zu können, sind effiziente und gut skalierbare Prozesse notwendig. Für den Einsatz im gentherapeutischen Bereich werden hohe Qualitätsanforderungen gestellt. Diesbezüglich müssen die Anforderungen regulatorischer Behörden (Food and Drug Administration, FDA/ European Medicines Agency, EMEA) eingehalten werden. Verfahren zur Herstellung von Plasmid-DNA bestehen aus der Kultivierung der plasmidreplizierenden Bakterien, einem Zellaufschluss zur Freisetzung der Plasmide und im Anschluss daran geeigneten Aufarbeitungsprozessen. Die bioverfahrenstechnische Herausforderung besteht in der Abtrennung der superspiralisierten Plasmid-DNA von strukturell ähnlichen Verunreinigungen wie RNA, chromosomaler DNA (chrDNA) und Lipopolysacchariden (LPS). In der Regel werden zur Aufreinigung von Plasmid-DNA chromatographische Verfahren eingesetzt. Die meisten dieser Methoden sind zeitaufwändig, mit hohen Produktverlusten verbunden oder es bestehen Schwierigkeiten in der Maßstabsvergrößerung. Eine Alternative stellen Extraktionsprozesse dar, da diese gut skalierbar sind und lediglich einfache und kostengünstige Chemikalien und Geräte benötigt werden. Es können sowohl wässrige als auch inversmizellare Zweiphasensystemen zur Aufarbeitung von Plasmid-DNA eingesetzt werden. Im Rahmen dieser Arbeit wurde die Verteilung von Nukleinsäuren in einem inversmizellaren Zweiphasensystem untersucht. Es wurde der Einfluss verschiedener Salze und Salzkonzentrationen sowie die Auswirkung unterschiedlicher Alkohole auf das Extraktionssystem betrachtet. Im Fokus der Untersuchungen stand die Trennleistung des Systems. Darüber hinaus wurde die Kapazität und die Möglichkeit zur Trennung verschiedener Plasmidformen geprüft. Die Ergebnisse konnten erfolgreich eingesetzt werden, um RNA, Proteine, chromosomale DNA und Endotoxine während der Extraktion der Plasmid-DNA aus einem konditionierten bakteriellen Klarlysat abzureichern.
The demand for plasmid DNA in large quantities at high purity and concentration is expected to escalate as more DNA vaccines are entering clinical trial status and becoming closer to market approval. This review outlines different methods for DNA vaccine manufacture and discusses the challenges that hinder large-scale production. Current technologies are summarized, focusing on novel approaches that have the potential to address downstream bottlenecks and adaptability for large-scale application. Product quality in terms of supercoiled percentage and impurity levels are compared at the different production levels.
Electroporation has been demonstrated as an effective technique for enhancing the delivery of plasmids coding for DNA vaccines and therapeutic proteins into skeletal muscle. Nevertheless, constant-voltage techniques do not take into account the resistance of the tissue and result in tissue damage, inflammation, and loss of plasmid expression. In the present study, we have used a software-driven constant-current electroporator to deliver plasmids to mice and small and large pigs. The voltage, amperage, and resistance of the tissue during pulses were recorded and analyzed. Optimal conditions of electroporation were identified in both species, and found to be highly dependent on the individual tissue resistance. Six- to 10-week-old pigs had higher muscle resistance compared to 1- to 2-year-old pigs, but both values were four to five times lower than the resistance of the mouse muscle. In mice, optimum amperage, pulse length, and lag time between plasmid injection and electroporation were identified to be 0.1 Amps, 20 msec and 0 sec. The electroporation pulse pattern among the electrodes also affected plasmid expression. These results indicate that age- and tissue-specific resistance, pulse pattern, and other variables associated with the electroporation need to be optimized for each separate species to achieve maximum plasmid expression.
The expectation has been that plasmid DNA vaccines may have use against a wide range of microbial and oncologic targets. However, attempts at their development have been hampered by the inability to achieve high, consistent levels of immunogenicity in large experimental species and humans. Successful development is probably contingent on a delivery method that provides robust, consistent antigen expression and immune responses. Electroporation (EP), a promising approach that dramatically enhances expression of the encoded antigen as well as the potency and immunogenicity of DNA vaccines, could facilitate clinical implementation of DNA vaccination. With the recent development of EP systems that enable safe, tolerable, reproducible and clinically acceptable administration, EP-based DNA vaccination has become a clinical reality. The technology is now being tested for safety and immunogenicity in several Phase I clinical trials.
