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

Enzyme and Microbial Technology 33 (2003) 865–883

Review

Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification

Kristala Jones Prather, Sangeetha Sagar, Jason Murphy, Michel Chartrain∗

Merck 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 especially 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 potential therapeutic applications [2,3]. Since then, intramuscular 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 therapy, cancer vaccine, and therapeutic vaccine clinical trials have been initiated [4–8]. Notably in gene therapy, the use of a plasmid encoding for VEGF, a vascular endothelial growth

∗ Corresponding author. Tel.: +1-732-594-4945; fax: +1-732-594-4400.

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

mailto:[email protected]

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 [11–13]. Subsequently, plasmid DNA vaccines have yielded very encouraging results in the fights against malaria [14] and AIDS [15–18]. Currently the use of plasmid DNA vaccines is being investi- gated against many other infectious diseases including hepatitis B and C, and tuberculosis [1,7,19].

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 viru- lent or attenuated infectious microorganisms (Fig. 1). These are the properties that make DNA vaccines extremely attractive and, in principle, potentially superior to protein-based 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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866 K.J. Prather et al. / Enzyme and Microbial Technology 33 (2003) 865–883

VACCINATION WITH PLASMIDS

MUSCLE

fibroblastsAPC

Internal synthesis of Internal synthesis offoreign peptide/protein 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 CELLULAR IMMUNE RESPONSE 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 na¨ıve 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 pe- riods 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 stability, 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 vaccine 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 (5–40 mg/l) volumetric titers. Additionally, the utilization of laboratory- scale 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 particular, during plasmid design and the development of a production process, it is crucial that the regulatory environment be constantly considered. DNA vaccines are

K.J. Prather et al. / Enzyme and Microbial Technology 33 (2003) 865–883 867

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) divi- sion 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 [23–30].

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.


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 occur. 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-studied Gram-negative bacterium Escherichia coli [31–33]. The reasons for this selection are three-fold. First, ColE1-derivatives have been most commonly employed as vectors useful for recombinant protein expression and are widely available. Second, they have successfully been modified to replicate at high copy numbers, thereby increasing the overall theoretical yield from a plasmid DNA production process. Last, production processes and scale-up conditions have been extensively studied using E. coli as host for recombinant protein production.

An extensive amount of literature exists detailing the selection and optimization of plasmid DNA vectors for recombinant protein expression (see the following reviews [34–36]). This literature can be very useful in choosing a replicon as the foundation of a DNA vaccine candidate, though the desire to produce plasmid DNA as the ultimate product of interest necessitates that a different set of cri- teria guide the final selection. Many possibilities exist for this choice; however, most naturally occurring replicons exist at low to moderate copy numbers (Table 1). Such low copy numbers were a limitation for the production of large amounts of recombinant protein, as the gene dosage became the limiting factor in maximizing expression. Likewise, the use of a low-copy number vector is undesirable for plasmid DNA vaccine production since the product yields will be unfavorably low. One solution to this potential problem can be found in the form of the multi-copy derivatives of the ColE1-type plasmid pBR322, which is derived from pMB1 [37,38].

ColE1-type plasmids require only host-encoded proteins for replication [39–41]. The necessary plasmid-based func-

Table 1Replicons commonly used in Escherichia coli

tions exist in the form of two RNA transcripts, known as RNAI and RNAII. The RNAII transcript is the pre-primer for DNA replication. It binds to the complementary plasmid DNA and is processed by ribonuclease H (RNase H) in a manner that depends on a stable DNA:RNA hybrid. Host-encoded DNA polymerase I then continues unidirec- tional replication from the active primer. RNAI encodes for an anti-sense strand complementary to a small region of RNAII, and the RNAI:RNAII hybrid prevents formation of the stable DNA:RNAII hybrid necessary for RNase H cleavage and the formation of the active primer. The interaction between RNAI and RNAII is enhanced by a plasmid-encoded protein, called Rop or Rom (for repressor of primer and RNA one modulator, respectively). Fig. 3 de- picts these various effectors that lead to the control of plasmid copy number.

The first elevated copy number derivatives of pBR322 were found to exclude the Rop/Rom protein [42–44]. These plasmids (e.g. pAT153) exhibited an approximatelythree-fold improved copy number of ∼150 per cell overpBR322. The pUC vectors, including the still commonly used plasmids pUC18 and pUC19 [45], were also constructed without a functional rop gene; however, these vectors were found to behave differently than other rop−

derivatives with respect to copy number. Minton [46] reported an increase in recombinant protein activity when expressed from a pUC8-derived vector that was ultimately correlated to an additional 3–4-fold increase in plasmid copy number (500–700 copies per cell) over pAT153-like plasmids [47]. This additional increase in copy number was attributed to a G → A point mutation in the pUC vectors relative to pBR322 that was not identified in the originally published sequence of pUC19 [48]. The point mutation maps to the RNAII sequence, impacts binding affinity of the RNAI:RNAII complex, and affects copy number in a temperature-dependent manner [49]. The copy number effects are suppressed at 30 ◦C and enhanced at 42 ◦C, and Rop/Rom suppresses the mutation at all temperatures. The addition of this point mutation to lower-copy number pBR322-derived constructs has been successfully employed to improve the yield of plasmid DNA for gene therapy purposes [31].

Because of the high copy numbers achievable with the pUC-like versions of ColE1-type plasmids, they have long been the replicons of choice for recombinant protein expression in cases where toxicity due to high expression is not an issue. As a result, much work has been done to create a vari- ety of useful expression vectors, many of which are commer-

Representativeplasmid

Replicon(incompatibility group)

Copy number(copies per cell) cially available. The high copy numbers and wide availabil-

ity have also made these vectors very attractive as the basispML31 F plasmid (FI) 1–4P1 prophage P1 (Y) 1–4 pSC101 R6 (FII) ∼6 pMMB66 RSF1010 (P4/Q) 12–13 pFF1 RK2 (P1) 10–15 pACYC184 p15A 18–22 pBR322 pMB1 (ColE1) 40–55

for DNA vaccine constructs, especially since other methods to increase yields of lower copy vectors, such as chloramphenicol or rifampicin addition are not viable for pharmaceutical products. Recently, a new class of high-copy plasmids comparable to pUC and derived from the ColE1-like and -compatible RSF1030 replicon has been described that


(a)RNA II

RNaseHcleavage

RNA Iori

(b)

RNA II

ori

(1) degradation of DNA:RNAIIhybrid to ori by RNaseH

(2) elongation by DNA polymerase

(c)Rop/Rom

RNA II RNA I

ori (3) formation of stable DNA:RNAII hybrid blocked by RNAI:RNAII hybrid

Fig. 3. Molecular control mechanisms for ColE1-type plasmid copy number in E. coli. (a) RNAI is transcribed from a complementary portion of the5 end of RNAII. Both transcripts display complex secondary structure formation. (b) RNAII forms a persistent hybrid with the complementary DNA, allowing RNaseH degradation back to the ori and extension by DNA polymerase. (c) Formation of the RNAI:RNAII hybrid prevents stable DNA:RNAII hybrid formation, enhanced by the Rop/Rom protein (illustration adapted from [114,154]).

may also prove useful for the high-yield production of plasmid DNA [50].

Although pUC-based plasmids are in widespread use as potential DNA vaccines, the “runaway-replication” plasmids derived from some low-copy replicons—so called because of the loss of control of replication usually induced by a temperature shift—would also seem an attractive op- tion from a process development perspective. The loss of replication control results in a large accumulation of plasmid DNA within the host cell, up to copy numbers near1000 [51,52]. Such vectors have been used successfully to achieve inducible high gene dosages for the production of recombinant proteins [53,54]. These vectors have caused some concerns due to the large metabolic burden typically encountered with recombinant gene expression from very high-copy number plasmids [55–58]. However, in the case where plasmid DNA is the ultimate product of interest and expression of the transgene in the host cell is minimal or non-existent, concerns about transcriptional and translational limitations typically associated with recombinant protein expression are no longer relevant. In spite of this apparent advantage of using runaway-replication vectors to produce high titers of plasmid DNA, these authors did not locate any examples of these vectors being used as the basis for DNA vaccines in the literature. It is not entirely clear why such vectors have not been exploited; however, one

may speculate that the primary reason lies in the traditional separation between basic research and process development activities. Proof of concept is readily obtained with small quantities of material, and considerations of process feasibil- ity may be postponed until after vectors have been chosen. However, once the vector has been used in safety assess- ment or clinical studies, redesigning the replication control region to modify plasmid copy number is not possible since this results in the creation of a new molecule, requiring such studies to be repeated at least in part if not in whole.

