Design of flowsheets for the recovery and purification of plasmids for gene therapy and DNA vaccination

Chemical Engineering and Processing 43 (2004) 615–630

Design of flowsheets for the recovery and purification of plasmids for gene therapy and DNA vaccination D.M.F. Prazeres a,∗ , G.N.M. Ferreira b a

Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, Lisbon 1049-001, Portugal b Centre for Structural and Molecular Biomedicine, University of Algarve, Algarve, Portugal Received 10 October 2002; received in revised form 28 January 2003; accepted 5 February 2003

Abstract Plasmids are covalently closed, double stranded, DNA molecules which carry genetic information. These macromolecules have been used for gene therapy and DNA vaccination, and belong to a new class of medicinal agents that contain genetic materials. The manufacturing of plasmids under current Good Manufacturing Practices (cGMP) compliance as required by the Food and Drug Administration (FDA) and European Medicines Evaluation Agency (EMEA) is crucial to obtain a product which is consistent in purity, potency, identity, efficacy and safety. This paper gives an overview of the manufacturing of plasmids and provides the basic concepts for designing flowsheets for recovery and purification. The focus is mainly on the downstream processing, which is designed to release plasmid molecules (less than 1% w/w) from the Escherichia coli host cells and to remove impurities such as genomic DNA, RNA, proteins and endotoxins until the desired level of purity and other specifications are met. Many of the host impurities have similar properties to plasmid DNA, and will thus behave identically in most of the downstream processing steps. This constraints the selection and ordering of the units-operations used for separation throughout the process. The downstream processing unit operations can be grouped in three different stages: primary recovery, intermediate recovery and final purification. The objective of each of these stages as well as the unit operations more commonly used is described. The specific problems encountered that are associated with the structural nature of plasmids (high molecular weight, charge and flexibility) are highlighted. Four flowsheets that are representative of the current panorama in downstream processing of plasmid DNA are described and their weaknesses highlighted. An alternative process solution is suggested that, although unproven, addresses many of the weaknesses of the existing processes. © 2003 Elsevier B.V. All rights reserved. Keywords: Gene therapy; DNA vaccines; Plasmid DNA; Bioprocess design; Process synthesis; Downstream processing

1. Gene therapy and DNA vaccines Gene therapy is a therapeutic strategy in which nucleic acids are introduced in human cells, modifying their genetic repertoire for therapeutic purposes [1]. Both viral and non-viral (plasmid DNA) vectors have been developed to efficiently deliver nucleic acids to target cells [2]. The use of viral vectors has raised safety and regulatory concerns after half a dozen deaths were reported in several separate clinical trials [3]. For these reasons, non-viral, plasmid DNA vectors constitute an attractive gene transfer system [2,4] in an overall market that is expected to reach US$ 45 billion by 2010 [5]. The number of approved ∗ Corresponding author. Tel.: +351-218-419-133; fax: +351-218-419-062. E-mail address: [email protected] (D.M.F. Prazeres).

0255-2701/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cep.2003.02.002

gene therapy protocols using plasmid DNA-based delivery vectors has increased exponentially since 1995, representing approximately 25% of the ongoing gene therapy trials (http://www.wiley.co.uk/genetherapy/clinical). Plasmid DNA has also been used to express specific antigens on cell membranes, stimulating and enhancing the immune system response and memory, as a newer and safer generation of vaccines [6,7]. Plasmid DNA vectors, however, are less effective in transfecting cells when compared with viral vectors [2]. It is estimated that only one in every 1000 plasmid molecules presented to the cells reaches the nucleus and is expressed [1]. Thus, full treatments may require milligram amounts of plasmid DNA. The development of large-scale plasmid DNA manufacturing processes must therefore accompany the increasing number of gene therapy and DNA vaccine applications that are moving from the laboratory to clinical trials and eventually to the market.

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2. Plasmid DNA: product properties The set-up of a bioprocess should always take into consideration the properties of the target molecule and associated impurities. This knowledge is important for instance in the design of adsorption techniques that are based upon specific interactions between the target molecule and an adsorbent matrix [8], or in the design of unit operations which separate molecules on the basis of size and shape. DNA is a nucleic acid that carries genetic information. Structurally, it is a polynucleotide in which each nucleotide monomer is joined to the adjacent through a phosphodiester bond. At pH > 4 phosphate groups in the molecule backbone are ionised and therefore each nucleotide contributes with one negative charge to the overall net charge. DNA (and RNA) is thus a poly-anionic molecule with an overall net charge equal to the number of nucleotides. The winding of two anti-parallel DNA strands about each other, results in a right-handed double helix structure, which is stabilised by hydrogen bonds between complementary nucleotides in opposing strands. The aromatic bases of the nucleotides are stacked over each other and oriented towards the interior of the molecule, perpendicularly to the sugar-phosphate backbone [9]. An important consequence of the nucleotide arrangement in DNA is the formation of continuous indentations in the molecule surface, termed grooves, which enable solvent and ligand molecules to access the nucleotides. Such accessibility is crucial if ligand-DNA binding and recognition are to be explored in the design of purification operations [10,11]. Plasmids are double stranded DNA molecules which have the particularity of being covalently closed. In cells they act as extra-chromosomal carriers of genetic information. Apart from their helical nature, plasmids display a higher-order structure named supercoiling, which occurs due to topological constraints imposed upon circular DNA molecules that are under-wound or over-wound around the helix axis. This higher order structure gives rise to different isomers (termed topoisomers), of which open circular (oc) and supercoiled (sc) plasmid DNA are the most common [9,12]. The sc plasmid DNA is more efficient in terms of gene expression and

is therefore the preferred form for gene delivery. The sc plasmid DNA is thought to be highly dynamic, with branches forming and retracting rapidly [12,13]. The practical consequence of such a dynamic behaviour is that the molecule dimensions and shape can change by changing the solvent conditions. For instance, sc plasmid DNA molecules can collapse or expand by increasing or decreasing the solvent ionic strength, respectively [13], or by changing the type of counter-ions (cations) [14]. Plasmids are usually hosted and produced in bacteria such as Escherichia coli that can be grown by fermentation.