In an effort to improve DNA vaccine immune potency electroporation has emerged as a method of delivery of plasmids to target tissues. However, few studies have examined the use of this technology to deliver plasmid vaccines to the skin. Here we studied the effect of electroporation on DNA vaccine potency and gene delivery using skin as a target tissue in larger animal species. Using a pig model, we determined that high plasmid concentrations resulted in improved gene expression for plasmid GFP delivered by the intradermal/subcutaneous (ID/SQ) route. In a macaque model, we observed higher cellular and humoral responses to an HIV DNA vaccine, which included plasmid-encoded IL-12, with electroporation compared to ID/SQ injection alone. The induced responses were TH1 mediated. These results support that skin electroporation may have importance as an immunization approach in larger animal models.
Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimal conditions for individual species and muscle group are not well defined. Using a muscle-specific plasmid driving the expression of a secreted embryonic alkaline phosphatase (SEAP) reporter gene, we have optimized the electroporation conditions in a large mammal model, i.e. pig. The parameters optimized include electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. Constant current pulses, between 0.4 and 0.6 A, applied 80 s after the injection of 0.5 mg SEAP-expressing plasmid in a total formulation volume of 2 mL produced the highest expression in semimembranosus muscle in pigs. These results could be extrapolated for a different muscle group in pigs, the biceps femoris, and may be an evaluation starting point for large muscle in veterinary species or humans (see
Note 1
).
Extending immunization coverage to underserved populations will require innovative immunization strategies. This study evaluated one such strategy: the use of a prefilled, single-use injection device for outreach immunization by village midwives. The device, UniJect, is designed to prevent refilling or reuse. Stored at ambient temperatures for up to 1 month in midwives' homes, vaccine-filled UniJect devices were immediately available for outreach. Between July 1995 and April 1996, 110 midwives on the Indonesia islands of Lombok and Bali visited the homes of newborn infants to deliver hepatitis B vaccine to the infants and tetanus toxoid to their mothers. Observations and interviews showed that the midwives used the device properly and safely to administer approximately 10,000 sterile injections in home settings. There were no problems with excessive heat exposure during the storage or delivery of vaccine. Injection recipients and midwives expressed a strong preference for the UniJect device over a standard syringe. Use of the prefilled device outside the cold chain simplified the logistics and facilitated the speed and efficiency of home visits, while the single-dose format minimized vaccine wastage.
Electroporation is believed to be a nonthermal phenomenon at the membrane level. However, the effects of associated processes, such as Joule heating, should be considered. Because electroporation of skin, specifically the stratum corneum (SC), occurs at highly localized sites, the heating is expected to conform locally to the sites of electroporation. Significant localized heating was found to be strongly dependent on the voltage and duration of the high-voltage pulses. Specifically, a localized temperature rise was predicted theoretically and confirmed by experiments, with only a small rise (about 17 degrees C) for short, large pulses (1 ms, 100 V across the SC), but was increased (about 54 degrees C) for long, large pulses (300 ms, 60 V across the SC). The latter case appears to result in irreversible structural changes like vesicularization of the lipid lattice. These results support the hypothesis that electroporation occurs within the SC and that additional processes, such as localized heating, may be important.
Electroporation, the increase in the permeability of bilayer lipid membranes by the application of high voltage pulses, has the potential to serve as a mechanism for transdermal drug delivery. However, the associated current flow through the skin will increase the skin temperature and might affect nearby epidermal cells, lipid structure or even transported therapeutic molecules. Here, thermal conduction and thermal convection models are used to provide upper and lower bounds on the local temperature rise, as well as the thermal damage, during electroporation from exponential voltage pulses (70 V maximum) with a 1 ms and a 10 ms pulse time constant. The peak temperature rise predicted by the conduction model ranges from 19 degrees C for a 1 ms time constant pulse to 70 degrees C for the 10 ms time constant pulse. The convection (mass transport) model predicts a 18 degrees C peak rise for 1 ms time constant pulses and a 51 degrees C peak rise for a 10 ms time constant pulse. The convection model compares more favorably with previous experimental studies and companion observations of the local temperature rise during electroporation. Therefore, it is expected that skin electroporation can be employed at a level which is able to transport molecules transdermally without causing significant thermal damage to the tissue.
Intramuscular injection of plasmid is a potential alternative to viral vectors for the transfer of therapeutic genes into skeletal muscle fibers. The low efficiency of plasmid-based gene transfer can be enhanced by electroporation (EP) coupled with the intramuscular application of hyaluronidase. We have investigated several factors that can influence the efficiency of plasmid-based gene transfer. These factors include electrical parameters of EP, optimal use of hyaluronidase, age and strain of the host, and plasmid size. Muscles of very young and mature normal, mdx, and immunodeficient mice were injected with plasmids expressing beta-galactosidase, microdystrophin, full-length dystrophin, or full-length utrophin. Transfection efficiency, muscle fiber damage, and duration of transgene expression were analyzed. The best transfection level with the least collateral damage was attained at 175-200 V/cm. Pretreatment with hyaluronidase markedly increased transfection, which was also influenced by the plasmid size and the strain and the age of the mice. Even in immunodeficient mice, there was a significant late decline in transgene expression and plasmid DNA copies, although both still remained relatively high after 1 year. Thus, properly optimized EP-assisted plasmid-based gene transfer is a feasible, efficient, and safe method of gene replacement therapy for dystrophin deficiency of muscle but readministration may be necessary.