3. Designing a DNA vaccine or gene therapy construct

The design of a plasmid DNA vaccine or gene therapy construct is conceptually straightforward [32]. The plasmid should contain elements required for maintenance and propagation in the bacterial host and for expression of the transgene in the human host (Fig. 4). The primary elements required for bacterial propagation include the origin of replication and any plasmid-encoded functions (e.g. RNAI/RNAII sequences in ColE1-type replicons) required for replication. These are determined by the initial choice of the replicon upon which the vector will be based. A selectable marker should be included as well to allow for selection of successful transformants upon initial introduction


promoter/enhancer (e.g., CMVIE, SV40,

EF-1, UbC, native)

selection mechanism(e.g., antibiotic resistance)

pathogen gene

transciption terminator/ polyadenylation signal (e.g., BGH, SV40,

human -globin)

bacterial replication elements(e.g., modified ColE1-type)

gene expression in mammalian cells include those from human cytomegalovirus/immediate-early (CMVIE), simian virus/early (SV40), human elongation factor-1 (EF-1 ), and human ubiquitin C (UbC). The CMVIE promoter has been shown to be more effective than both SV40 [60,61] and UbC [62], and inclusion of intron A can further improve CMVIE-directed expression [61]. A survey of commercial suppliers of expression vectors will show that those containing the CMVIE promoter/enhancer are most widely available, further increasing the likelihood that this promoter will be used for commercial development of plasmid

Fig. 4. Map of a typical DNA vaccine construct. The vector includesprokaryotic elements for plasmid replication, maintenance and selection in the bacterial host, and eukaryotic elements for expression in the mammalian host.

of the plasmid into the bacterial host and to create selective pressure against a predominance of plasmid-free segregants arising in the population upon subsequent growth of clones. The choices that exist for the selectable marker can be grouped into two categories: (i) those that require both plasmid- and host-based genetic manipulations, and (ii) those that require only plasmid-encoded sequences. Exam-

ples of the former include the use of auxotrophic strains that can be complemented by plasmid-encoded gene

functions, and strains with thermo-sensitive mutations in either essen- tial genes (e.g. those involved in translation) or toxic genes that can be complemented or repressed,

respectively, by plasmid-based genes at permissive temperatures. Recently, a selectable strain-plasmid system

was described in which the host genome contained a kanamycin resistance gene under the control of the lac operator and promoter and the plasmid contained lacO

[59]. In the presence of kanamycin, the transformed strains could only grow if host-encoded LacI repressor

molecules were successfully titrated by plasmid-based lacO, creating selective pressure for plasmid presence. In each of these cases, the options for growth media may

be limited by the nature of the marker, and the construction of special hosts is required, limiting the ability to rapidly evaluate several strains to determine the best host for propagation of the desired construct. Additionally,

plasmid-encoded gene products may accumulate in the cytoplasm, allowing plasmid-free segregants to continue

growth for a finite number of generations in the absence of the marker gene [35]. Most researchers have therefore

settled on markers that only require plasmid-based sequences, the most common of these conferring

antibiotic resistance. As discussed in the following sections, the choice of antibiotic and the decision to include this component in the final production vessel is governed

in part by regulatory considerations. The expression elements should be designed to maximize transient

production of the transgene upon entry into the human host. These elements include a eukaryotic promoter, a

transcription termination/polyadenylation signal, and the transgene of interest. Promoters available for recombinant

DNA vaccines. Promoters derived from the antigen source may also be used for plasmid-based expression, though this approach would limit the ability to construct a single plasmid backbone for the expression of multiple antigens [63]. Transcription terminators/polyadenylation signals that are commonly used include those from bovine growth hormone (BGH), SV40, and human -globin.

In addition to the basic elements of a DNA vaccine construct, it may be possible to further optimize the plasmid backbone by exploiting the immunostimulatory effects of the bacterial DNA. In particular, it has been observed that bacterial CpG dinucleotide motifs can enhance the immuno- genicity of plasmid DNA vaccines [32,64–66]. These dinucleotides occur in bacterial DNA at an expected frequency and are generally un-methylated. Conversely, mammalian DNA contains CpG motifs at a suppressed frequency, some10–20-fold lower than expected, in a largely methylated-C form. It is believed that this difference in frequency and methylation patterns results in an enhanced immune response following injection of plasmid DNA vaccines with a bacterial backbone. The magnitude of the immune response is also affected by the identity of the flanking dinucleotides [64]. Accordingly, it may be possible to optimize codon use in the plasmid vector to increase the occurrence of stimula- tory CpG motifs or the nature of the surrounding nucleotides and thereby improve the natural adjuvant properties of the plasmid DNA.

During selection and construction of a plasmid vector suitable for vaccine usage, the regulatory environment must be closely considered. A major regulatory concern, potential DNA integration, should be addressed during the design of the plasmid. Care should be taken in the choice of the insert, and in avoiding homologous sequences between the human genome and the plasmid DNA. Promoters and terminator regions should also be carefully selected to re- strict their biological activities on the sequences inserted on the plasmid. When selecting an antibiotic resistance marker, one should avoid the use of beta-lactams and rather select the aminoglycosides neomycin or kanamycin [28]. Both of these aminoglycoside antibiotics are appropriate as they are seldom used in clinics and have a low incidence of adverse effects such as ototoxicity and nephrotoxicity. In addition, all steps that lead to the construction of the plasmid must be well recorded, with detailed descriptions of cell lines, in- termediates manipulation, and substrates used. Finally, the


complete sequence of the plasmid must be provided prior to the initiation of phase I clinical studies. Host selection requires that the source of the microbial strain be characterized and free of any adventitious agents, and that the strain be genotypically and phenotypically well characterized. It is also important that the methods leading to the selection of the clone be well documented [23–30].

4. Plasmid production

Economic large-scale plasmid production from E. coli requires the concomitant optimization of plasmid copy number (specific yield) and of biomass concentration. Achieving elevated specific yields is particularly important as it also positively impacts downstream processing and ultimately purification yields. Fermentation methods for the production of recombinant protein by E. coli have been extensively studied, successfully optimized, and implemented at the commercial scale [67,68]. It is highly probable that a substantial fraction of the knowledge gained by experimentalists during studies directed at optimizing recombinant protein production will be translatable to processes aimed at the over-production of plasmid. However, conditions leading to the optimization of protein expression may differ somewhat from the circumstances necessary for achieving high plasmid copy number. For example, it is well established that the metabolic burden of high plasmid copy number and elevated foreign protein synthesis is far greater than that of the presence of a high copy number plasmid only [69]. It is therefore likely that achieving optimal plasmid production will require several paradigm shifts to occur in the cultivation methods.

4.1. Medium formulation

The cultivation medium formulation can dramatically influence the performance of microbial processes. E. coli is a non-fastidious microorganism that grows in both rich complex organic media as well as in salt-based chemically defined media providing that a source of organic carbon is provided. Through the type and concentration of ingredients used, cultivation medium composition directly dictates the amount of biomass produced, and is therefore likely to influence plasmid volumetric yield. In addition, it is probable that medium composition will directly bear on the physiology of the microorganisms by influencing their intricate regulatory systems, and therefore will control plasmid copy number or specific yield. It is also generally understood that plasmid copy number of ColE1-type plasmids in E. coli varies inversely with growth rate in batch culture. Lin-Chao and Bremer [70] observed that the copy number of pBR322 changes from 15 to 23 plasmids per genome as the growth rate decreases from 1.7 to 0.4 h−1 as a result of growth in complex Luria Bertani (LB) medium versus glycerol minimal medium. These data were subsequently confirmed by determining both plasmid copy number and the expression

of plasmid-encoded cat as a function of growth rate in LB and minimal media [71]. A similar trend was observed with ColE1-type plasmids of altered copy number [72]. This experiment also suggested that there was an upper limit to plasmid content per cell, as the growth rate was further suppressed to ∼0.25 h−1 by adding glucose uptake inhibitors to minimal medium.

Cultivation media suitable for E. coli can be purchased or their formulations can be obtained from the literature. Com- positions of both complex and chemically defined media are abundantly discussed in many publications [73–77]. Due to the simplicity of use associated with off-the-shelf cultivation media, a number of laboratory-scale DNA vaccine production schemes rely on the use of un-optimized small-scale processes employing commercially available complex formulations [78–80]. Generally, these simple processes are based on the growth of E. coli in either large shake flasks or small laboratory fermentors, usually employing simple media formulations such as Luria Bertrani (LB), or brain heart infusion (BHI). These processes yield low cell mass, ranging from 1 to 7 g dry cell weight/l, which in turn support modest volumetric plasmid yields (Table 2). Such processes are, however, sufficient for the production of amounts of plasmids needed for studies employing a limited number of small animals. Interestingly, the data presented in Table 2 show that although employing different E. coli strains and plasmids, the specific yields achieved by several research groups when using LB broth are all remarkably similar, with all reaching about 4–6 g of plasmid/mg dry cell weight.

Modification of pre-existing media, leading to a better control of the physiology of the host cells, may result in enhanced process yields. When E. coli DH5 harboring the plasmid pSV was cultivated in a semi-defined medium, a higher plasmid specific yield of 9.12 g plasmid/mg dry cell weight, was achieved over the standard complex LB formulation which only supported 6.0 g plasmid/mg dry cell weight [81]. Increasing medium strength, by either adding additional nutrients (LB plus glucose) or by employing richer formulations (BHI, Terrific Broth), supports higher volumetric yields without compromising specific yields (Table 2). In these specific examples, the benefits observed can be entirely attributed to the increase in biomass caused by employing a richer cultivation medium. When modifying a semi-defined cultivation medium, O’Kennedy et al. [81] demonstrated the existence of an optimum carbon/nitrogen (C:N) ratio. They found that a C:N ratio of2.78:1 was optimal for plasmid production. Higher ratios of up to 12:1, which are more in line with standard media formulations, yielded lower plasmid specific yields. Because no differences were observed in the growth kinetics and final biomass production when testing variable C:N ratio media, the authors speculate that the changes in C:N ratio may have influenced the physiology of the microbes in a fashion that privileged the synthesis of plasmid DNA.