3. Product specifications, quality control and monitoring Biopharmaceuticals based on plasmid DNA are chemically highly defined and, therefore, can be analysed by chemical, biochemical and physical assays [15]. The monitoring of a manufacturing process performance, as well as the assessment of the final product quality in comparison with product specifications, is a key issue in process development, validation and product approval [11,15,16]. The exact final product specifications (identity, efficacy, safety, potency and purity) usually depend on the intended therapeutic use, and thus are defined during clinical trials [17]. However, regulatory agencies such as the Food and Drug Administration (FDA) in USA and the European Medicines Evaluation Agency (EMEA) in the European Union provide guidelines and quality standards that are helpful in product and process development (Table 1) [18–20]. The set-up of analytical methodologies and procedures capable of fully characterising the product and process performance is thus a crucial task that must be considered during the design stage.

4. Plasmid manufacturing A process for the manufacture of plasmid DNA consists of a number of activities aimed at the production of

Table 1 Specifications and recommended assays for assessing plasmid DNA purity, safety and potency (adapted from [8,56]) Specification

Acceptance criteria

Analytical method

Plasmid Appearance Identity of plasmid Plasmid homogeneity Plasmid concentration Potency

Clear, colourless solution Restriction map as expected, sequence homology >90% supercoiled form According to application According to application

Visual inspection Restriction mapping sequencing, PCR Agarose gel + densitometry A260, HPLC, fluorescence Cell transfection

Impurities Proteins RNA gDNA Endotoxins

Not detectable, < 0.01 ␮g /dose Not detectable < 0.05 ␮g /␮g plasmid, < 0.01 ␮g /dose < 0.1 EU/␮g plasmid, < 5 EU/kg body weight

Bicinchoninic acid (BCA) assay, SDS-PAGE 0.8% agarose gel Hybridisation blots, PCR, fluorescence Limulus ameobocyte lysate (LAL) assay

D.M.F. Prazeres, G.N.M. Ferreira / Chemical Engineering and Processing 43 (2004) 615–630 Upstream

Cell banks Raw materials

Cell culture

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Downstream

Primary recovery

Intermediate recovery

Final Purification

Fill &Finish

Quality control and monitoring

Fig. 1. Manufacturing of plasmid DNA.

a certain amount (measured as mass or biological activity) of the target product at an acceptable cost and quality. The preparation of cell banks and the selection and testing of raw materials are at the forefront of these activities. Upstream and downstream processing unit-operations are selected, arranged, designed and operated to manufacture a bulk product. After filling and finishing, the product can be distributed and shipped (Fig. 1). Although the recovery and isolation of other biopolymers such as proteins is well established, downstream processing of plasmid DNA presents specific problems on its own. These are related to the peculiar shape and conformation of plasmids, to the unusually high viscosity of some process streams, and to the presence of impurities with similar properties [17,21]. This paper will focus mainly on the downstream processing part of the process. 4.1. Process considerations A flowsheet for the recovery and purification of plasmid DNA is usually established with a number of rules of thumb or heuristics in mind (Table 2), and on the basis of accumulated experience with the target product [22,23]. Simulation tools have also been developed for the systematic synthesis of bioprocesses which can be used to rapidly evaluate alternatives and speed up development [23–25]. The specificity’s of plasmid purification were first addressed by molecular biologists which have developed a range of efficient lab-scale protocols for the purification of plasmid DNA [26]. Unfortunately, many of these protocols Table 2 Heuristics for setting up a plasmid recovery and purification flowsheet [22,23] 1 2 3 4 5 6

Remove the most plentiful impurities first Remove the easiest to remove impurities first Make the most difficult and expensive separations last Select processes that make use of the greatest differences in the properties of the product and impurities Select and sequence processes that exploit different separation driving forces Just because it works in the lab does not mean it’s right for the factory

cannot be scaled-up or use reagents that are not acceptable for the manufacturing of a biological pharmaceutical. Nevertheless, many of the existing process solutions correspond to modifications or adaptations of these laboratory procedures. This mode of action should be carefully taken since the realities of factory and research lab are completely different (check heuristic 6) [22]. Fermentation is usually optimised to obtain high cell densities and plasmid mass [10,15,17,21]. Next, a sequence of unit operations must be set up to recover plasmid and eliminate host cell impurities (Table 3) until the desired level of purity is met (Table 1). For a product with an intended use in humans this removal of impurities is mandatory to avoid side effects once administered to patients. The downstream processing unit operations can be grouped in three different stages: primary recovery, intermediate recovery and final purification (Figs. 1 and 2). Ideally, the overall process should have a limited number of high-recovery steps, so that processing costs are reduced [27]. The process should also favour the use of reagents that are Generally Regarded As Safe (GRAS) by the FDA. Furthermore, lengthy operations and processes should be avoided in order to cut costs from overhead, amortisation of equipment and direct labour charges [22].

Table 3 Characterisation of nucleic acids and other components in an E. coli cell (adapted form [90]) Species

Amount (% w/w)

Water

70

Nucleic Acids Genomic DNA Transfer RNA Ribosomal RNA Messenger RNA Plasmid DNA

0.5 4.8 0.9 0.3 <1

Proteins Endotoxins Small molecules and ions

15 5 3

a b

Different species/cell 1 1a 40 3 400–800 1 1100 800–2000

Average MW (kDa) 18 2.8 × 106 28 500–1000 660–990 3300b 8–200 10 <1

Rapid growing E. coli cells have on average four molecules of gDNA. A plasmid molecule with an average 5 kbp is considered.

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Fig. 2. Generic block of downstream processing of plasmid showing unit operations options.

4.2. GMPs and validation Plasmid DNA, such as all products that are to be administered to humans or animals, must be manufactured in accordance with current Good Manufacturing Practices (cGMP) requirements as clearly stated by the FDA guideline on the preparation of investigational new drug products [28]. GMPs regulations cover all aspects of the production, from choosing and testing raw materials to utilities, packaging, shipping and transferring final products to the clinic [29]. If these items are not in conformity with cGMPs, the product is deemed to be adulterated and cannot be legally approved [30].