We evaluated the immunogenicity of hepatitis B (HB) vaccine in UniJect, a pre-filled, non-reusable injection device, stored at tropical temperatures for up to one month and used to give the first dose of HB vaccine to newborns. Infants in Tabanan district, Bali, Indonesia, were given their first dose of HB vaccine with UniJect stored out of the cold chain, UniJect stored in the cold chain; or standard syringe, needle and multidose vial stored in the cold chain. Subsequent doses were given by usual means and blood samples drawn 4–6 weeks after the third dose. No significant differences were found in seroconversion rates or geometric mean titres of HB surface antibody between the three groups.
A procedure for extracting plasmid DNA from bacterial cells 1s described. The method 1s simple enough to permit the analysis
by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction
enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently
closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization,
chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs
have been extracted by this method.
The objective of this study was to evaluate women's acceptance of and ability to self-administrate the injectable contraceptive Cyclofem using prefilled UniJect devices. A total of 102 women were invited to participate in the study. Fourteen women (13.7%) refused to participate. Of the remaining 88 women, 32 women (31.4%) consented to participate and were trained using oranges but were still afraid of the procedure and ultimately refused to self-administer the injections. Only 56 women (55%) ultimately self-injected Cyclofem with UniJect. They performed a total of 144 injections, all of them on the ventral side of the thigh. When nurses evaluated women's ability to activate the devices, they found that more than 80% were successful in both the group of women that later self-administered the injections and the group that did not. The evaluation of the self-administered injection technique showed that more than 90% of the women correctly self-administered the contraceptive using UniJect. With respect to the opinion of the women about the self-administration of the contraceptive, more than 50% (32 of 56) of women who self-injected preferred to self-administer the injection and said that they wished to continue with the self-administration, one-third (17) reported that they were afraid, and seven women (12.5%) expressed the opinion that the injection in the thigh was more painful than the administration in the buttocks or arm. In conclusion, our study showed that women can be trained to successfully self-administer the monthly injectable contraceptive Cyclofem and generally respond positively to UniJect.
This study evaluated the performance, acceptability, and appropriateness of a new, single-use, prefilled injection device called UniJect for an outreach immunization application. Between April and June 1995, UniJect devices were used by 36 traditional birth attendants to administer tetanus toxoid injections to 2,240 pregnant women during routine, antenatal home visits in the Northern, Ichilos, and Warnes Districts of Santa Cruz, Bolivia. Because tetanus toxoid is relatively heat stable, the traditional birth attendants were able to keep the tetanus toxoid-filled UniJect devices in their homes for up to one month without refrigeration. The devices were stored, transported, and disposed of in an outreach carrier designed to reduce the risks of improper handling and disposal. Data were collected from injection recipients, traditional birth attendants, and supervisors via observation, questionnaires, and post-study interviews. The performance of the UniJect device and its acceptability among all groups was very high. The traditional birth attendants used UniJect properly and safely; there were no reports or observations of device misuse, reuse, or needle-stick. Advantages cited included the fact that the device required no assembly, offered assured sterility, and reduced vaccine wastage sometimes associated with multi-dose vials. The ability to store and transport the vaccine-filled devices without ice also greatly simplified logistics.
We describe a process for the commercial manufacture of therapeutic grade plasmid DNA. The industrially scaleable unit operations employed in this process are: (i) optimized alkaline lysis; (ii) bag filtration; (iii) expanded bed anion exchange chromatography; (iv) ultrafiltration, and (v) size exclusion chromatography. These steps are scaleable alternatives to current approaches to plasmid DNA isolation such as high speed centrifugation for feedstock clarification and solvent precipitation for plasmid concentration, and an efficient alternative to conventional low through-put packed bed chromatography.
The process produces plasmid DNA characterized by low level chromosomal DNA, RNA and endotoxin contamination without the use of flammable solvents or toxic reagents and is suitable for therapeutic administration.