Nutritional requirements and cellular composition of E. coli are well defined and this information can be advanta-


Table 2Description and performance of plasmid DNA production processes

Process type and scale Cell yield at harvest Plasmid yield Reference

Shake flask cultivation at 2-l scale (LBbroth with ampicillin 30 g/ml)

Small laboratory fermentor (4.5-l working volume) (terrific broth with 0.1 g/l ampicillin)

OD600 = 2.6 late log phase 7.74 g/ml (lysate) [78]74 mg/l (5.95 g/mg cell)a

OD600 = 14 174 mg/4.5-l or 38.66 mg/l [79]5.52 g/mg cella

Sake flask process (500 ml working volume) (LB broth) NA NA [80]

14-l fermentor (BHI broth) NA NA [153]

10-l fermentor (tryptic broth with 50 g/ml of kanamycin) OD600 = 30 4 mg/l [95]0.26 g/mg

10-l fermentor (complex medium with 59 g/ml kanamycin) NA 7.5 mg/l [31] Temperature shift 37–42 ◦ C 5.1 g/l 34.1 mg/l (6.7 g/mg) " Temperature shift 37–45 ◦ C 4.8 g/l 49.3 mg/l (10.3 g/mg) " Fed-batch (with step increase) and 37–42 ◦ C shift NA 220 mg/l "

Fermentor fed-batch 55 g/l 225 mg/l (4.1 g/mg) [103]

Fermentor 2.5 l batchLB broth 0.8 g/l 4 mg/l (5 g/mg) [86]LB plus glucose 5.0 g/l 20 mg/l (4 g/mg) " Defined MW1 3.5 g/l 60.0 mg/l (17.1 g/mg) " Defined MW2 4.0 g/l 30 mg/l (7.5 g/mg) "

Shake flask (500 ml working volume)LB broth NA 0.56 mg/l (6.0 g/mg) [81] Semi-defined (SDCAS) NA 5.2 mg/l (9.12 g/mg) "

Fermentor (7- and 80-l scale) semi definedFed-batch (manual) 10–12 g/l 5–8 mg/l (0.7 g/mg) [104] Fed-batch (DO-stat) 60 g/l 100 mg/l (1.7 g/mg)

Fermentor (10-l working volume) (LB broth with 100 g/ml ampicilin and with chloramphenicol amplification)

NA 4.5 mg/l [101]

a Dry cell weight estimated based on 1 OD600 = 0.5 g/l dry cell weight.

geously used in the design of cultivation media formulations [73,82–85]. Employing this approach, Wang et al. [86] reported the rational design of a defined medium optimized for plasmid production by E. coli strain JM109 harboring the pcDNA3S plasmid. After identifying six key amino acids (aspartate, glutamine, glycine, histidine, leucine and trypto- phan) to add to a glucose-basal salt medium, the designed formulation supported higher plasmid specific yield when compared with those routinely achieved in the complex LB medium (7.5 versus 5.0 g plasmid/mg dry cell weight). When the defined medium was supplemented with ribonu- cleotides (adenosine, guanosine, cytidine, and thymidine), the specific plasmid yield was further enhanced by about2.5-fold and reached a value of about 17.1 g plasmid/mg dry cell weight.

The effects of medium composition on plasmid production are closely intertwined with those affecting plasmid segregational stability. An important factor contributing to plasmid stability is the presence of an antibiotic, selecting for cells containing the desired plasmid, in the cultivation medium. Antibiotic presence in the cultivation medium is not problematic since its clearance, or clearance of its inactivated form, can be readily achieved during the down-

stream steps [68]. However, the mechanisms of resistance encoded by the plasmid usually lead to either deactivation or degradation of the antibiotic. In liquid culture, selective pressure may diminish with time as deactivation of the antibiotic occurs [87,88]. Therefore, a potential for plasmid loss exists due to inherent instability, even when antibiotics are added to the cultivation medium. The recombinant protein over-production literature, with the caveat that ex- periments were performed in the context of concomitant protein synthesis, has reported extensively on the effect of nutrition on plasmid stability. The mechanisms of plasmid instability observed, dimer formation, segregational instability, and degradation, probably remain valid [89–91]. Using frequent serial transfers, O’Kennedy et al. [81] evaluated plasmid stability when employing several media (complex LB versus semi-defined formulations) and found that a semi-defined medium supported the highest plasmid stability, while the complex LB medium offered the low- est plasmid stability. Media that supported similar growth rates between plasmid-bearing and plasmid-free cells also supported high plasmid stability. The authors concluded that higher plasmid instability resulted from a wider growth rate difference achieved when using certain formulations,


which conferred an advantage to the plasmid-free cells. In other studies, employing continuous cultivation experi- ments, Wang et al. [86] showed that when compared to two complex richer media, plasmid stability was higher, by up to 63%, when employing defined media. These authors also speculate that higher stability in the defined media may result from a lower growth advantage for plasmid-free cells, when compared with richer cultivation media.

4.2. Cultivation methods

Both batch and fed-batch technologies have been successfully employed for plasmid over-production by E. coli [92–94]. Batch cultivation, although logistically simple, is severely limited with respect to achieving elevated biomass.

DNA production processes that employ simple batch cultivation methodology yield relatively low biomass, with cell mass values ranging from 1 to 8 g/l, and correlatively support low plasmid volumetric yields (Table 2). For example, Horn et al. [95] report reaching an optical density, measured at 600 nm (OD600 ) of 30 when employing a rich complex cultivation medium (TB medium). The harvested cells, in late log phase, yielded about 4 mg of plasmid per liter. In another example, a volumetric yield of 38 mg/l was achieved by Diogo et al. [79] when cultivating E. coli in a rich complex medium and harvesting the cells in late log

phase at an OD600 of about 14. Generally, volumetric yields achieved in batch cultivation tend to be relatively

low, with values ranging from 4 to 40 mg/l (Table 2). When employing rich cultivation media, the fermentor oxygen transfer capacity is eventually exceeded, resulting in the creation of an oxygen-limited environment. This lack of oxygen triggers the fermentative metabolism of E. coli,

rapidly leading to the production of toxic by-products, mostly acetic acid, that

severely limit growth and even lead to cell death [96–98]. Fed-batch technology delivers nutrients over an extended

period of time, thereby achieving the control of nutrient availability to a level compatible with the oxygen transfer capacities of the bioreactor, offering a reasonable solution that has been amply explored. The feeding of nutrients, usually glucose, has been extensively researched, and in- corporates a range of approaches that span from simple to very elaborate, with each presenting its own advantages and disadvantages. Feeding regimens based on feed rate increase, either simple or following sophisticated algo- rithms aimed at maintaining a more constant environment thus maintaining a desired growth-rate, have been successfully implemented. In general, these strategies lead to the accumulation of biomass in the 60–120 g dry cell/l range, with each recombinant construct presenting its own limitations. These limitations are probably the result of the extra metabolic burden caused by the over-expression of plasmid and recombinant product [92–94].

In addition to influencing biomass production, cultivation methods, especially the manipulation of the growth rate, greatly influence plasmid yield and stability. Just as altering

growth rate by changing medium composition affects plasmid copy number, several investigators have clearly demonstrated that plasmid content (yield) is directly affected by the dilution rate in continuous culture. Using continuous culture, Seo and Bailey [99] established that fast growing cultures (0.6–0.8 h−1 ) contain less plasmid than those growing at much slower rates (0.3–0.4 h−1 ). However, their data suggest that maintaining lower dilution rates (below 0.2 h−1 ) would result in lower plasmid copy number. Reinikainen and Virka- järvi [100] reported similar trends, employing a different plasmid. These low plasmid copy numbers may be reflective of elevated stress caused by excessive nutritional limitations. In another study, Reinikainen et al. [101] demonstrated that pH and temperature greatly influenced plasmid copy number. The authors speculate that these environmental factors (elevated pH and reduced temperature), positively influenced plasmid copy number by down-regulating the growth rate. Several investigators have observed that plasmid copy number increases during both the late exponential and early stationary phases of growth [101,102]. This phenomenon has been coined plasmid amplification in these publications and these observations also support a strong correlation between growth rate and plasmid accumulation. Higher plasmid content achieved under reduced growth rate conditions is attributed to both higher plasmid stability (due to effective equivalent growth rates of plasmid-free and plasmid-bearing cells) and to privileged plasmid synthesis over other biochemical pathways. The following examples show that these findings in conjunction with fed-batch technology have been successfully applied for the production of plasmid DNA.

Schmidt et al. [103] used a feeding strategy that maintained a slow growth rate (about 0.15 h−1 ) for the most part of the cultivation (10–30 h). Plasmid concentration continued to increase during the first 5 h of stationary phase after which it reached a plateau. This strategy allowed the authors to achieve a biomass of about 50 g/l of dry cell weight and a final plasmid yield of about 225 mg/l.