Validation, which is one of the cornerstone provisions of cGMPs, has been introduced to assure product consistency [31,32]. According to a well-known definition, validation is “the process of establishing documentary evidence that provides a high degree of assurance that any product, process, activity, procedure, system, equipment, or software used in the control and manufacture consistently performs to or meets its predetermined specifications” [32]. Downstream processing operations must thus be validated to prove that they are capable of consistently removing impurities such as host cell components, process-related materials and adventitious agents to an acceptable level [30]. Additionally, acceptance limits and operating ranges for each step must be

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determined. Another extremely important aspect that needs to be validated is the cleaning of individual equipment items [32]. Validation studies usually lead to optimised processes with reduced variability and, as a consequence, to a decrease in the number of failed batches. Thus, developing a bioprocess (and associated facility) with validation in mind not only helps to improve quality assurance and accelerate approval, but usually leads to a reduction of costs [33,34]. 4.3. Environmental and safety issues An assessment of the environmental impact of a process is an essential part of the design [23,25]. The costs associated with the treatment and disposal of the waste generated by a particular process solution should be estimated and used as one of the key elements in the final decision on which process to select. Environmentally-unfriendly operations such as those that generate large amounts of hazardous solvents and materials, which are usually costly to dispose off, should definitely be avoided. Other common components of biological waste streams such as buffers, acids, alkalis and E. coli fermentation derived organic-chemicals (e.g. cell debris and residual media components) usually pose limited risk to the environment once inactivated. Nevertheless, process optimisation should always be geared towards minimisation of waste generation [23,25]. Unit operations that need special safety precautions (e.g. explosion proof tanks, blow out walls, emission containment, personnel protection, etc.) to operate should also be carefully considered, since these requirements can dramatically increase the cost of equipment, building design and construction [35]. Overall, environmental and safety issues require an intimate knowledge of the process technology solutions available.

5. Downstream processing E. coli fed-batch fermentations have been optimised and automated to deliver a broth with high cell density (up to an optical density at 600 nm of 100) and plasmid concentration (e.g. 100 mg/l) [36]. This broth, with a cell concentration close to 0.45 g/(OD600 l), constitutes the starting point for downstream processing. 5.1. Primary recovery In the primary recovery stage cells are harvested from the fermentation broth and plasmid DNA is released from the cells together with other impurities (Table 3). A significant reduction in volume occurs and the most plentiful impurities such as extracellular liquid, proteins, genomic DNA (gDNA) and lipids are removed (Heuristic 1). Cell harvesting is well established in the bioindustry and is usually accomplished by centrifugation, rotary-drum filtration or cross-flow microfiltration. When concentrating small cells such as E. coli, microfiltration is often the most attractive option due to cell

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recoveries close to 100% [23] and lower capital and operating costs [24]. The disruption of cells to release plasmid DNA is probably the most critical and troublesome of all unit operations in the downstream processing. High amounts of intact, sc plasmid DNA must be released to the surrounding medium in order to guarantee high overall process yields. Other intracellular components such as RNA, gDNA, endotoxins and proteins are also released (Table 3). Shear and chemical sensitivity of plasmid and gDNA molecules [37], as well as the high viscosity of the process streams essentially attributed to the large concentration of nucleic acids [38], are of major concern during this stage [17]. Prior to disruption, the cell paste is usually re-suspended with an adequate lysis buffer. Cells in this buffer are typically concentrated 10–15 times relatively to the original fermentation broth [39]. Mechanical disruption of cells with unit operations such as sonication, bead milling microfluidisation and homogenisation are avoided since the associated plasmid damage is usually significant [11,40]. The best plasmid recoveries (74%), of which 94% in the sc conformation, were obtained in bead mills with 50% cell disruption [40]. Increasing cell disruption yields led to the drop-off of the sc plasmid recovery to less than 10% [40]. Cell disruption by alkaline lysis, originally described by Birnboim and Doly [41] and extensively used by molecular biologists, has been one of the most used procedures. An alkaline solution of a detergent (e.g. NaOH + sodium dodecyl sulphate, SDS) is used to solubilise cell membranes and to promote the irreversible denaturation of proteins and nucleic acids while keeping plasmid denaturation reversible. The process is fast at the microscopic level, with almost all cells disrupted after 30–60 s [38,42], but the high viscosity and non-Newtonian properties of the resulting lysate makes flow and handling of the material difficult. The use of SDS as a detergent translates directly from laboratory practice. From a manufacturing point of view SDS presents some disadvantages, namely its propensity to be hydrolysed under alkaline conditions [43]. Alternative detergents such as Tween® (a polysorbate) and Triton® (a polyoxyethylene ether) have been suggested [35,44]. The alkyldimethylphosphine oxide (APO) detergent has been heralded by its users as an excellent alternative due to its stability on alkaline solution and its definable nature that makes it compatible with GMP procedures [43]. Mixing during alkaline lysis should be efficient to guarantee a uniform distribution of pH around 12.2–12.4, thus avoiding the irreversible denaturation of plasmid DNA. Simultaneously it should be gentle in order to keep gDNA and RNA with the highest molecular weight possible so as to maximise precipitation and filtration in the subsequent steps. Upon neutralisation of the alkaline lysate (e.g. with chilled potassium acetate), an ill-defined mass of solids containing cell debris, denatured proteins and nucleic acids is generated which must be removed by low shear, solid–liquid unit operations. A fraction of these solids floats

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while another sediments, making separation more difficult. Continuous industrial centrifuges are usually excluded since the high shear in the incoming liquid stream can break the precipitate material both at the macroscopic and molecular level. Dead-end filtration is a convenient choice [45,46]. Up to 67% plasmid recovery can be achieved after removing 99% of the solid mass with a 5 ␮m pore diameter filter [46]. Filter layers of glass, silica gel, diatomaceous earth, aluminium oxides and hydroxyapatite can be used as filtration aids to assist in the separation of solids from liquids [46–48]. Apart from a reduction in back-pressure and shearing of solids, materials such as diatomaceous earth have the additional ability to adsorb host impurities such as RNA and endotoxins [47]. However, plasmid loss due to co-adsorption and absorption may constitute a significant problem, as demonstrated by Theodossiou et al.[49]. The performance of filtration can be improved by first removing the bulk of the solids by floatation [49]. Lysates are left to age for 1 h at 10 ◦ C and the flocs further aggregate, forming a more compact bed of floating material. Air can be injected to promote a low shear mixing and improve floatation. The plasmid containing liquid is then drained from underneath and subjected to filtration to remove the remaining solids. Overall, the clearance capacity of a complete alkaline lysis disruption process can reach 98% for gDNA, 97% for proteins and 98% for suspended solids [50]. As alternative to alkaline lysis, cells can be disrupted using a buffer containing detergent (e.g. Triton® ) and lysozyme at high temperature. By heating the cells up to 70–77 ◦ C in a flow through heat exchanger it has been possible to induce cell lysis and precipitate gDNA, proteins and other debris while keeping the plasmid in solution [51]. The use of a flow through device for disruption has the advantage of enabling a continuous and more reproducible processing when compared with the usual batch alkaline lysis carried in tanks. The improved consistency of heat lysis also means that validation is easier. The presence of endogenous nucleases in plasmid preparations obtained at the end of the alkaline lysis step can be advantageously used to remove high molecular weight RNA. Monteiro et al. [52] demonstrated that RNA levels can be reduced by 40% when the lysates are incubated at 37 ◦ C with only 9% plasmid loss. Exogenous enzymes such as lysozyme and ribonuclease (RNase) are sometimes added during or shortly after lysis to improve degradation of proteins and RNA [51,53]. Although very effective, the use of these animal derived enzymes is expensive and constitutes a source of mammalian pathogens (retroviruses and prions) which presents significant safety and regulatory issues [17]. 5.2. Intermediate recovery The intermediate recovery stages are designed to concentrate and further purify the product. In plasmid processing, the starting point is usually a complex and viscous lysate obtained after cell lysis. The plasmid in this stream is usually