Since the first reports of plasmid vaccines, there have been substantial changes made to the design of plasmid backbones, as well as to the antibiotic resistance markers chosen for clinical vectors compared with first generation vectors. These changes aid manufacturing, production and scale up and at the same time aid conceptual safety by limiting the ability of the vaccines to transfer useful genetic selection genes to other bacterial infectious agents. In contrast, there has been little change to the original promoters or polyadenlyation tracts in the last decade. We have learned that these first generation plasmid vaccines for HIV-1 appear very well tolerated in humans. However, while safe and immunogenic, improving the immune potency of DNA vaccines is a critical goal for this technology. The combination of antigens used should be carefully examined for possible immune interference. Such interference may only become apparent when each component of the vaccine is tested individually. This interference also suggests one mechanism of immune pathogenesis possibly by HIV-1. Optimization of the immune response can come through manipulation of the transfection efficiency, expression or through the use of various T cell and B cell plasmid adjuvants. It is likely that the combination of such advancements will significantly improve the clinical phenotype of this important vaccine modality.
The nonviral gene transfer technologies include naked DNA administration, electrical or particle-mediated transfer of naked DNA, and administration of DNA-synthetic macromolecule complex vectors. Each method has its advantage, such as low immunogenicity, inexpensiveness, ease in handling, etc., but the common disadvantage is that the transfection efficiency has been relatively poor as far as conventional plasmid vectors are involved. To improve the nonviral gene transfer systems, Epstein-Barr virus (EBV)-based plasmid vectors (also referred to EBV-based episomal vectors) have been employed. These vectors contain the EBNA1 gene and oriP element that enable high transfer efficiency, strong transgene expression and long term maintenance of the expression. In the current article, I review recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, drastic tumor suppression was achieved by gancyclovir administrations following an intratumoral injection with an EBV plasmid vector encoding the HSV1-TK suicide gene. Equiping the plasmid with carcinoembryonic antigen (CEA) promoter sequences enabled targeted killing of CEA-positive tumor cells, which was not accomplished by conventional plasmid vectors without the EBV genetic elements. Transfection with an apoptosis-inducing gene was also effective in inhibiting tumors. Interleukin (IL)-12 and IL-18 gene transfer, either local or systemic, induced therapeutic antitumoral immune responses including augmentation of the cytotoxic T lymphocyte (CTL) and natural killer (NK) activities, while an autologous tumor vaccine engineered to secrete Th1 cytokines via the EBV system also induced growth retardation of tumors. Non-EBV conventional plasmids were much less effective in eliciting these therapeutic outcomes. Intracardiomuscular transfer of the beta-adrenergic receptor gene induced a significant elevation in cardiac output in cardiomyopathic animals, suggesting the usefulness of the EBV system in treating heart failure. The EBV-based nonviral delivery also worked as genetic vaccine that triggered prophylactic cellular and humoral immunity against acute lethal viral infection. All the nonviral delivery vehicles so far tested showed an improved transfection rate when combined with the EBV-plasmids. Collectively, the EBV-based plasmid vectors may greatly contribute to nonviral gene therapy against a variety of disorders, including malignant, congenital, chronic and infectious diseases.
Electroporation designates the use of short high-voltage pulses to overcome the barrier of the cell membrane. By applying an external electric field, which just surpasses the capacitance of the cell membrane, transient and reversible breakdown of the membrane can be induced. This transient, permeabilized state can be used to load cells with a variety of different molecules, either through simple diffusion in the case of small molecules, or through electrophoretically driven processes allowing passage through the destabilized membrane--as is the case for DNA transfer. Initially developed for gene transfer, electroporation is now in use for delivery of a large variety of molecules: From ions to drugs, dyes, tracers, antibodies, and oligonucleotides to RNA and DNA. Electroporation has proven useful both in vitro, in vivo and in patients, where drug delivery to malignant tumours has been performed. Whereas initial electroporation procedures caused considerable cell damage, developments over the past decades have led to sophistication of equipment and optimization of protocols. The electroporation procedures used in many laboratories could be optimized with limited effort. This review (i) outlines the theory of electroporation, (ii) discusses factors of importance for optimization of electroporation protocols for mammalian cells, (iii) addresses particular concerns when using electroporation in vivo, e.g. effects on blood flow and considerations regarding choice of electrodes, (iv) describes DNA electrotransfer with emphasis on use in the in vivo setting, and (v) sums up data on safety and efficacy of electroporation used to enhance delivery of chemotherapy to tumours in cancer patients.
The demand for larger quantities of high-purity plasmids continues to increase. Substantial quantities of plasmid DNA are needed to support viral and plasmid-based gene-therapy programmes, including drug delivery, preclinical and clinical studies and production of DNA vaccines. Reliance on fermentation, which generates large lysate volumes, for producing the needed quantities of plasmid DNA is becoming more widespread. Development of an efficient capture-unit operation for very large plasmid DNA molecules from these large lysate volumes can present a considerable challenge. Use of conventional chromatographic beaded media for plasmid capture is problematic, owing to their restrictive pores, which limits access of the large DNA molecules to only those binding sites on the beads' outer surface. Anion-exchange membranes, on the other hand, have large convective pores that allow the plasmid DNA to readily access all of the membrane's anionic binding sites, even at very high flow rates. The convenience of anion-exchange membranes, configured in ready-to-use capsules, can greatly simplify large-scale plasmid purification strategies. The effectiveness of membrane-based technology for the capture of a pCAT reporter plasmid from large volumes of clarified Escherichia coli lysate was demonstrated. The captured and eluted plasmid pool was found to have been concentrated 10-fold with a reduction in endotoxin of four orders of magnitude.