Chen et al. [104] report the use of several fed-batch strategies, employing a semi-defined medium, used for the cultivation of E coli DH10B carrying a plasmid coding for HIV pEnv. When glucose was fed, employing a predetermined feeding algorithm designed to match consumption rate, a high specific growth rate of about 0.7 h−1 was maintained for about 8 h. However, during the course of experiment, the feed rate of glucose exceeded the anticipated consumption rate and caused glucose accumulation and oxygen limitation which triggered the accumulation of acetic acid. A limited biomass of about 10–12 g/l was achieved and plasmid volumetric yield reached a value of 8–10 mg/l. To avoid the accumulation of glucose, a feeding strategy based on feed-back control using the input from the pH and DO control loops was designed. The nutrient solution, a mixture of glucose and yeast extract, was fed when either the DO rose above 50% (indicative of a lack of nutrient) or the pH rose above 7.2 (caused by the consumption of alternative carbon sources such as fatty acids). Both conditions were believed


to accurately reflect a lack of glucose in the medium. This fed-batch strategy was initiated after about 8 h of cultivation. During the initial batch portion a growth rate of 0.69 h−1 was achieved while a lower specific growth rate of 0.13 h−1 was maintained during the fed-batch portion. Glucose accumulation was never observed, and final biomass concentrations ranging from 70 to 100 g dry cell/l were achieved. Plasmid volumetric and specific yields were improved over those obtained with the glucose fed-batch strategy (70–90 mg plasmid/l versus 8–10 mg plasmid/l and 1.7 g plasmid/mg dry cell weight versus 0.7 g plasmid/mg dry cell weight, respectively). The authors clearly attribute the specific yield improvements to achieving a slower growth rate, conducive to plasmid amplification and higher stability, during the second portion of the fermentation process. The scale-up of this strategy was demonstrated from 7 to 80 l.

As mentioned previously in the description of E. coli replicons, a point mutation in the RNAII primer sequence of pUC19 relative to the parental plasmid pBR322 results in a temperature-dependent elevation of plasmid copy number. Lahijani et al. [31] introduced this G → A mutation into a pBR322-derived plasmid in an attempt to improve plasmid DNA yields. Initially, E. coli was batch cultivated in

a semi-defined medium. When the temperature was shifted in mid-log phase (OD of 12) from 37 ◦C to either 42 or45 ◦C, plasmid volumetric yields of 34 and 49 mg/l were respectively achieved. These volumetric yields compared very favorably with the 7.5 mg/l achieved without temperature shift. A fed-batch approach employing the feeding of a mixed nutrient solution (glucose, yeast extract, MgSO4 , and l-leucine) coupled with a pre-determined program that kept the culture growing with a specific growth rate of0.25 h−1 , was implemented in conjunction with the temperature shift strategy. At mid log phase (OD 56) the temperature was shifted to 45 ◦C and the batch continued until the OD value dropped. A volumetric plasmid yield of 220 mg/l was achieved under these conditions. Close examination of the data show that the specific yield was comparable between the fed-batch and batch cultivation.

In terms of regulatory guidelines, the rules pertaining to the cultivation of the microbial host, E. coli, are similar to those in place for the production of recombinant proteins, and are captured in the FDA good manufacturing practices for vaccine production documents [105]. These guidelines are in place to ensure the quality, potency, and consistency of each lot, and require that all operations be consistent

Fig. 5. Major factors controlling plasmid productivity in bioreactors.

Bio

mas

s ()

Plas

mid

DN

A (

)


Fed-Batch Cultivation

Slow Growth

Rapid Growth

Time

Fig. 6. Representative plot of cell growth and plasmid accumulation in a typical fed-batch process. Commonly used units for biomass are either optical density measured at 600 nm or dry cell weight expressed in g/l. Plasmid DNA production is usually expressed either as volumetric (mg/l) or specific ( g/mg of dry cell weight).

with current good manufacturing practices (cGMP). The cultivation of the microbial strain should be performed under controlled conditions as to reproducibly yield a product of high quality [23–30]. It should be well understood that while it is acceptable to use an antibiotic for the selection of plasmid-bearing cells, the antibiotic should not be used to maintain the culture axenic in replacement of proper sterile techniques [106].

Medium composition and cultivation conditions play an important role by controlling plasmid copy number, stability and the amount of biomass produced. Important intertwin- ing inputs that lead to an efficient plasmid DNA production process are depicted in Fig. 5. It is apparent from the examples reviewed here that media formulations specifically designed for the over-production of plasmid DNA can support higher yields and increase plasmid stability. While perhaps more difficult and time consuming to initially complete, the design and development of an optimized cultivation medium can provide an extensive pay-off in the long-term. Coupled with the powerful tool of statistical experimental design, the design of chemically defined cultivation medium will likely yield highly optimized media formulations [107,108]. Chemically defined formulations offer the possibility to per- form extensive analytical investigations, which in turn can support metabolic studies and quality control purposes. In addition, the use of chemically defined media eliminates most of the uncertainty facing the origin and composition

of the raw complex ingredients. In the context of developing processes for the commercial production of vaccines, the use of chemically defined cultivation media will also help in achieving a better position with respect to the regulatory environment by supporting safety and reproducibility claims [109]. Although the available information is limited in comparison with the extensive recombinant protein production literature, some general rules and methodologies pertaining to the production of plasmid DNA by cultivation of E. coli are beginning to emerge. As anticipated, while many of the conditions that lead to elevated recombinant protein production can be directly translated, several modifications are necessary in order to achieve maximum yields. Productive plasmid DNA processes tend to employ a two-phase strategy. Namely, a phase of biomass buildup where the cells grow exponentially followed by a phase of slow growth, achieved via fed-batch technology, where plasmid amplification takes place (Fig. 6). Additional studies in this field should revolve around the optimization of these steps and will bring much needed additional information.

5. Plasmid DNA purification

When a molecular biologist thinks of “large-scale” plasmid DNA production, the range of 10–100 mg of DNA usually comes to mind. However, at a pharmaceutical


Fig. 7. Large-scale plasmid DNA purification steps. Each important step is listed here along with the choices (left) and scale-up issues (right) for each applicable technology.

production-scale, plasmid DNA requirements may exceed50 g per batch. In extreme cases, many kilograms of plasmid DNA per year will be needed to fill the ultimate marketing demand for DNA vaccines currently in clinical trials. The following section is to be considered in conjunction with several reviews recently published [110–112]. Here, we specifically emphasize the practicality and potential scale-up issues facing existing plasmid DNA purification technologies.

Post cultivation, the purification of plasmid DNA at the large scale (>10 g) typically involves the steps outlined in the diagram shown in Fig. 7. The order of steps is often as fol- lows: cell harvest, cell lysis, cell debris removal, affinity precipitation, adsorption, and buffer exchange/concentration. At first, and to better understand the pitfalls of large-scale plasmid DNA purification, it is relevant to consider the diffi-

culties associated with the direct scale-up of some common laboratory procedures.

Historically, plasmid DNA purification is accomplished through the use of cesium chloride/ethidium bromide (CsCl/EtBr) buoyant density gradient separation [113,114]. This method allows the separation of plasmid DNA by buoyant density into purified bands of different forms (e.g. supercoiled, open circular, linear, multimeric) when the CsCl/EtBr/plasmid DNA mixture is subjected to ultracentrifugation. The covalently closed plasmid DNA band is then withdrawn from the ultracentrifuge bottle(s) and further extracted and alcohol precipitated to remove both CsCl and EtBr. While it yields highly purified plasmid, this approach is not scaleable because of personnel safety issues and the hazardous waste considerations associated with the use of cesium chloride and ethidium bromide. In addition, the use of ultracentrifugation is also a major impediment to the scale-up of this technology. However, this procedure is still considered the purity benchmark against which all other plasmid DNA purification techniques are evaluated.

Current bench-scale purification methods employ kits marketed by commercial companies that take advantage of several of the techniques reviewed below, including alkaline lysis and the use of disposable chromatography columns. However, these purification conditions do not lend them- selves to scale up because they may employ hazardous chemicals, such as phenol and chloroform, require the use of large amounts of chromatographic resin per milligram of plasmid purified, or necessitate the use of relatively expensive enzymes in order to degrade contaminants (e.g. RNase and protease K). Finally, the use of fairly dilute solutions would lead to extremely large process equipment on the>50 g plasmid DNA production scale and would translate into considerable capital investment. Due to their simplicity, these purification methods are however the method of choice for the purification of small amounts of plasmid DNA (<20 mg) and are extensively used in research laboratories. For a more in-depth discussion of small-scale plasmid DNA purification, Sambrook and Russell [114] offer an extensive and up-to-date review of this technology.

Beginning with cell harvest, the next sections follow the progression shown in Fig. 7 and detail each step in typical large-scale plasmid DNA purification. In addition, commonscale-up issues are summarized.