diluted, and although most gDNA has been removed, large amounts of RNA and proteins (>90% total solute mass) still remain in the clarified lysates [53,54]. Volume reduction by concentration is commonly performed by precipitating the plasmid with agents such as isopropanol [55], polyethylene glycol [35,48,56] or cetyl trimethylammonium bromide (CTAB) [57]. The removal of low molecular weight nucleic acids and the possibility of performing a buffer exchange are additional advantages of this operation. Ethanol and isopropanol precipitation work very well at lab scale, but its use is problematic at large scale. The need to maintain a low temperature (−20 ◦ C) closely controlled in order to obtain reproducible results, the requirement of explosion proof tanks and processing areas and the generation of large amounts of hazardous waste which needs to be disposed off can dramatically increase costs [35]. Precipitation with CTAB affords a novel selectivity by removing gDNA and even the more closely related relaxed and denatured forms of plasmid [57]. PEG and CTAB precipitation are therefore advantageous options. After concentration of plasmid by precipitation, a large fraction of impurities (proteins, endotoxins and higher molecular weight RNA) can be removed with an additional precipitation step using salts such as ammonium sulphate [55], ammonium acetate [56], calcium chloride [58] and lithium chloride [59]. For instance, addition of ammonium sulphate up to 2.5 M results in a substantial reduction of the protein (undetected after precipitation) and endotoxin (20,000 times) content, increasing plasmid purity 6.5 fold [55,60]. Precipitation, whether used for concentrating the plasmid or for removing impurities always imply the subsequent use of a solid–liquid separation, usually centrifugation or filtration. The clarified and neutralised lysates can also be subjected to liquid-liquid extraction using aqueous two-phase systems (ATPS) such as polyethylene glycol (PEG)/K2 HPO4 [61]. Plasmid recovery yields of 39%, 42% and 100% have been obtained with PEG molecular weights of 300, 600 and 1000 respectively, with considerable improvements in purity. Ultrafiltration and microfiltration have also been considered an option for an intermediate purification of clarified lysates [44,62,63]. The use of tangential flow filtration with UF polyethersulfone membranes with nominal weight cut-offs of 1,000,000 and 500,000 Da, has been claimed to remove more than 99% of RNA and 95% of protein from clarified lysates obtained after an extended alkaline lysis with lysozyme [44,62]. Nitrocellulose filtration membranes (0.45 ␮m) have been used to selectively remove gDNA, RNA and endotoxins (at high salt), not only from clarified lysates [63], but also from re-suspended PEG precipitates and anion-exchange eluates [64]. Although the different steps, briefly described above, produce a cleaner and smaller process stream, there is evidence that these operations can be by-passed, proceeding directly to chromatography with increases in process yield [54]. This strategy, however, has the disadvantage of reducing the life

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time of chromatographic media due to fouling. Furthermore, if unclarified lysates are used, column performance will decrease as a consequence of bed clogging. Expanded bed adsorption may thus be preferred to fixed bed chromatography [53,65]. 5.3. Final purification A pharmaceutical product such as plasmid DNA requires a high degree of purity. This level of purity is usually achieved with a combination of chromatography and filtration. The goal is to separate sc plasmid DNA from related impurities (in terms of composition, structure and electrostatics), such as oc and denatured plasmid DNA, gDNA, RNA and endotoxins that still persist in the streams. The use of an expensive and high-resolution operation such as chromatography in the later part of the downstream processing is in agreement with Heuristic 3 (Table 2). The chromatographic operations described in the literature for plasmid purification explore properties such as size, charge, hydrophobicity, conformation and accessibility of specific molecular groups. One of the major limitations of plasmid purification by chromatography is the current lack of capacity of existing adsorbents. This is related to the large size of plasmid molecules that can only bind to the external surface and to a thin outer layer of adsorbent particles [66–68]. Anion-exchange (AEX) chromatography explores the interaction between the negatively charged DNA backbone and stationary phases bearing positively charged ligands such as quaternary amines [68,69]. Bound nucleic acids are displaced with a salt gradient and elute as a function of increasing charge density. Nucleotide sequence and composition may affect the elution pattern [70,71]. In some anion-exchangers, the more compact sc plasmid forms, which have a higher charge density, elute later than the open circular forms, which have a lower, overall, charge density [68]. AEX is ideal for removing RNA, oligoribonucleotides and some proteins. However, other polyanionic molecules, such as gDNA fragments [8] and endotoxins [72], may co-purify with the plasmid due to their similar binding affinities and constrained diffusion of the macromolecules inside the adsorbent pores [73,74]. Some high molecular weight gDNA fragments and endotoxins can adhere tenaciously to the anion-exchange resins, decreasing the binding capacity for plasmid. In spite of the limitations described above, AEX chromatography has been used as a first purification step to capture and concentrate plasmid DNA [53,54,68,75]. An overall process that uses two AEX columns in the purification section has even been proposed, although this clearly contradicts common sense as expressed in Heuristic 5 [58]. Although AEX has been performed mostly in fixed-bed mode, AEX expanded-bed adsorption (EBA) has been recently reported [39,53,65]. This mode of operation is particularly attractive if the clarity of feedstock is insufficient for a reliable processing in fixed-beds without further processing.