Separation and purification of large quantities of plasmid DNA (pDNA) is a particularly difficult manufacturing issue because of the relatively low capacity, flow rate and purity observed using traditional bead-based chromatography. The objective of the present study was to evaluate the performance of anion-exchange membranes for the purification of pDNA from Escherichia coli lysate solution. The fate of host-cell protein and endotoxin relative to pDNA was measured and used to calculate recoveries, mass balances, dynamic capacities and purification factors as a function of the flow rate and loading volume of the lysate solution. Breakthrough curves were not sigmoidal and symmetric in shape. They rose sharply at first, and then slowly towards, but never reaching, saturation. Conversely, elution curves were independent of flow rate. pDNA bound tightly to the membranes, whereas protein and endotoxin did not. Dynamic binding capacity for pDNA was 20-25 times greater, and the flow rate was 55-550 times greater, than values observed for beads. However, some pDNA bound irreversibly to the membrane surface and was not removed completely during elution. The intrinsic rate of pDNA adsorption to the membrane was found to be rate-limiting, whereas effects of liquid-phase mass transfer and flow non-idealities were negligible. These results were interpreted using models of adsorption that included steric effects using the 'car-parking-problem' model, and surface residence time effects using the spreading model. This work demonstrated the advantages of ion-exchange membranes compared with beads for the purification of large biomolecules such as pDNA.
The objectives of this study were to evaluate the effects of immunization site and antigen presenting cell targeting on cattle immune responses to DNA immunization. Cows were vaccinated with the plasmid expression vector pCI alone, pCI encoding the bacterial antigen beta-galactosidase (pCI-beta-gal) or pCI encoding bCTLA 4 fused to beta-gal (pCI-bCTLA-hIgG-beta-gal). The plasmids were delivered by intramuscular, intradermal, intramammary gland, or intra supramammary lymph node needle-injection. Both vaccines induced significant humoral and cellular immune responses. pCI-beta-gal elicited a higher IgG response than immunization with pCI-bCTLA-hIgG-beta-gal. Cows injected intramuscularly and intramammary had higher IgG and IgG-1 humoral responses than cows immunized intradermaly or in the lymph nodes. The injection site did not significantly affect the magnitude of the IgG2 and IgM antibody responses, although a trend similar to the IgG results was observed. The lymphocyte proliferation index was higher with pCI-beta-gal but was not affected by the injection site. These results suggest that in bovine, the injection site can affect immune responses but they do not provide evidence that bCTLA-4-hIgG-antigen targeting is effective in cattle.
Injection of plasmid DNA encoding pseudorabies virus (PRV) glycoproteins into pig muscle has been shown to result in protective immunity against lethal infection. Nevertheless, such DNA vaccines are still less efficient than some attenuated or killed live vaccines. One way to increase DNA vaccine efficacy is to improve the vectorisation system at the molecular level, thereby enhancing the rate of in vivo-produced immunogen protein and consequently specific acquired immunity. The present study compared the effectiveness of the protein expression system depending on Sindbis virus (SIN) replicase [J. Virol. 70 (1996) 508] with that of more classical pcDNA3 plasmid. Pigs were vaccinated twice at 3-week interval with a mixture of three pcDNA3 plasmids expressing gB, gC and gD (designated as PRV-pcDNA3) or a mixture of three SIN plasmids expressing the same glycoproteins (PRV-pSINCP), and were challenged with a highly virulent PRV strain. The two DNA vaccines induced PRV-specific T cell-mediated immune response characterized by very low levels of IFN-gamma mRNA in PBMC after in vitro antigen-specific stimulation. Very low levels of neutralizing antibodies (NAb) were also obtained in sera following DNA injection(s). A second DNA injection did not boost immune responses. After a lethal challenge, high levels of IFN-gamma mRNA and high NAb response were induced in all DNA-vaccinated pigs, regardless of the vector used. Therefore, the two eukaryotic expression systems showed comparable efficacy in inducing antiviral immunity and clinical protection against PRV in pigs. This suggests that SIN DNA-based vector immunizing potential may differ according to antigen and/or host.