5.1. Cell harvest

Upon completion of cell cultivation, nearly all cell lysis steps require the use of a concentrated cell slurry produced via either microfiltration [115] or continuous centrifugation [116]. In addition to providing concentrated cell slurry, this step removes the majority of the spent fermentation broth, which can interfere with most common downstream lysis and/or purification steps. Scale-up issues include potential membrane fouling and cleaning of continuous centrifuges.


5.2. Cell lysis

Most current bacterial cell lysis techniques (homogeniza- tion, freeze/thaw, detergent-based extraction, etc.) have been optimized for the purification of recombinant proteins and most are not applicable to plasmid DNA purification. Since large-scale plasmid DNA production is in its infancy, currently only a handful of lysis techniques are used. By far, the most common technique used in the production of plasmid DNA is that of alkaline lysis [117]. This procedure relies on the use of sodium hydroxide and the detergent sodium dode- cyl sulfate (SDS) whose combined effects result in the disruption of the cell membrane, leading to lysis and release of the intracellular components. Subsequent neutralization with sodium acetate precipitates protein and genomic DNA. Upon neutralization, supercoiled plasmid DNA anneals from its pH-denatured state while large molecular weight (∼200 kb) genomic DNA cannot diffuse and anneal properly, resulting in single-stranded genomic DNA precipitation. In addition, at high pH, RNA degrades, which benefits downstream separation steps. However, this lysis is rather delicate as plasmid DNA can be degraded at high pH if high shear conditions are produced during this procedure [118]. Since the DNA does denature during this lysis, some of the supercoiled DNA is converted to alternative forms (e.g. denatured supercoiled, multimeric, open circle, and linear). Also, alkaline lysis can cause fragmentation of genomic DNA, which can make downstream removal more difficult.

Another method of lysis used in the production of plasmid DNA is that of boiling cell lysis first introduced by Holmes and Quigley [119]. This lysis method utilizes a lysozyme digestion to break down the peptidoglycan in cell walls (through hydrolysis of glycosidic linkages) followed by heating to the point that the membranes disassociate, leading to the release of plasmid DNA from its cytosolic location. In addition, undesirable DNAses are inactivated and proteins are denatured and precipitated during this heat step. Scale-up issues with heat lysis include heat exchanger design and cleaning. The prime scale-up example of this lysis technique utilizes a continuous heat exchanger with rapid heating and cooling [120]. This lysis has been carried out at the multiple-gram scale and is highly scaleable.

In addition, lysozyme alone can be used to lyse cells in the presence of a detergent (e.g. Triton X-100) [114]. How- ever, it should be noted that lysozyme is traditionally produced from hen-egg whites and is not a desirable raw material in the cGMP production of plasmid DNA due to the risk of contamination from this avian-derived material. An alternative to hen egg lysozyme, although slightly more costly, has been the use of a recombinant lysozyme, purified from E. coli [121,122]. Scale-up challenges include minimizing the use of relatively high cost lysozyme; however, the lysis is very simple and can be done in a single process tank with very high plasmid DNA recoveries. A major advantage of a lysozyme-based lysis, beside simplicity, is that product degradation is not an issue (e.g. formation of denatured

supercoiled, linear and open circle plasmid). A major disad- vantage of the common lysozyme versus alkaline lysis procedure is that lysozyme lysis releases cellular contents and does not aid in purification.

The use of mechanical lysis is a fairly new development in the field of nucleic acid separation. A common problem, and an advantage of mechanical lysis for protein purification, is that high shear degrades nucleic acids [123]. It has been shown that relatively low shear induced by a bead mill can release plasmid DNA from E. coli while keeping it up to 90% intact [124]. Currently, the only published work utilizing mechanical lysis in plasmid DNA purification is at a relatively small scale; however, mechanical lysis is used in large-scale protein manufacture and equipment is readily available.

5.3. Affinity precipitation of plasmid DNA

Most large-scale production processes utilize an upstream affinity precipitation of plasmid DNA. Alcohols, namely ethanol and isopropanol, are well-known DNA precipitation agents. In addition, polyethylene glycol [125], cationic detergents, for example CTAB, [122,126], certain salts such as LiCl and CaCl2 [127], polyamines such as spermidine [128], and polyethylene imine [129], are used to remove the majority of contaminants in a large-scale preparation without resorting to costly chromatographic separations.

A practical example of affinity precipitation is the use of CTAB. When employing this technology, plasmid DNA yields are normally greater than 90% and this precipitation method has been used on the multiple gram scale by Lander et al. [122]. An additional example of affinity precipitation in practice is the use of polyamines, specifically spermidine. Again yields are commonly greater than 90% and Murphy et al. [128] have applied this purification strategy at the gram scale.

In addition, DNA-binding ligands (triplex interaction) affixed to temperature sensitive polymers have been used in the purification of plasmid DNA. These polymers are soluble at low temperature but are insoluble at higher temperatures resulting in a temperature controllable affinity precipitation agent [130]. Most recently, the company Dr. Muller AG has applied this approach and is marketing a large-scale plasmid DNA separation strategy based on triplex affinity ligands attached to a stimulus responsive polymer.

Scaleability issues with affinity precipitations mainly in- volve proper mixing, separation of the precipitants, washing of the precipitants, and the method of addition of the affinity precipitation agent. Temperature, pH, degree of mixing, and the mixing time can affect the precipitant particle size and the extent of any precipitation. The mixing and method of addition of the affinity precipitation agent are also important because high local concentration of affinity agents can cause coprecipitation of impurities. Additionally, the quantity of residual liquid remaining prior to re-suspension can greatly affect the levels of impurities.


5.4. Solid–liquid separation in plasmid DNA production

The separation of solids from liquids is a necessity in the production of plasmid DNA. With plasmid DNA doses on the order of milligrams per dose and the solubility limit of crude DNA-containing lysates at 1–2 mg/ml, extremely large solution volumes with moderate levels of solids volume need to be processed. The most economical means of clearing solids is by a centrifugation-based separation; however, this method of solid–liquid separation cannot produce a highly clarified solution without substantial residence times, partly due to the high viscosity of plasmid DNA in solution. Large residence times require either extremely large centrifuges or long processing times, neither of which is optimal in a manufacturing setting. In addition, cleaning concerns with current industrial centrifuges have been reported [131].

The most attractive filtration-based solid–liquid separation for plasmid DNA large-scale processing is that of a body feed filtration (e.g. addition of a filter aid such as diatomaceous earth (DE)). This cake-building filtration decreases the amount of filter fouling as the filtration surface is continuously regenerated as filter aid is deposited, allowing for the removal of the large amounts of cell waste resulting from E. coli lysis. In addition, DE is known to bind RNA [132], aiding downstream purification steps. However, a major drawback in the scale-up of body feed filtrations is solidwaste disposal. With common DE slurries at ∼50 g DE/l,large-scale production methods can produce tons of contam- inated solid waste per year.

Tangential flow filtration (TFF) of plasmid DNA-containing alkaline lysates has been demonstrated as a feasi- ble purification unit operation. TFF can be accomplished for buffer exchange and small ion removal. However, at a high loading, large bio-molecules cannot be adequately removed during TFF without use of expensive nucleases, long term exposure to high pH, and costly proteases due to the formation of a DNA gel layer [133]. In addition, upon scale up, the elevated liquid flow rates achieved necessitate the use of high shear pumps that can degrade the shear-sensitive plasmid DNA and the necessary membrane area can become excessive.

5.5. Adsorbent-based plasmid DNA separation

Chromatography, a hallmark of bioseparation, turns out to be a very tedious step in the purification of plasmid DNA. Common chromatography resins used in the separation of nucleic acids are anion-exchange (e.g. functional groups of quaternary amine (Q) and diethyl aminoethyl (DEAE)), reverse phase, hydrophobic interaction, hydroxyapatite, size exclusion, and immobilized metal affinity chromatography [78,95,134,135]. Most chromatographic separation media currently produced are optimized for the production of proteins. The average pore size of common chromatographic supports is smaller than or approximately the same size as the radius of gyration of a plasmid DNA molecule [136].

Thus, plasmid DNA cannot access pores in standard chromatographic media and can only bind to the outer surface of the particles; hence, only approximately 0.2–2 g plasmid DNA bind per liter of resin in contrast to proteins where the loading can range from 10 to 100 g per liter of resin. This means a substantial amount of chromatography media is needed in a large-scale plasmid DNA separation (∼500–2000 l of resin/kg plasmid DNA processed). Sagar et al. [121] goes into further detail about the application of chromatographic techniques to the purification of plasmid DNA.

Immobilized Metal Affinity Chromatography has recently been applied to plasmid DNA purification. For example, an iminodoacetic acid (IDA) resin charged with Cu(II) was found to bind free purine bases in solution. Thus, damaged DNA and RNA bind to an IMAC resin in flow-through mode while plasmid DNA is not retained, thereby allowing downstream polishing to handle >100 g of plasmid DNA per liter of IMAC resin (for a feed with 2% (w/w) RNA contamination) [135]. IMAC has only been applied to plasmid DNA at the 10 mg-scale to this point; however, due to the scaleability of flow-through chromatography (no gradients or steps), scale-up issues should be minimal.