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With EBA it is possible to by-pass clarification and concentration operations such as the ones described in the previous section [53,65]. Due to its early use in the downstream processing, EBA can be considered as an intermediate recovery unit operation. Hydrophobic interaction chromatography (HIC) has also been used to purify plasmid [55,60,76]. With matrices derivatised with mildly hydrophobic ligands it is possible to separate sc and oc plasmid DNA from RNA, gDNA, endotoxins, denatured plasmid forms and oligonucleotides. HIC takes advantage of the higher hydrophobicity of single stranded nucleic acids that show a high exposure of the hydrophobic aromatic bases when compared with double stranded nucleic acids. In double stranded plasmid molecules the hydrophobic bases are packed and shielded inside the helix and thus interaction with the HIC support is minimal. On the other hand, the high content of single strands in RNA and gDNA impurities enables hydrophobic interactions to take place. Endotoxins interact even more strongly with the HIC media via the lipidic moiety [55]. Scale-up of HIC is straightforward with no losses in purity and only marginal losses in yield, and run-to-run consistency is high, even after sanitation and cleaning cycles of caustic washing [76]. Reverse-phase chromatography (RPC) also explores interaction of hydrophobic, non-polar regions of the molecules, with non-polar immobilised ligands. Bound molecules are eluted with decreasing polarity gradients, by adding organic modifiers to the column eluent [8,77]. Therefore, the solutes are eluted in the order of decreasing polarity or increasing hydrophobicity [8,77]. When the target molecules are polar, which is the case of nucleic acids, however, reverse-phase principles are achieved by adding amphiphilic organic ions to the buffers—a technique termed ion-pair chromatography. These ions establish ionic interactions with the target molecules (e.g. plasmid molecules), forming hydrophobic non-polar ion pairs that bind to reverse-phase resins [74,78]. RPC and ion-pair chromatography have been used to purify sc plasmid DNA from crude cell lysates [79,80] and from AEX eluates [51]. Low molecular weight RNA and linear and oc plasmid forms are usually well separated from the late eluting sc plasmid DNA [51,79,80]. Endotoxins and gDNA remain bound to the column, and are removed after sanitising the column with 1 M NaOH [79]. The need to use organic solvents and mixtures thereof to elute plasmid molecules in RPC constitutes a significant disadvantage. Affinity chromatography is based on the recognition of a particular structure in the target plasmid molecule by an immobilised ligand. The formation of triple helices between oligonucleotides linked to a chromatographic matrix and duplex sequences present on the plasmid molecule has been explored, but only at lab-scale [81,82]. The technique is claimed to purify plasmid DNA in one-step, while significantly reducing the levels of impurities. Although highly selective, triple helix affinity chromatography has a low versatility since each ligand targets a specific base sequence.

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Fig. 3. Plasmid production flowsheet I (based on [56]). Kac: potassium acetate; PEG: polyethylene glycol; A. acetate: ammonium acetate; SEC: size exclusion chromatography.

D.M.F. Prazeres, G.N.M. Ferreira / Chemical Engineering and Processing 43 (2004) 615–630 Fig. 4. Plasmid production flowsheet II (based on [55,60]). Kac: potassium acetate; SEC: size exclusion chromatography; A. sulphate: ammonium sulphate; HIC: hydrophobic interaction chromatography.

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Furthermore, the costs associated with large scale processing are likely to be prohibitive. Purification based on affinity interactions between plasmids and stationary phases mediated by a zinc finger-glutathione S-transferase linker is a recent development [83]. Size-exclusion chromatography (SEC) fractionates and purifies plasmids on the basis of size and conformation. SEC can be used alone [48,56,84–87] or sequentially to other chromatographic steps such as anion exchange [53,54]. It constitutes an ideal finishing operation, which enables the removal of residual contaminants such as gDNA and RNA, the partial separation of sc from oc plasmid variants and a substantial reduction in endotoxin loads [56]. It further offers the possibility of exchanging the plasmid into an adequate formulation or storage buffer. Typically the different DNA molecules exit the column as a broad, non-Gaussian peak: gDNA elutes first as the leading edge, followed by oc and then sc plasmid. Smaller solutes such as RNA, oligonucleotides and salts are easily separated from the leading DNA peak. A judicious choice of the fractions to be collected enables the recovery of almost pure sc plasmid. Disadvantages of SEC are its limited capacity and the dilution of the plasmid. Furthermore, few SEC gels in the market have the adequate selectivity to be used in plasmid purification.

6. Existing process synthesis solutions In this section, four different flowsheets (hereafter named I, II, III and IV) are presented (Figs. 3–6) to illustrate the synthesis of large-scale plasmid DNA downstream processes. These flowsheets are representative of the current panorama in downstream processing of plasmid DNA. They were adapted from the literature and drawn in a simplified way using the biochemical process simulator BioProDesigner (Intelligen, Scotch Plains, NJ, USA). Other process synthesis solutions have been reported in the literature or patented, but are not presented here due to the lack of space ([39,54,58,75,88,89]). In the examples presented, the unit operations described previously and summarised in Fig. 2 are put together differently, but with the same final objective of producing a plasmid DNA product within the specifications (Table 1). In the figures shown, the different unit operations are grouped in stages (primary recovery, intermediate recovery and final purification) as described. In all cases centrifugation is used to harvest cells, even though a microfiltration unit would probably be a better choice as was previously described. Although not reported in the associated references, cell loss (and hence plasmid) during centrifugation could typically reach 1–5% [23]. In the first three examples (Figs. 3–5), primary recovery is accomplished by alkaline lysis, while in the fourth case (Fig. 6) cell disruption is accomplished by the joint action of heat, detergent and lysozyme in a flow through heat exchanger. This later mode of cell disruption is claimed to be

more reproducible and consistent than alkaline lysis [51]. In flowsheet III, RNase is added during lysis to digest RNA and thus improve the purification performance during the subsequent steps. The size of the intermediate recovery stage decreases from flowsheet I to flowsheet IV. In the first case, this section is quite extensive, involving three different precipitation steps and associated solid/liquid operations (total 7 unit operations). In flowsheet II two precipitation steps are included (total 4 unit operations), while in flow sheet III the inclusion of expanded bed adsorption avoids clarification steps (total 3 unit operations). Flowsheet IV presents a process synthesis solution that includes an RNA digestion step using RNase plus two membrane operations in the intermediate recovery section (total 3 unit operations). With the exception of flowsheet III, all flowsheets include two chromatographic steps in the final purification stage. An interesting characteristic is that in all cases the two types of chromatography are different, in a clear intention of exploring different separation driving forces as recommended in Heuristic V (Table 2). As for flowsheet III, although only one chromatographic step is used in the final purification, expanded bed chromatography is used upstream in the intermediate recovery stage. Size exclusion chromatography is used in three flowsheets (I–III) as a final purification/buffer exchange step. In flowsheet IV, buffer exchange is taken care off by diafiltration with an UF operation, which has the advantage over SEC of enabling the concentration of the product. In the case of flowhseet I, concentration is performed by ethanol precipitation followed by resuspension of the precipitated plasmid with the final buffer. A final sterilisation step should be performed in all cases prior to filling, for instances with a 0.2 ␮m pore size membrane filtration. The reported overall plasmid yields of processes I and IV are 51 [56] and 54% [51] respectively, while the overall yield in process II prior to SEC is 70% [55]. No yield data is available for process III. The use of RNase in flowsheets III and IV, constitutes a disadvantage since large scale RNase reactions are costly and difficult to perform. Furthermore, the use of this enzymes poses significant safety and regulatory issues as previously mentioned. A common weakness of the four processes is the use organic solvents (isopropanol and/or ethanol) in the operations designed to precipitate plasmid. These process solutions will thus require safety measures such as the design of explosion proof facilities or use of appropriate protection masks [45].

