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8 Industrial Scale Production of Plasmid DNA for Vaccine and Gene Therapy

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Text of 8 Industrial Scale Production of Plasmid DNA for Vaccine and Gene Therapy

Enzyme and Microbial Technology 33 (2003) 865883

Review

Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purificationKristala Jones Prather, Sangeetha Sagar, Jason Murphy, Michel ChartrainMerck Research Laboratories, Department of Bioprocess R&D, RY80Y-105, Rahway, NJ 07065, USA Received 9 January 2003 ; received in revised form 16 June 2003 ; accepted 24 June 2003

Abstract The past several years have witnessed a rapidly increasing number of reports on utilizing plasmid DNA as a vector for the introduction of genes into mammalian cells for use in both gene therapy and vaccine applications. Naked DNA vaccines allow the foreign genes to be transiently expressed in transfected cells, mimicking intracellular pathogenic infection and triggering both the humoral and cellular immune responses. While considerable attention has been paid to the potential of such vaccines to mitigate a number of infections, substantially less consideration has been given to the practical challenges of producing large amounts of plasmid DNA for therapeutic use in humans, for both clinical studies and, ultimately, full-scale manufacturing. Doses of naked DNA vaccines are on the order of milligrams, while typical small-scale Escherichia coli fermentations may routinely yield only a few mg/l of plasmid DNA. There have been many investigations towards optimizing production of heterologous proteins over the past three decades, but in these cases, the plasmid DNA was not the final product of interest. This review addresses the current state-of-the-art means for the production of plasmid DNA at large scale in compliance with existing regulatory guidelines. The impact of the nature of the plasmid vector on the choice of fermentation protocols is presented, along with the effect of varying cultivation conditions on final plasmid content. Practical considerations for the large-scale purification of plasmid DNA are also discussed. 2003 Elsevier Inc. All rights reserved.Keywords: Plasmid DNA; Gene therapy; Vaccine; DNA vaccine

1. Plasmid DNA in vaccines and gene therapy The use of plasmid DNA as vector for gene therapy or es- pecially vaccination has gained considerable interest during the last decade. While it was known that genetic transfec- tion affects cell physiology, reviewed by Chattergoon et al. [1], the report of uptake and extended in vivo expression of transgenes inserted on a plasmid injected into the leg muscle of mice by Wolf et al. [2] generated tremendous interest. The expressed heterologous proteins were detectable for up to 60 days after injection, indicating that the genes were expressed in vivo over an extended period of time, thus suggesting po- tential therapeutic applications [2,3]. Since then, intramus- cular injection of plasmid DNA has been extensively used in gene therapy and in the design of novel DNA-based vaccines. To date, well over 600 plasmid DNA-based gene ther- apy, cancer vaccine, and therapeutic vaccine clinical trials have been initiated [48]. Notably in gene therapy, the use of a plasmid encoding for VEGF, a vascular endothelial growth

E-mail address: [email protected] (M. Chartrain).

Corresponding author. Tel.: +1-732-594-4945; fax: +1-732-5944400.

factor that has been shown to promote revascularization in ischemic limbs and hearts of patients, has generated extensive interest and publicity [5,9,10]. With regard to vaccines, several investigators have demonstrated that the injection of plasmid DNA containing selected genes from pathogens can elicit a protective immune response [1113]. Subsequently, plasmid DNA vaccines have yielded very encouraging re- sults in the fights against malaria [14] and AIDS [1518]. Currently the use of plasmid DNA vaccines is being investi- gated against many other infectious diseases including hep- atitis B and C, and tuberculosis [1,7,19].0141-0229/$ see front matter 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0141-0229(03)00205-9

It is believed that plasmid DNA vaccination mimics the natural intracellular pathogen gene expression pathways, which triggers both cellular and humoral responses, and therefore can achieve the same protection afforded by virulent or attenuated infectious microorganisms (Fig. 1). These are the properties that make DNA vaccines extremely attrac- tive and, in principle, potentially superior to proteinbased vaccines which generally only elicit humoral response [7,20]. In general, DNA-based vaccines are considered very safe due in part to the lack of genetic integration and to the absence of specific immune response to the plasmid itself