Perfusion chromatography offers higher flow rates and moderately higher binding capacities of plasmid DNA [121]. The typical perfusion media for example has ∼4000 Å con-vective pores along with the standard ∼100–1000 Å pores of a typical media. The convective flow through the media (typically ∼5% of the overall fluid flow) decreases pressure drop and the larger convective pores can allow some plasmid DNA to enter the chromatography media, increasing plasmid DNA-binding capacity. In addition, linear velocities of 1000–5000 cm/h are achievable using a perfusion chromatography media (as opposed to 50–400 cm/h for conven- tional chromatography media).

Tentacle chromatographic supports have also been used in the large-scale production of plasmid DNA. These resins contain long poly-electrolyte chains with large linkers that extend from the surface of the chromatographic resin. This increases the number of charged sites on the resin, and pos- sibly the loading capacity. To this point, however, there have been only limited publications detailing the use of tentacle resins for plasmid DNA purification [137].

Studies have been completed using additives to increase the loading of plasmid DNA on columns by reducing the effective size of plasmid DNA molecules in solution [121,138]. However, even with condensed particles, the binding of DNA to chromatography media is quite low (typically less than 5 mg plasmid DNA/ml of resin).

In addition to classic column chromatography, the use of expanded bed chromatography has been to be applied to plasmid DNA separation [139,140]. The expanded bed chromatographic design is an offshoot of a standard flu- idized bed, however the expanded bed is operated in a range where the particles are supported and do not back mix, al-


lowing for plates of separation to still exist. Also, this type of chromatographic separation allows lysates to be purified with limited particulates and allows for linear velocities in

Table 3Example of purity requirements for DNA vaccines [121]

Contaminant Levelexpanded bed mode of ∼300 cm/h. The major drawbackof expanded bed adsorbents for plasmid DNA purification

Percentage of covalently closed plasmidDNA (gel electrophoresis)

>90%

is that of loading capacity. Since plasmid does not pene-trate the pores of most chromatographic media, surface area (and thus particle size) scales with loading capacity. Be- cause expanded bed adsorbents typically have large particle

Percentage E. coli genomic DNA (qPCR) <1% Percentage RNA (HPLC ribose assay) <0.1%Endotoxin (LAL assay) <0.5 EU (endotoxin)/mg

plasmid DNAProtein (BCA Assay) <1%

sizes, loading capacities are typically much lower that that of classic packed bed chromatography columns. To com- bat this inherent problem with expanded bed adsorbents,prototype adsorbents with smaller particle sizes and chromatographic ligands based on polyethylene imine (that act as a tentacle ligand) have been utilized with some success [141].

With the inherent issues with chromatographic purification of plasmid DNA, other forms of adsorptive separation have been attempted. To this end, batch adsorption of process contaminants can be performed as opposed to column chromatography, when a low cost adsorbent is utilized. One example of this approach was demonstrated with the use of a hydrated calcium silicate-based batch adsorption of DNA and other contaminants [142]. In this approach hydrated calcium silicate is used to bind genomic DNA, open circle plasmid, endotoxin, proteins, and residual detergents leav- ing supercoiled plasmid in solution.

Adsorptive membrane separations also show some promise with increased plasmid DNA loading capacity (∼10 mg/ml of membrane), but relatively high cost of themembranes prohibits their use at large scale [143]. In addition, the adsorption of E. coli genomic DNA on nitrocellulose based filters has been discussed but the low loading capacity of genomic DNA on nitrocellulose makes them impractical at the large scale [144].

5.6. Buffer exchange/concentration

Membrane-based TFF can be used for buffer exchange and small ion removal; however, at a high loading, large biomolecules cannot be permeated due to the formation of a DNA gel layer. Possible scale-up issues facing this technology include both the requirements for large membrane surface area and high fluid flow rates in order to keep membranes from fouling prematurely.

Buffer exchange and concentration can also be accomplished by using an alcohol-based precipitation. This allows for the precipitation of DNA and the production of a vacuum- dried solid bulk, which is more stable and takes up signifi- cantly less storage space than a typical <10 mg/ml plasmid DNA solution [95,145].

5.7. Final product

Final DNA vaccine or gene therapy product must be0.22 m sterile filtered and must meet minimum release

specifications after purification. Release specifications mayvary due to the fact that regulatory agencies have not set defined values for key contaminants in DNA vaccine prepa- rations. An example of a current product purity specification is shown in Table 3.

Final formulation and delivery of DNA vaccines are an extremely important area of research to improve DNA vaccines as a whole. While naked DNA vaccines have been shown to be effective, an efficient delivery system could re- duce dose requirements and increase product effectiveness, while reducing product cost. For more information Luo and Saltzman [146] and Lian and Ho [147] give in-depth reviews of some current DNA delivery strategies.

6. Conclusion

As advances continue in the field of DNA vaccines and/or plasmid-based gene therapies, and as commercial products emerge, factories capable of producing kilograms of plasmid DNA per year must be constructed. Advances in vector design, fermentation, purification and formulation of plasmid DNA are being driven by this new need for large quantities of plasmid DNA and the current regulatory environment.

For commercial uses, considerations of intellectual property restrictions must be made when selecting specific vectors, host cell lines, and production technologies. A large number of patents and patent applications that specifically or potentially cover this field have been issued for vector design [148,149], cultivation methods [104], and purification procedures [150–152]. These few examples are listed here to remind the reader that attention must be paid to this field. However, it must be remembered that patents have only a temporal duration. Additionally, while published applications are often quite broad in scope, the granted claims upon issue can be much narrower, and claims may be further limited (i.e. invalidated) through court challenge. Therefore, working in close relationship with a legal team to carefully review the applicable intellectual property environment is the most appropriate path forward.

The pharmaceutical-scale plasmid DNA fermentation and process technologies reviewed here are a good start toward the goal of cost effective large-scale DNA vaccine production.


References

[1] Chattergoon M, Boyer J, Weiner D. Genetic immunization: a new era in vaccines and immune therapeutics. FASEB 1997;11:753–63.

[2] Wolf J, Malone R, Williams P, Chong W, Acsadi G, Jani A,et al. Direct gene transfer into mouse muscle in vitro. Science1990;247:1465–8.

[3] Danko I, Wolff J. Direct gene transfer into muscle. Vaccine 1994;12:149–1502.

[4] Mountain A. Gene therapy: the first decade. TIBTECH 2000;18:119–28.[5] Ferber D. Gene therapy: safer and virus free. Science

2001;294:1638–42.

[6] Gurunathan S, Wu C, Friedag B, Seder R. DNA vaccines: a key for inducing long-term cellular immunity. Curr Opin Immunol2000;12:442–7.

[7] Shroff K, Smith L, Baine Y, Higgins T. Potential for plasmid DNAs as vaccines for the new millenium. PSTT 1999;2:205–12.

[8] Restifo N, Rosenberg S. Developing recombinant and syntheticvaccines for the treatment of melanoma. Curr Opin Oncol1999;11:50–7.

[9] Baumgartner I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K, et al. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 1998;97:1114–23.

[10] Losordo D, Vale P, Symes J, Dunnington C, Esakof D, Maysky

M, et al. Gene therapy for myocardial angiogenensis. Circulation1998;98:2800–4.

[11] Tang D, DeVit M, Johnston S. Genetic immunization is a simple method for eliciting an immune response. Nature 1992;356:152–4.

[12] Robinson H, Hunt L, Webster R. Protection against a lethal influenza

virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vaccine 1993;11:957–60.

[13] Ulmer J, Donnelly J, Parker S, Rhodes G, Felgner P, Dwarki V,

et al. Heterelogous protection against influenza by injection of DNAencoding a viral protein. Science 1993;259:1745–9.

[14] Doolan D, Hoffman S. DNA-based vaccines against malaria: status and promises of the multi-stage malaria DNA vaccine operation. Int J Parasitol 2001;31:753–62.

[15] Mascola J, Nabel G. Vaccines for the prevention of HIV-1 disease.Curr Opin Immunol 2001;13:489–95.

[16] Shiver J, Fu T, Chen L, Casimiro D, Davies M, Evans R, et al.

Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature 2002;415:331–5.

[17] Barouch D, Craiu A, Kuroda M, Schmitz J, Zheng X, SantraS, et al. Augmentation of immune responses to HIV-1 and simian immunodeficiency virus DNA vacines by IL-2/Ig plasmid administration in rhesus monkeys. PNAS 2000;97:4192–7.

[18] Barouch D, Santra S, Schmitz J, Kuroda M, Fu T, Wagner W,

et al. Control of verimia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination. Science2001;290:486–92.

[19] Liljeqvist S, Stahl S. Production of recombinant subunit vaccines: protein immunogens, live delivery systems and nucleic acid vaccines. J Biotechnol 1999;73:1–33.

[20] Leitner W, Ying H, Restifo N. DNA and RNA-based vaccines:

principles, progress and prospects. Vaccine 2000;18:765–77.[21] Robinson H. DNA vaccines. Clin Microbiol Newslett 2000;23:17–

22.

[22] Herweijer H, Wolf J. Progress and prospects: naked DNA gene transfer and therapy. Gene Therapy 2003;10:453–8.