7. An alternative process synthesis solution In this section, an alternative process synthesis solution is presented based on the authors experience and knowledge, and also on the perception of the environmental, safety and regulatory issues involved. The flowsheet presented has not been tested experimentally, nor has it been assessed by any

D.M.F. Prazeres, G.N.M. Ferreira / Chemical Engineering and Processing 43 (2004) 615–630 Fig. 5. Plasmid production flowsheet III (based on [53,65]). Rnase: ribonuclease; Kac: potassium acetate; EBA: expanded bed chromatography; SEC: size exclusion chromatography; UF: ultrafiltration.

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Fig. 6. Plasmid production flowsheet IV (based on [51]). Rnase: ribonuclease A; AEX: anion exchange chromatography; RPC: reversed phase chromatography.

D.M.F. Prazeres, G.N.M. Ferreira / Chemical Engineering and Processing 43 (2004) 615–630 Fig. 7. An alternative but unproven plasmid production flowsheet. A. sulphate: ammonium sulphate; HIC: hydrophobic interaction chromatography; SEC: size exclusion chromatography; UF: ultrafiltration.

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particular process synthesis procedure (Fig. 7). Whether it can compete or not with other process alternatives is a matter for extensive process analysis (including experimental work, process optimisation, cost analysis and economical evaluation). Following fermentation, cells are harvested by microfiltration, a process that is likely to be more efficient than centrifugation. Cell disruption by heat and detergent in a flow through heat exchanger, as suggested by Lee and Sagar [51], is used instead of alkaline lysis in view of its claimed reproducibility and consistency, and probable less problematic validation. The need to use lysozyme in this step is a drawback since it could increase costs. The processing of the resulting cell lysate by filtration with inert particles (e.g. diatomaceous earth) is probably a more effective clarification operation when compared to centrifugation. After mixing in the diatomaceous earth, the slurry is processed through a bag filter or equivalent equipment. Apart from the removal of the mass of solids, significant removal of soluble RNA can be expected. Plasmid loss due to co-adsorption should nevertheless be carefully assessed. Volume reduction of the resulting filtrate stream could be accomplished by precipitating the plasmid with PEG instead of isopropanol or ethanol. After separation of the precipitated plasmid by filtration (e.g with a Nutsche filter) and resuspension in an adequate buffer, extensive removal of impurities such as RNA and endotoxins is performed by an ammonium sulphate precipitation step. A tangential flow filtration through 0.45 ␮m pore size nitrocellulose membranes is suggested as the next step. Apart from removing precipitated material, this operation could provide a significant reduction in the levels of soluble gDNA, RNA, endotoxin and protein still present [63]. Hydrophobic interaction chromatography is then used to purify the plasmid DNA from the remaining impurities, particularly due to its ability to reduce the endotoxin burden to levels well within the specifications [60]. Size exclusion is used to fractionate plasmid, if required, and to exchange the buffer for an adequate formulation or storage buffer. Final sterilisation prior to filling is done with a 0.2 ␮m pore size membrane filtration. The proposed process does not use or generates significant amounts of hazardous materials and no special safety requirements are envisaged. Thus, environmental or safety associated costs are kept to a minimum. The reagents used do not pose any special regulatory concern since they are non-toxic, non-mutagenic and non-flammable.

8. Concluding remarks The increasing demands on the production of pharmaceutical-grade supercoiled plasmid DNA require the development of plasmid DNA purification processes and their optimisation in terms of selectivity, yield, efficiency and

productivity, in order to provide sufficient material for the ongoing gene therapy and DNA vaccine clinical trials. The conceptual design of process flowsheets for plasmid processing under cGMPs should be based on a profound knowledge of the target molecule and associated impurities, and of the performance of the available unit operations for plasmid processing. Although a number of well established processes have been documented in the literature, new developments and process solutions are likely to surface within the next years as a result of increased investments from academia and industry in the area. Acknowledgements Financial support from the Portuguese Ministry of Science and Technology (project POCTI/1999/BIO/43620) is acknowledged. References [1] R.G. Crystal, The gene as the drug, Nat. Med. 1 (1995) 15–17. [2] D. Luo, W.M. Saltzman, Synthetic DNA delivery systems, Nat. Biotechnol. 18 (2000) 33–37. [3] J.L. Fox, Gene therapy safety issues come to fore, Nat. Biotechnol. 17 (1999) 1153. [4] A. Mountain, Gene therapy: the first decade, Trends Biotechnol. 18 (2000) 119–128. [5] V. Glaser, Promise of gene therapy for treating disease remains on trial in the clinic, Genet. Eng. News, 14 (1997), pp. 14, 35, & 38. [6] H. Tighe, et al., Gene vaccination: Plasmid DNA is more than just a blueprint, Immunol. Today 19 (1998) 89–97. [7] J. Reimann et al., Genetic vaccination with plasmid vectors. in: M. Schleef (Ed.), Plasmids for Therapy and Vaccination, Wiley, Weinheim, Germany, 2001, pp. 45–73. [8] G. Sofer, L. Hagel, (Eds.), Handbook of Process Chromatography: A Guide to Optimisation, Scale-Up and Validation, Academic Press, San Diego, USA, 1997. [9] R.R. Sinden, (Ed.), DNA Structure and Function, Academic Press, San Diego, USA, 1994. [10] G.N.M. Ferreira et al., Plamid manufacturing-an overview. in: M. Schleef (Ed.), Plasmids for Therapy and Vaccination, Wiley, Weinheim, Germany, 2001, pp. 193–236. [11] D.M.F. Prazeres et al., Purification of plasmids for gene therapy and DNA vaccination, in: M. R. El-Gewely (Ed.), Biotechnology Annual Review, Elsevier, The Netherlands, 2001, pp. 1–30. [12] T.C. Boles, et al., Structure of plectonemically supercoiled DNA, J. Mol. Biol. 213 (1990) 931–951. [13] A.V. Vologodskii, et al., Conformational and thermodynamic properties of supercoiled DNA, J. Mol. Biol. 227 (1992) 1224–1243. [14] J.C. Murphy, et al., Purification of plasmid DNA using selective precipitation by compactation agents, Nat. Biotechnol. 17 (1999) 822–823. [15] M. Schleef, Issues of large-scale plasmid DNA manufacturing. in: A. Mountain, U. Ney, D. Schomburg (Eds.), Recombinant Proteins, Monoclonal Antibodies and Therapeutic Genes, Wiley, Weinheim, 1999, pp. 443–469. [16] M. Marquet et al., Characterization of plasmid DNA vectors for use in Human gene therapy, Part 1, Biopharmaceutics (May) (1997), pp. 42–50. [17] D.M.F. Prazeres, et al., Large-scale production of pharmaceuticalgrade plasmid DNA for gene therapy: Problems and bottlenecks, Trends Biotechnol. 17 (1999) 169–174.