We assessed the use and acceptability of an injection device (Uniject) prefilled with oxytocin, as part of active management of third-stage labor (AMTL) by Indonesian midwives attending home births. We interviewed 140 village midwives (bidan di desa) and 2220 mothers whose deliveries they attended during the intervention period. We completed baseline and post-intervention assessments to determine their experiences and views of oxytocin Uniject use. Delivery logs and supervisory reports were reviewed. The assessment was done in three rural districts and one municipality in Lombok. Injection practices and oxytocin availability did not change dramatically, although dose accuracy, use of sterile injection equipment, and proper disposal improved when the Uniject device was used. Midwives had little difficulty using the Uniject device properly; they overwhelmingly preferred it to standard needles and syringes. Postpartum hemorrhage rates did not change substantially. Oxytocin via Uniject holds promise for safer, more convenient use of oxytocin by trained midwives attending home deliveries, thereby potentially reducing the incidence of postpartum hemorrhage.
DNA vaccines offer considerable promise for improvement over conventional vaccines. For the crucial step of delivering DNA vaccines intracellularly, electroporation (EP) has proven to be highly effective. This method has yielded powerful humoral and cellular responses in various species, including nonhuman primates. In an attempt to further improve DNA vaccination we used micron-size gold particles (which do not bind or adsorb DNA) as a particulate adjuvant which was coinjected with DNA intramuscularly into mice, followed by EP of the target site. The presence of gold particles accelerated the antibody response significantly. Maximum titers against hepatitis B surface antigen (HBsAg) were reached after one boost in 6 weeks, whereas 8 weeks were required without particles. These immunizations were effective in protecting mice against tumor challenge with cancer cells expressing HBsAg as a surrogate cancer antigen. Computer modeling of electric fields and gene expression studies indicate that gold particles do not stimulate EP and subsequent antigen expression. The particles may act as an attractant for immune cells, especially antigen presenting cells. We conclude that particulate adjuvants combined with DNA vaccine delivery by EP reduces the immune response time and may increase vaccine efficacy. This method may become valuable for developing prophylactic as well as therapeutic vaccines. The rapid response may be of particular interest in countering bio-terrorism.
Early vaccination is necessary to protect pigs against postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). However, at present no commercial vaccine allows successful vaccination. This is partly due to the presence of maternally derived antibodies. Since DNA vaccines are suggested to be superior to protein vaccines in young animals with maternal antibodies, we determined whether the fimbrial adhesin (FaeG) of F4ac(+) ETEC could be used as a plasmid DNA vaccine to prime piglets in a heterologous prime-boost approach. Hereto, pcDNA1/faeG19 was constructed and expression of rFaeG in Cos-7 cells was demonstrated. Thereafter, pigs were immunised (days 0, 21 and 42) intramuscularly by injection or intradermally by gene gun and humoral and cellular immune responses were analysed. Even though responses were low, results demonstrated that intramuscular injection was superior to gene gun delivery for priming the humoral immune response since higher antibody titres were raised, whereas gene gun delivery better induced a cellular response, evaluated by a lymphocyte proliferation assay. Effective priming of the humoral immune response was evidenced by high IgG titres 1 week after a protein boost with purified F4. The low responses to the pcDNA1/faeG19 DNA vaccination suggest that delivery of the DNA and/or the expression of the faeG gene should be improved.
Anion-exchange is the most popular chromatography technique in plasmid DNA purification. However, poor resolution of plasmid DNA from RNA often results in the addition of bovine-derived ribonuclease (RNase) A to degrade RNA impurities which raises regulatory concerns for the production of pharmaceutical-grade plasmid DNA. Low capacity for plasmid of most commercial media is another issue affecting the suitability of anion-exchange chromatography for large-scale processing. This study reports the use of anion-exchange chromatography to remove RNA in an RNase-free plasmid purification process. Resolution was achieved through careful selection of adsorbent and operating conditions as well as RNA reduction steps before chromatography. Dynamic capacity for plasmid was significantly increased (to 3.0mg/ml) so that it is now possible to envisage the large-scale manufacturing of therapeutic-grade plasmid DNA in the absence of added RNase using anion-exchange chromatography as a polishing step.
One potential reason for the enhancement of immune responses to DNA vaccines following electroporation is increased gene expression. However, the inflammatory response and accompanying cellular infiltration stimulated by electroporation may also be essential for enhancing immune responses to DNA vaccines. These parameters were investigated in pigs, using different electroporation conditions to induce different levels of gene expression and inflammation. Results indicated that the least effective strategy was conventional intramuscular injection where there was low gene expression and low inflammatory cell infiltration. The most efficacious strategy was plasmid administration immediately followed by electroporation. This latter set of conditions elicited a combination of high gene expression and high cellular infiltration. This indicates that electroporation enhances immune responses to DNA vaccines through increased gene expression and inflammatory cell infiltration.