[23] FDA. Points to consider on plasmid DNA vaccines for preventive

infectious diseases. 1996. Docket no. 96N-0400.

[24] W.H.O., WHO guidelines for assuring the quality of DNA vaccines.

Biologicals 1998;26:205–12.

[25] Falk L, Ball L. Current status and future trends in vaccine regulation- USA. Vaccine 2001;19:1567–72.

[26] Cichutek K. DNA vaccines: development, standardization and regulation. Intervirology 2000;43:331–8.

[27] Robertson J, Cichutek K. European union guidance on the quality,safety and efficacy of DNA vaccines and regulatory requirements. Dev Biol 2000;104:53–6.

[28] Smith H. Regulatory considerations for nucleic acid vaccines.Vaccine 1994;12:1515–28.

[29] Smith H. Regulation and review of DNA vaccine products. DevBiol 2000;104:57–62.

[30] Smith H, Klinman D. The regulation of DNA vaccines. Curr OpinBiotechnol 2001;12:299–303.

[31] Lahijani R, Hulley G, Soriano G, Horn N, Marquet M. High-yield production of pBR322-derived plasmids intended for human gene therapy by employing a temperature controllable point mutation. Human Gene Therapy 1996;7:1971–80.

[32] Gurunathan S, Klinman D, Seder R. DNA vaccines: immunology,application, and optimization. Annu Rev Immunol 2000;18:927–74.

[33] Musacchio A, Herrera ERodriguezA, Quintana D, Muzio V.Multivalent DNA-based immunization against Hepatitis B virus with plasmids encoding surface and core antigens. Biochem Biophys Res Commun 2001;282(2):442–6.

[34] Makrides S. Strategies for achieving high-level expression of genesin Escherichia coli. Microbiol Rev 1996;60(3):512–38.

[35] Balbas P, Bolivar F. Design and construction of expression plasmid vectors in Escherichia coli. In: Goeddel DD, editor. Methods in enzymology. San Diego, CA: Academic Press; 1990. p. 14–37.

[36] Venetianer P. Possibilities of increasing the expression of clonedforeign genes in E. coli. Acta Biotechnol 1991;11(2):129–33.

[37] Bolivar F, Rodriguez R, Betlach M, Boyer H. Construction and characterization of new cloning vehicles: I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene 1977;2:75–93.

[38] Bolivar F, Greene P, Betlach M, Heyneker H, Boyer H, Crosa J, etal. Construction and characterization of new cloning vehicles: II. A multipurpose cloning system. Gene 1977;2:95–113.

[39] Davison J. Mechanism of control of DNA replication andincompatibility in ColE1-type plasmids—a review. Gene 1984;28:1–15.

[40] Lacatena R, Banner D, Castagnoli L, Cesareni G. Control of initiation of pMB1 replication: purified Rop protein and RNA I affect primer formation in vitro. Cell 1984;37:1009–14.

[41] Tomizawa J, Som T. Control of ColE1 plasmid replication:enhancement of binding of RNA I to the primer transcript by theRom protein. Cell 1984;38:871–8.

[42] Twigg A, Sherratt D. Trans-complementable copy-number mutants of plasmid ColE1. Nature 1980;283:216–8.

[43] Vieira J, Messing J. The pUC plasmids, an M13mp7-derived systemfor insertion mutagenesis and sequencing with synthetic universal primers. Gene 1982;19:259–68.

[44] Balbás P, Soberón X, Merino E, Zurita M, Lomeli H, Valle F, etal. Plasmid vector pBR322 and its special-purpose derivatives—a review. Gene 1986;50:3–40.

[45] Norrander J, Kempe T, Messing J. Construction of improved M13vectors using oligodeoxynucleotide-directed mutagenesis. Gene1983;26:101–6.

[46] Minton N. Improved plasmid vectors for the isolation of translational lac gene fusions. Gene 1984;31:269–73.

[47] Chambers S, Barstow D, Minton N. The pMTL nic-cloning vectors.I. Improved pUC polylinker regions to facilitate the use of sonicatedDNA for nucleotide sequencing. Gene 1988;68:139–49.

[48] Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 1985;33:103–19.

[49] Lin-Chao S, Chen W, Wong T. High copy number of the pUCplasmid results from a Rom/Rop-suppressible point mutation inRNA II. Mol Microbiol 1992;6(22):3385–93.

[50] Phillips G, Park S, Huber D. High copy number plasmids compatible with commonly used cloning vectors. Biotechniques2000;28(3):400–8.


[51] Uhlin B, Nordström K. A runaway-replication mutant of plasmid R1drd-19: temperature-dependent loss of copy number control. Mol Gen Genet 1978;165:167–79.

[52] Remaut E, Tsao H, Fiers W. Improved plasmid vectors with

a thermoinducible expression and temperature-regulated runaway replication. Gene 1983;22:103–13.

[53] Ansorge M, Kula M. Investigating expression systems for the stablelarge-scale production of recombinant l-leucine-dehydrogenase from Bacillus cereus in Escherichia coli. Appl Microbiol Biotechnol2000;53:668–73.

[54] Chao Y, Chern J, Wen C. Coupling the T7 A1 promoter to the runaway-replication vector as an efficient method for stringent control and high-level expression of lacZ. Biotechnol Prog2001;17:203–7.

[55] Peretti S, Bailey J, Lee J. Transcription from plasmid genes, macromolecular stability, and cell-specific productivity in Escherichia coli carrying copy number mutant plasmids. Biotechnol Bioeng 1989;34:902–8.

[56] Wood T, Peretti S. Depression of protein synthetic capacity due tocloned-gene expression in E. coli. Biotechnol Bioeng 1990;36:865–78.

[57] Vind J, Sorensen M, Rasmussen M, Pedersen S. Synthesis of proteins in Escherichia coli is limited by the concentration of free ribosomes. J Mol Biol 1993;231:678–88.

[58] Dong H, Nilsson L, Kurland CG. Gratuitous overexpression ofgenes in Escherichia coli leads to growth inhibition and ribosome destruction. J Bacteriol 1995;177(6):1497–504.

[59] Williams S, Cranenburgh R, Weiss A, Wrighton C, Sherratt D,Hanak J. Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids. Nucleic Acids Res1998;26(9):2120–4.

[60] Davis M, Huang E. Transfer and expression of plasmids containing human cytomegalovirus immediate-early gene 1 promoter-enhancer sequences in eukaryotic and prokaryotic cells. Biotechnol Appl Biochem 1988;10:6–12.

[61] Chapman B, Thayer R, Vincent K, Haigwood N. Effect of intron

A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells. Nucleic Acids Res1991;19(14):3979–86.

[62] Garapin A, Ma L, Pescher P, Lagranderie M, Marchal G.Mixed immune response induced in rodents by two naked DNAgenes coding for mycobacterial glycosylated proteins. Vaccine2001;19:2830–41.

[63] Beyer J, Chebloune Y, Msell-Lakhal L, Hötzel I, Kumpula- McWhirter N, Cheevers W. Immunization with plasmid DNA expressing the caprine arthritis-encephalitis virus envelope gene: quantitative and qualitative aspects of antibody response to viral surface glycoprotein. Vaccine 2001;19:1643–51.

[64] Krieg A. Direct immunologic activities of CpG DNA andimplications for gene therapy. J Gene Med 1999;1:56–63.

[65] Krieg A, Ae-Kyung Yi, Schorr J, Davis H. The role of CpGdinucleotides in DNA vaccines. Trends Microbiol 1998;6(1):23–7.

[66] Chen Y, Lenert P, Weeratna R, McCluskie M, Wu T, Davis H, et al. Identification of methylated CpG motifs as inhibitors of the

immune stimluatory CpG motifs. Gene Therapy 2001;8:1024–32. [67] Swartz J. Escherichia coli recombinant DNA technology. In:

Neidhardt F, editor. Escherichia coli and Salmonella. Cellular and molecular biology. Washington DC: ASM Press; 1996. p. 1693–711.[68] Swartz J. Advances in Escherichia coli production of

therapeuticproteins. Curr Opin Biotechnol 2001;12:195–201.

[69] Jones K, Keasling J. Construction and characterization of F plasmid- based expression vectors. Biotechnol Bioeng

1998;59:659–65.[70] Lin-Chao S, Bremer H. Effect of the bacterial growth rate on

replication control of plasmid pBR322 in Escherichia coli. MolGen Genet 1986;203:143–9.

[71] Klotsky R, Schwartz I. Measurement of cat expression from growth- rate-regulated promoters employing -lactamase activity as an indicator of plasmid copy number. Gene 1987;55:141–6.

[72] Seo J, Bailey J. Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli. Biotechnol Bioeng 1985;27:1668–74.

[73] Riesenberg D, Menzel K, Schultz V, Schumann K, Veith G, Zuber G,et al. High cell density fermentation of recombinant Escherichia coliexpressing human interferon alpha 1. Appl Microbiol Biotechnol1990;34:77–82.

[74] Zabriskie D, Wareheim D, Polanski M. Effects of fermentation feeding strategies prior to induction of expression of a recombinant malaria antigen in Escherichia coli. J Ind Microbiol 1987;2:87–95.