D.M.F. Prazeres, G.N.M. Ferreira / Chemical Engineering and Processing 43 (2004) 615–630 [18] USFDA (Ed.), Points to consider on plasmid DNA vaccines for preventive infectious disease indications, US FDA Center for Biologics Evaluation and Research, Rockville, MD, USA, 1996. [19] USFDA (Ed.), Addendum to the points to consider in Human somatic cell and gene therapy (draft), US FDA Center for Biologics Evaluation and Research, Rockville, MD, USA, 1996. [20] EMEA (Ed.), Note for guidance on the quality, preclinical and clinical aspects of gene transfer medicinal products, The European Agency for the Evaluation of Medicinal Products, 1999. [21] G.N.M. Ferreira, et al., Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications, Trends Biotechnol. 18 (2000) 380–388. [22] S.M. Wheelwright, Designing downstream processes for large-scale protein purification, Nat. Biotechnol. 5 (1987) 789–793. [23] D. Petrides, Bioprocess design. in: R.G. Harrison, P.W. Todd, S.R. Rudge, D. Petrides (Eds.), Bioseparations Science and Engineering, Oxford University Press, Oxford, UK, 2003, pp. 319–372. [24] M.A. Steffens, et al., Synthesis of bioprocesses using physical properties data, Biotechnol. Bioeng. 68 (2000) 218–230. [25] D.P. Petrides, et al., The role of process simulation in pharmaceutical processes development and product commercialization, Pharm. Eng. 22 (2002) 1–8. [26] T. Maniatis et al. (Ed.), Molecular Cloning: Laboratory Manual, Cold Spring Harbor, New York, 1982. [27] P. Knight, Downstream processing, Nat. Biotechnol. 7 (1989) 777– 782. [28] USFDA (Ed.), Guideline on the preparation of investigational new drug products (human and animal), US FDA Center for Biologics Evaluation and Research, Rockville, MD, USA, 1991. [29] W.S. Kletch, Regulatory considerations. in: E. Goldberg (Ed.), Handbook of Downstream Processing, Blackie, London, 1997, pp. 557–568. [30] O. Doblhoff-Dier, R. Bliem, Quality control and assurance from the development to the production of biopharmaceuticals, Trends Biotechnol. 17 (1999) 266–270. [31] B. Eckman, Validation. in: E. Goldberg (Ed.), Handbook of Downstream Processing, Blackie, London, 1997, pp. 569–600. [32] G. Sofer, Validation of biotechnology products and processes, Curr. Opin. Biotechnol. 6 (1995) 230–234. [33] W.R. Tolbert, et al., Designing an initial gene therapy manufacturing facility, Biopharmaceutics, 1996, pp. 32–40. [34] J. Akers, et al., Biotechnology product validation, part 2: A logical plan, Pharm. Technol. Europe 6 (1994) 230–234. [35] M. Marquet, et al., Production of pharmaceutical-grade plasmid DNA, US Patent 5,561,064, (1996). [36] W. Chen, Automated high-yield fermentation of plasmid DNA in Escherichia coli US Patent 5,955,323 (1999). [37] M.S. Levy, et al., Effect of shear on plasmid DNA solution, Bioprocess Eng. 20 (1999) 7–13. [38] L.A. Ciccolini, et al., Time course of SDS-alkaline lysis of recombinant bacterial cells for plasmid release, Biotechnol. Bioeng. 60 (1998) 768–770. [39] D.R. Thatcher, et al., Method of plasmid DNA production and purification US Patent 5,981,735 (1999). [40] A. Carlson, et al., Mechanical disruption of Escherichia coli for plasmid recovery, Biotechnol. Bioeng. 48 (1995) 303–315. [41] H.C. Birnboim, J. Doly, A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucleic Acids Res. 7 (1979) 1513–1523. [42] L.A.S. Ciccolini, et al., Rheological properties of chromosomal and plasmid DNA during alkaline lysis reaction, Bioprocess Eng. 21 (1999) 231–237. [43] G.C. Kresheck, M. Altschuler, Method of isolating purified plasmid DNA using a nonionic detergent solution, US Patent 5,625,053 (1997). [44] M.D. Butler, et al., Purification of plasmid DNA, US Patent 6,313,285 (2001).