The objective of this study was to determine whether a DNA vaccine encoding bovine CD154 linked to a truncated version of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD-CD154) induces enhanced tgD-specific immune responses in cattle. In vitro characterization demonstrated that tgD and tgD-CD154 both bind to cultured bovine B cells, whereas only tgD-CD154 induces interleukin-4-dependent proliferation, suggesting that tgD-CD154 specifically binds the CD40 receptor and induces receptor signalling. Calves were immunized with plasmid encoding either tgD or tgD-CD154 by intradermal injection with a needle-free device. After two immunizations, tgD-specific immune responses were observed in both vaccinated groups and after challenge with BHV-1 these responses further increased. Animals immunized with plasmid encoding tgD-CD154 had significantly higher tgD-specific serum titres of immunoglobulins G and A but significantly lower numbers of tgD-specific interferon-gamma-secreting cells than animals immunized with plasmid encoding tgD after BHV-1 challenge. This suggests that the expression of an antigen as a chimeric protein with CD154 can qualitatively alter immune responses in cattle. Since we previously showed that plasmid encoding tgD-CD154 induces significantly enhanced secondary tgD-specific antibody responses in sheep, there appear to be interspecies differences in the immune responses induced by tgD-CD154, which suggests that both proteins in the chimeric molecule may influence protein targeting and the induction of an immune response.
Naked plasmid DNA can be used to introduce genetic material into a variety of cell types in vivo. However, such gene transfer and expression is generally very low compared with that achieved with viral vectors and so is unsuitable for clinical therapeutic application in most cases. This difference in efficiency has been substantially reduced by the introduction of in vivo electroporation to enhance plasmid delivery to a wide range of tissues including muscle, skin, liver, lung, artery, kidney, retina, cornea, spinal cord, brain, synovium, and tumors. The precise mechanism of in vivo electroporation is uncertain, but appears to involve both electropore formation and an electrophoretic movement of the plasmid DNA. Skeletal muscle is a favored target tissue for three reasons: there is a pressing need to develop effective therapies for muscular dystrophies; skeletal muscle can act as an effective platform for the long-term secretion of therapeutic proteins for systemic distribution; and introduction of DNA vaccines into skeletal muscle promotes strong humoral and cellular immune responses. All of these applications are significantly improved by the application of in vivo electroporation. Importantly, the increased efficiency of plasmid delivery following electroporation is seen in larger species as well as rodents, in contrast to the decreasing efficiencies with increasing body size for simple intramuscular injection of naked plasmid DNA. As this electroporation-enhanced non-viral gene delivery system works well in larger species and avoids the vector-specific immune responses associated with recombinant viruses, the prospects for clinical application are promising.
This study examined whether increased antigen expression resulted in enhanced antigen-specific immune responses in the context of DNA vaccines. To increase antigen expression, two copies of antigen expression cassettes were arranged in a plasmid pDX. BALB/c mice were intramuscularly immunized with various constructs that express influenza antigens and analysed for DNA-raised immunity. The plasmid pDX that expresses two copies of the antigen gene induced stronger antigen-specific immune responses than the plasmid pGA which expresses single antigen gene. To explore the in vivo transgene expression by pDX and pGA, luciferase activity was measured in the muscles transduced with luciferase expression plasmids. The pDX expressing two copies of luciferase induced the highest luciferase activity, which corresponded to the results from vaccination. We concluded that increasing the number of antigen expression cassettes in a vaccine construct improved antigen expression in the transduced tissue, which induced stronger DNA-raised immune responses.
Several non-Watson Crick DNA structures have been discovered to date, which may be incorporated into future plasmid constructs for gene therapy and DNA vaccine products. In this study, intrinsic DNA structures were included at a defined point in a 2.9 kb plasmid, and their effects on cell growth rate, total plasmid yield, and topology (i.e. the relative proportions of supercoiled plasmid, open circular and linear forms), were determined. The stability of the inserted sequences were assessed using gel electrophoresis. Z-DNA was shown to be unstable in a batch Escherichia coli DH1 production system grown in complex medium. Encouragingly other sequences studied (triplex, bend and quadruplex) did not cause spontaneous deletions, and no detrimental effect was found on growth rate or on total plasmid yield; indicating that such sequences could be included in future DNA products without any detrimental effect on plasmid yields; although the intra molecular triplex studied significantly decreased the proportion of supercoiled species.
