Automated alkaline lysis for industrial scale cGMP production of pharmaceutical grade plasmid-DNA

Urthaler, Jochen; Ascher, Christine; Wöhrer, Helga; Necina, Roman

doi:10.1016/j.jbiotec.2006.08.018

Cited by 40 publications

(41 citation statements)

References 32 publications

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“…Rapid progresses in the field of gene-based technology, such as DNA vaccination or gene therapy, require large quantities (milligrams or grams) of clinical-grade plasmid [28] . Large-scale production and purification of plasmid is usually a result of combination and optimization of several unit operations.…”

Section: Discussionmentioning

confidence: 99%

Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries

Yang

Zhang

et al. 2009

Acta Pharmacol Sin

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Aim: To prepare a clinical-grade anti-caries DNA vaccine pGJA-P/VAX and explore its immune effect and protective efficacy against a cariogenic bacterial challenge. Methods: A large-scale industrial production process was developed under Good Manufacturing Practices (GMP) by combining and optimizing common unit operations such as alkaline lysis, precipitation, endotoxin removal and column chromatography. Quality controls of the purified bulk and final lyophilized vaccine were conducted according to authoritative guidelines. Mice and gnotobiotic rats were intranasally immunized with clinical-grade pGJA-P/VAX with chitosan. Antibody levels of serum IgG and salivary SIgA were assessed by an enzyme-linked immunosorbent assay (ELISA), and caries activity was evaluated by the Keyes method. pGJA-P/VAX and pVAX1 prepared by a laboratory-scale commercial kit were used as controls. Results: The production process proved to be scalable and reproducible. Impurities including host protein, residual RNA, genomic DNA and endotoxin in the purified plasmid were all under the limits of set specifications. Intranasal vaccination with clinical-grade pGJA-P/ VAX induced higher serum IgG and salivary SIgA in both mice and gnotobiotic rats. While in the experimental caries model, the enamel (E), dentinal slight (Ds), and dentinal moderate (Dm) caries lesions were reduced by 21.1%, 33.0%, and 40.9%, respectively. Conclusion: The production process under GMP was efficient in preparing clinical-grade pGJA-P/VAX with high purity and intended effectiveness, thus facilitating future clinical trials for the anti-caries DNA vaccine.

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Section: Discussionmentioning

confidence: 99%

Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries

Yang

Zhang

et al. 2009

Acta Pharmacol Sin

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“…In several cases, mixing is achieved by static [18] or active mixers [19]; although simple T-mixers have proven to be very efficient when using sufficient flow rates [20,21]. The removal of the precipitated debris and impurities is achieved by centrifugation and subsequent filtration, settling on suitable filter aids [18], or froth flotation [19][20][21]. In the latter case, an efficiently cleared lysate was obtained that could be applied to subsequent downstream processing steps without any further clarification.…”

Section: Cell Disruptionmentioning

confidence: 99%

Downstream Processing of Plasmid DNA for Gene Therapy and Genetic Vaccination

Voss

2008

Chem Eng & Technol

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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 multiand 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.

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“…For the cell disintegration and the clarification step automated proprietary systems, built in house were used [76,77]. During development of the chromatography steps, XK columns from Amersham Biosciences were used for the particulate adsorbents and CIM tubes of 8 mL and 80 mL volume in the case of the monolithic adsorbent (BIA Separations).…”

Section: Instrumentationmentioning

confidence: 99%

“…The cGMP-compliant process consists of a gentle, automated and continuous lysis and clarification procedure [76] and three consecutive chromatography steps, based on different principles, as key steps of the purification.…”

Section: Introductionmentioning

confidence: 99%

Industrial Scale cGMP Purification of Pharmaceutical Grade Plasmid‐DNA

2005

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The advances in gene therapy and genetic vaccination based on plasmid DNA result in an increasing demand of pharmaceutical grade plasmids produced under cGMP. A generic process consisting of a cell disintegration step followed by three different chromatography steps has been developed. Cell lysis is performed by an automated continuous reactor. The clarified lysate is further purified by hydrophobic interaction, anion exchange, size exclusion chromatography and by an ultra-filtration step. The produced plasmid DNA was up to 98 % in the supercoiled form. The final genomic DNA content was lower than 10 lg/mg plasmid DNA, RNA was not detectable by agarose gel electrophoresis, the protein content was lower than 1 lg/mg plasmid DNA and the endotoxin content lower than 0.1 EU/mg plasmid DNA. An overall yield of 50 % and higher in the desired final buffer could be obtained. This process is scalable and does not require animal derived materials, detergents or organic solvents, meeting current standards for therapeutic applications.

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Automated alkaline lysis for industrial scale cGMP production of pharmaceutical grade plasmid-DNA

Cited by 40 publications

References 32 publications

Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries

Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries

Downstream Processing of Plasmid DNA for Gene Therapy and Genetic Vaccination

Industrial Scale cGMP Purification of Pharmaceutical Grade Plasmid‐DNA

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