629

[45] M. Marquet, et al., Process development for the manufacture of plasmid DNA vectors for use in gene therapy, Biopharmaceutics (September) (1995), pp. 26–37. [46] I. Theodossiou, et al., The processing of plasmid based gene from Escherichia coli. Primary recovery by filtration, Bioprocess Eng. 16 (1997) 175–183. [47] N. Horn, Process for reducing RNA concentration in a mixture of biological material using diatomaceous earth, US Patent 5,576,196 (1996). [48] N. Horn, et al., Purification of plasmid DNA during column chromatography. US Patent 5,707,812 (1998). [49] I. Theodossiou, et al., Methods of enhancing the recovery of plasmid genes from neutralised cell lysate, Bioprocess Eng. 20 (1999) 147– 156. [50] L.A.S. Ciccolini, et al., A mass balance study to assess the extent of contaminant removal achieved in the operations for the primary recovery of plasmid DNA from Escherichia coli cells, Biotechnol. Bioeng. 77 (2002) 796–805. [51] A.L. Lee, S. Sagar, Method for large scale plasmid purification, US Patent 6,197,553 (2002). [52] G.A. Monteiro, et al., Analysis and use of endonuclease activities in Escherichia coli lysates during the primary isolation of plasmids for gene therapy, Biotechnol. Bioeng. 66 (1999) 189–194. [53] D.L. Varley, et al., Production of plasmid DNA for human gene therapy using modified alkaline cell lysis and expanded bed anion exchange chromatography, Bioseparation 8 (1998) 209–217. [54] G.N.M. Ferreira, et al., Development of process flow sheets for the purification of plasmid vectors for gene therapy applications, Biotechnol. Prog. 15 (1999) 725–731. [55] M.M. Diogo, et al., Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography, Biotechnol. Bioeng. 68 (2000) 576–583. [56] N.A. Horn, et al., Cancer gene therapy using plasmid DNA: purification of DNA for human clinical trials, Hum. Gene Ther. 6 (1995) 565–573. [57] R.J. Lander, et al., Fractional precipitation of plasmid DNA from lysate by CTAB, Biotechnol. Bioeng. 79 (2002) 776–784. [58] R. Bhikhabhai, Plasmid DNA purification using divalent alkaline earth metal ions and two anion exchangers, US Patent 6,410,274 (2002). [59] A. Chakrabarti, et al., A procedure for large-scale plasmid isolation without using ultracentrifugation, Biotechnol. Appl. Biochem. 16 (1992) 211–215. [60] M.M. Diogo, et al., Production, purification and analysis of an experimental DNA vaccine against rabies, J. Gene Med. 3 (2001) 577–584. [61] S.C. Ribeiro, et al., Isolation of plasmid DNA from cell lysates by aqueous two-phase systems, Biotechnol. Bioeng. 78 (2002) 376–384. [62] D.W. Kahn, et al., Purification of plasmid DNA by tangential flow filtration, Biotechnol. Bioeng. 69 (2000) 101–106. [63] D. Kendall, et al., Purification of plasmid DNA by an integrated operation comprising tangential flow filtration and nitrocellulose adsorption, Biotechnol. Bioeng. 79 (2002) 816–822. [64] M.S. Levy, et al., Removal of contaminant nucleic acids by nitrocellulose during pharmaceutical-grade plasmid DNA processing, J. Biotechnol. 76 (2000) 197–205. [65] G.N.M. Ferreira, et al., Anion-exchange purification of plasmid DNA using expanded bed adsorption, Bioseparation 9 (2000) 1–6. [66] A. Ljunglöf, et al., Direct visualisation of plasmid DNA in individual chromatography adsorbent particles by confocal scanning laser microscopy, J. Chromatogr. 844 (1999) 129–135. [67] G.N.M. Ferreira, et al., Studies on the batch adsorption of plasmid DNA onto anion-exchange chromatographic supports, Biotechnol. Prog. 16 (2000) 416–424. [68] D.M.F. Prazeres, et al., Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography, J. Chromatogr. A 806 (1998) 31–45.

630

D.M.F. Prazeres, G.N.M. Ferreira / Chemical Engineering and Processing 43 (2004) 615–630

[69] G. Chandra, et al., Large-scale purification of plasmid DNA by fast protein liquid chromatography using Hi-Load Q Sepharose column, Anal. Biochem. 203 (1992) 169–172. [70] W. Müller, Fractionation of DNA restriction fragments with ion-exchangers for high-performance liquid chromatography, Eur. J. Biochem. 155 (1986) 203–212. [71] H. Yamakawa, et al., Sequence-dependent DNA separation by anion-exchange high-performance liquid chromatography, Anal. Biochem. 240 (1996) 242–250. [72] I.P. Wicks, et al., Bacterial lipopolysaccharides copurifies with plasmid DNA: Implications for animal models and human gene therapy, Hum. Gene Ther. 6 (1995) 317–323. [73] A. Lyddiatt, D.A. O’Sullivan, Biochemical recovery and purification of gene therapy vectors, Curr. Opin. Biotechnol. 9 (1998) 177–185. [74] C.G. Huber, Micropellicular stationary phases for high-performance liquid chromatography of double-stranded DNA, J. Chromatogr. 806 (1998) 3–30. [75] M. Colpan, et al., Process for the separation and purification of nucleic acids form biological sources, US Patent 5,990,301 (1999). [76] M.M. Diogo, et al., Scale-up of hydrophobic interaction chromatography for the purification of a DNA vaccine against rabies, Biotechnol. Lett. 22 (2000) 1397–1400. [77] P.R. Brown, A.M. Krstulovic, Practical aspects of reversed-phase liquid chromatography applied to biochemical and biomedical research, Anal. Biochem. 99 (1979) 1–21. [78] V.R. Meyer (Ed.), Practical High-Performance Liquid Chromatography, Wiley, West Sussex, UK, 1994. [79] A.P. Green, et al., Preparative purification of supercoiled plasmid DNA for therapeutic applications, Biopharmaceutics (May) (1997), pp. 52–61.

[80] S. Colote, et al., Analysis and purification of plasmid DNA by reversed-phase high-performance liquid chromatography, Anal. Biochem. 154 (1986) 15–20. [81] P. Wils, et al., Efficient purification of plasmid DNA for gene transfer using triple-helix affinity chromatography, Gene Ther. 4 (1997) 323– 330. [82] T. Schluep, C.L. Cooney, Purification of plasmid DNA by triplex affinity interactions, Nucleic Acids Res. 26 (1998) 4524– 4528. [83] J. Woodgate, et al., Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker, Biotechnol. Bioeng. 79 (2002) 450–456. [84] M. Bywater, et al., A novel chromatographic procedure for purification of bacterial plasmids, Anal. Biochem. 132 (1983) 219– 224. [85] G.N.M. Ferreira, et al., A comparison of gel filtration chromatographic supports for plasmid purification, Biotechnol. Tech. 11 (1997) 417–420. [86] J.K. McClung, R.A. Gonzales, Purification of plasmid DNA by fast protein liquid chromatography on Superose 6 preparative grade, Anal. Biochem. 177 (1989) 378–382. [87] G.J. Raymond, et al., Large-scale isolation of covalently closed circular DNA using gel filtration chromatography, Anal. Biochem. 173 (1988) 125–133. [88] R.J. Lander, et al., Process for the scaleable purification of plasmid DNA, US Patent application 0,012,990 A1 (2002). [89] S. Nochumson et al., Process and equipment for plasmid purification, US Patent application 0,034,435 A1 (2001). [90] B. Atkinson, F. Mavituna (Eds.), Biochemical Engineering and Biotechnology Handbook, Macmillan, New York, USA, 1991.