An integrated process for purifying a 6.1 kilo base pair (kbp) plasmid from a clarified Escherichia coli cell lysate based on an ultra/diafiltration step combined with polymer/polymer aqueous two-phase system and a new type of chromatography is described. The process starts with a volume reduction (ultrafiltration) and buffer exchange (diafiltration) of the clarified lysate using a hollow fibre membrane system. The concentrated and desalted plasmid solution is then extracted in a thermoseparating aqueous two-phase system, where the contaminants (RNA and proteins) to a large extent are removed. While the buffer exchange (diafiltration) is necessary in order to extract the plasmid DNA exclusively to the top phase, experiments showed that the ultrafiltration step increased the productivity of the aqueous two-phase system by a factor of more than 10. The thermoseparated water phase was then subjected to a polishing step using lid bead chromatography. Lid beads are a new type of restricted access chromatography beads, here with a positively charged inner core that adsorbed the remaining RNA while its inert surface layer prevented adsorption of the plasmid DNA thus passing in the flow-through of the column. Differently-sized plasmid DNA in the range of 2.7-20.5 kbp were also partitioned in the aqueous two-phase system. Within this size range, all plasmid DNA was exclusively extracted to the top phase. The complete process is free of additives and easy scalable for use in large scale production of plasmid DNA. The overall process yield for plasmid DNA was 69%.
The demand for efficient production methods of plasmid DNA (pDNA) has increased vastly in response to rapid advances in the use of pDNA in gene therapy and in vaccines since the advantageous safety concerns associated with non-viral over viral vectors.
A prerequisite for the success of plasmid-based therapies is the development of cost-effective and generic production processes of pDNA. However, to satisfy strict regulatory guidelines, the material must be available as highly purified, homogeneous preparations of supercoiled circular covalently closed (ccc) pDNA. Large-scale production of pDNA for therapeutic use is a relatively new field in bioprocessing. The shift from small-scale plasmid production for cell transfection to large-scale production sets new constraints on the bacterial fermentation, processing of bacterial lysate and final purification and formulation of the plasmid DNA.
The choice of bacterial strain used for plasmid cultivation affects the plasmid yield, the proportion of different isoforms and the amount of endotoxins in the starting material. The choice of bacterial strain will be greatly influenced by the production and purification procedures of pDNA. Master and working cell banks need to be characterised and established. Alkaline lysis of the bacteria damages the pDNA, resulting in a reduced recovery of ccc pDNA and an increase in partially denaturated ccc pDNA and open circular (oc) forms. Shear stress in these processes needs to be tightly controlled, and buffer composition and pH need to be optimised. To obtain a homogeneous plasmid DNA preparation, different pDNA purification strategies aim at capturing ccc pDNA and eliminating the oc isoform. A highly purified final product corresponding to the stringent recommendations set forth by health and regulatory authorities can be achieved by (i) different chromatography techniques integrated with ultra/diafiltration to achieve optimal purification results; (ii) the formulation of the final pDNA product, that requires a detailed study of the plasmid structure; and (iii) the development of sensitive analytical methods to detect different impurities (proteins, RNA, chromosomal DNA, and endotoxins).
We present here a revue of the whole process to obtain such a plasmid DNA, and report an example of RNAse-free purification of ccc pDNA that could be used for gene therapy. Copyright
Clinical trials for gene-therapy products began in the 1990s. Currently, most of the 630 completed or on-going trials are in Phase I and II. Because of the safety issues surrounding the use of viral vectors, approx. 25% of the ongoing clinical trials use DNA plasmids to carry the corrective gene (http://www.wiley.co.uk/genmed). The purpose of this perspective (and this issue) is to highlight some of the unique technical challenges and potential solutions associated with the manufacture and expression of a plasmid-based gene-therapy product.
Perspectives on plasmid-based gene therapy: challenges for the product and the process Issues for improving multiplasmid DNA vaccines for HIV-1
- Wj Kelly
- K Muthumani
- S Kudchodkar
- D Zhang
- Ml Bagarazzi
- Jj Kim
- Boyer
- Jd
Kelly WJ. Perspectives on plasmid-based gene therapy: challenges for the product and the process. Biotechnol Appl Biochem 2003;37(Pt. 3):219–23. [6] Muthumani K, Kudchodkar S, Zhang D, Bagarazzi ML, Kim JJ, Boyer JD, et al. Issues for improving multiplasmid DNA vaccines for HIV-1. Vaccine 2002 May 6;20(15):1999–2003.
Electrokinetic enhancement of plasmid delivery in vivo
- R Draghia-Akli
- Smith
- Lc
Draghia-Akli R, Smith LC. Electrokinetic enhancement of plasmid delivery in vivo. In: Templeton NS, Lasic DD, editors. Gene therapy – therapeutic mechanisms and strategies. 2003 ed. New York: Marcel Dekker Inc.; 2003. p. 245–63.
Electrokinetic enhancement of plasmid delivery in vivo Gene therapy – therapeutic mechanisms and strategies
- R Draghia-Akli
- Lc Smith
Draghia-Akli R, Smith LC. Electrokinetic enhancement of plasmid
delivery in vivo. In: Templeton NS, Lasic DD, editors. Gene therapy
– therapeutic mechanisms and strategies. 2003 ed. New York: Marcel
Dekker Inc.; 2003. p. 245–63.
Electrokinetic enhancement of plasmid delivery in vivo
- Draghia-Akli