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VACCINATION WITH PLASMIDS

MUSCLE fibroblasts APC

Internal synthesis of foreign peptide/protein

Internal synthesis of foreign peptide/protein

Release of foreign peptide/protein

Presentation of foreign foreign peptide/protein

Stimulation of naive B cells

Stimulation of naive CD 8+ cells

HUMORAL IMMUNE RESPONSE

CELLULAR IMMUNE RESPONSE

Fig. 1. Immune response to plasmid DNA vaccination. Post injection, the plasmid DNA reaches the antigen presenting cells (APC) present in the muscle, where the transgene is transcribed. The foreign protein, which is synthesized in the cytoplasm, is further processed via the major histocompatibility complex of class I (MHC-I) pathway, and eventually displayed on the APC MHC-I present on the cell surface. The APC then migrate to the draining lymph nodes where they elicit a cellular immune response by triggering the maturation and proliferation of CD8+ lymphocytes or cytotoxic T-lymphocytes (CTL). The foreign antigen can also be released in the extra-cellular environment, via either shedding from the MHC-I sites or from the lysis of myocytes. Once in the extracellular milieu, the solubilized antigen comes in contact with CD4 T cells and nave B-cells, and initiates a classical humoral immune response cascade.

[3,21]. Although the lack of genetic integration may appear as a drawback for gene therapy applications, plasmids can be maintained episomally and transcribed for extended periods of time prior to being eliminated. This property makes their use very attractive where transient trans gene expression is desired such as for example, cardiac revascularization [9,10,22]. In addition, unlike live attenuated vaccines, plasmid DNA vaccines do not carry the hypothetical risk of reversion and causing illness [1]. On a small scale, plasmids are viewed as relatively easy to produce and purify. Plasmid DNA also offers extended sta- bility, thus presenting an invaluable advantage in vaccinating populations in areas where sophisticated storage technology may not be available. On the other hand, while this new vac- cine technology is very attractive, injection of naked DNA usually leads to a weak immune response unless relatively

large amounts are administered (up to several milligrams per dose in humans) [20]. However, plasmid production under non-optimized laboratory conditions invariably leads to very low (540 mg/l) volumetric titers. Additionally, the utilization of laboratoryscale purification methods at larger scales leads to poor yields. For vaccination of a potentially large population, and assuming dosages ranging from one to a few milligrams, it is readily apparent that these un-optimized processes and low productivities are inadequate at the industrial scale. In addition, commercial scale processes will have to support not only the economics but also the regulatory standards required for commercial DNA vaccine production. In par- ticular, during plasmid design and the development of a production process, it is crucial that the regulatory en- vironment be constantly considered. DNA vaccines are

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considered by the Food and Drug Administration (FDA) as biological products and are therefore regulated under the well-established vaccine review process controlled by the Office of Vaccine Research and Review (OVRR) division of the Center for Biologics Evaluation and Research (CBER). In order to offer additional clarification with respect to DNA vaccines, both the FDA and the WHO have issued points to consider guidelines and a guideline for assuring the quality of DNA vaccines, respectively. Both publications specifically and extensively address the production and testing of experimental DNA vaccines [23,24].

In addition, several publications offer comments and advice on the interpretation of these guidelines [2330]. The understanding, optimization, and validation of steps, from plasmid design and host strain selection to mass-cultivation and purification, are crucial if this novel vaccine technology is to be commercially successful. To date, while there are not yet any commercial applications of DNA vaccines, several clinical trials are advancing and are accompanied by research and development activities occurring in both industry and academia. The flow diagram presented in Fig. 2 outlines the steps involved in the devel-

Fig. 2. Process steps for the development of a plasmid DNA vaccine.

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opment and production of a DNA vaccine at the industrial scale, which are discussed herein.

2. The choice of a replicon for DNA vaccine or gene therapy constructs The choice of a replicon for plasmid DNA constructs is an important one, as it provides an appropriate framework in which production process development decisions should oc- cur. Most researchers have settled on the ColE1-type vectors as the basis for DNA vaccine or gene therapy constructs to be propagated in the well