[75] Nakagawa S, Oda H, Anazawa H. High cell density cultivationand high recombinant protein production of Escherichia coli strain expressing uricase. Biosci Biotech Biochem 1995;59:2263–7.

[76] Korz D, Hellmurh R, Sanders E, Deckwer W. Simple fed-batchtechnique for high cell density cultivation of Escherichia coli. J Biotechnol 1995;39:59–65.

[77] Strandberg L, Enfors S. Batch and fed-batch cultivations forthe temperature induced production of a recombinant protein inEscherichia coli. Biotechnol Lett 1991;13.

[78] Diogo M, Queiroz J, Monteiro G, Martins S, Ferreira G, Prazeres D. Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography. Biotechnol Bioeng2000;68(5):576–83.

[79] Diogo M, Ribeiro S, Queiroz J, Montiero G, Tordo N, Perrin P, et al. Production, purification and analysis of an experimental DNA vaccine against rabies. J Gene Med 2001;3:577–84.

[80] Drew D, Lighttowlers M, Strugnell R. Humoral immune responsesto DNA vaccines expressing secreted, membrane bound and non-secreted forms of the Taenia ovis 45W antigen. Vaccine2000;18:2522–32.

[81] O’Kennedy R, Baldwin C, Keshavarz-Moore E. Effects of growth medium selection on plasmid DNA production and initial processing steps. J Biotechnol 2000;76:175–83.

[82] Harris R, Adams S. Determination of the carbon-bound electronelectron composition of microbial cells and metabolites by dichromate oxidation. Appl Environ Microbiol 1979;37:237–43.

[83] Paalme T, Tiisma K, Kahru A, Vanatalu K, Vilu R. Glucose-limitedfed-batch cultivation of Escherichia coli with computer-controlled fixed growth rate. Biotechnol Bioeng 1990;35:312–9.

[84] Reiling H, Laurila H, Fiechter A. Mass culture of Escherichiacoli: medium development for low and high density cultivation ofEscherichia coli B/r in minimal and complex media. J Biotechnol1985;2:191–206.

[85] Fieschko J, Ritch T. Production of human alpha consensus interferon in recombinant Escherichia coli. Chem Eng Commun 1986;45:229–40.

[86] Wang Z, Le G, Shi Y, Wegrzyn G. Medium design for plasmid

DNA production based on stoichiometric model. Process Biochem2001;36:1085–93.

[87] Bech Jensen E, Carlsen S. Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate and salts. Biotechnol Bioeng 1990;36:1–11.

[88] Dennis K, Srienc F, Bailey J. Ampicillin effects on five recombinantEscherichia coli strains: implications for selection pressure design. Biotechnol Bioeng 1985;27:1490–4.

[89] Bentley W, Quiroga O. Investigation of subpopulation heterogeneityand plasmid stability in recombinant Escherichia coli via a simple segregated model. Biotechnol Bioeng 1993;42:222–34.

[90] Summers D. The kinetics of plasmid loss. TIBTECH 1991;9:273–8.[91] Zabriskie D, Arcuri E. Factors influencing the

productivity of fermentations employing recombinant microorganisms. Enz Microbiol Technol 1986;8:706–17.

[92] Riesenberg D. High-cell density cultivation of Escherichia coli.Curr Opin Biotechnol 1991;2:380–4.

[93] Yee L, Blanch H. Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli. Biotechnology1992;10:1550–6.


[94] Lee Y. High cell density culture of Escherichia coli. TIBTECH1996;14:98–105.

[95] Horn N, Meek J, Budahazi G, Marquet M. Cancer gene therapy using plasmid DNA: purification of DNA for human clinical trials. Hum Gene Therapy 1995;6:565–73.

[96] Pan J, Rhee J, Lebeault J. Physiological constraints in increasing biomass concentration of Escherichia coli B in fed-batch culture. Biotechnol Lett 1987;9:89–94.

[97] Luli G, Strohl W. Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl Environ Microbiol 1990;56:1004–11.

[98] Cherrington C, Hinton M, Chopra I. Effect of short-chain organic acids on macromolecular synthesis in Escherichia coli. J Bacteriol1990;68:69–74.

[99] Seo J, Bailey J. Continuous cultivation of recombinant Escherichia coli: existence of an optimum dilution rate for maximum plasmid and gene product concentration. Biotechnol Bioeng 1986;28:1590–4.

[100] Reinikainen P, Virkajärvi I. Escherichia coli growth and plasmid copy numbers in continuous cultivations. Biotechnol Lett1989;11(4):222–30.

[101] Reinikainen P, Korpela K, Nissinen V, Olkku J, Soderlund H, Markkanen P. Escherichia coli production in fermentor. Biotechnol Bioeng 1989;33:386–93.

[102] Namdev P, Irwin N, Thompson B, Gray M. Effect of oxygen fluctuations on recombinant Escherichia coli fermentation. Biotechnol Bioeng 1993;41:660–70.

[103] Schmidt T, Friehs K, Schleef M, Voss C, Flaschel E. In-process analysis of plasmid copy number for fermentation control. Pacesetter2001;5(1):4–6.

[104] Chen W, Graham C, Ciccarelli R. Automated fed-batch fermentation with feed-back controls based on dissolved oxygen (DO) and pH for production of DNA vaccines. J Ind Microbiol Biotechnol1997;18:43–8.

[105] United States Code of Federal Regulations, Title 21 part601.25(d)(4). Washington DC: US government printing office;

1999. [106] European Agency for the Evaluation of Medicinal Products, Note on guidance on pharmaceutical and biological aspects of

combinedvaccines; 1998.

[107] Greasham R, Inamine E. Nutritional improvement of processes. In: Demain A, Solomon N, editors. Manual for industrial microbiology and biotechnology. Washington DC: ASM; 1986. p. 41–8.

[108] Greasham R, Herber W. Design and optimization of growth media.In: Applied microbial physiology—a practical approach. Oxford: Oxford University Press; 1997. p. 53–74.

[109] Zhang J, Greasham R. Chemically defined media for commercial fermentations. Appl Microbiol Biotechnol 1999;51:407–21.

[110] Levy M, O’Kennedy R, Ayazi-Shamlou P, Dunnill P. Biochemical engineering approaches to the challenges of producing pure plasmid DNA. Trends Biotechnol 2000;18(7):296–305.

[111] Marquet M, Horn N, Meek J. Process development for the manufacture of plasmid DNA vectors for use in gene therapy. Biopharmacy; 1995. p. 26–37.

[112] Prazeres D, Ferreira G, Monteiro G, Cooney C, Cabral J. Large- scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks. TIBTECH 1999;17:169–74.

[113] Clewell D, Helinski D. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an open circular DNA form. PNAS 1969;62(4):1159–66.

[114] Sambrook J, Russell D. Molecular cloning: a laboratory manual,3rd ed., vol. 1. Cold Spring Harbor: Cold Spring Harbor LaboratoryPress; 2001.

[115] Riesmeier B, Kroner K, Kula M. Tangential filtration of microbial suspensions: filtration resistances and model development. J

Biotechnol 1989;12:153–72.[116] Tinnes R, Hoare M. The biocontainment of a high speed disk bowl

centrifuge. Bioprocess Eng 1992;8:165–72.

[117] Birnboim H, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res1979;7(6):1513–23.

[118] Chamsart S, Patel H, Hanak J, Hitchcock A, Nienow A. The impact of fluid-dynamic-generated stresses on chDNA and pDNA stability during alkaline cell lysis for gene therapy products. Biotechnol Bioeng 2001;75(4):387–92.

[119] Holmes D, Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 1981;114(1):193–7.

[120] Sagar S, Lee A. Production of a crude lysate from which plasmid DNA can be isolated, comprises passing cells from a large-scale microbial culture through a heat exchanger. Merck and Co. Inc., US Patent 6,197,553; 2001.

[121] Sagar S, Watson M, Lee A. Chromatography-based purification of plasmid DNA. In: Rathore AS, Vella G, editors. Scale-up and optimization in preprative chromatography: principles and biopharmaceutical applications. New York: Marcel Dekker; 2003. p. 251–72.

[122] Lander R, Winters M, Meacle F, Buckland B, Lee A. Fractional precipitation of plasmid DNA from lysate by CTAB. Biotechnol Bioeng 2002;79(7):776–84.

[123] Lengsfeld C, Anchordoquy T. Shear-induced degradation of plasmidDNA. J Pharm Sci 2002;91(7):1581–9.

[124] Carlson A, Signs M, Liermann L, Boor B, Jem K. Mechanical disruption of Escherichia coli for plasmid recovery. Biotechnol Bioeng 1995;48:303–15.

[125] Lis J, Schleif R. Size fractionation of double-stranded DNAby precipitation with polyethylene glycol. Nucleic Acids Res1975;02(3):383–9.

[126] Ishaq M, Wolf B, Ritter C, Theodossiou I, Sondergaard M, Thomas O. Large-scale isolation of plasmid DNA using cetyltrimethylammonium bromide. Biotechniques 1990;9:19–24.

[127] Kondo T, Mukai M, Kondo Y. Rapid isolation of plasmid DNA byLiCl-ethidium bromide treatment and gel filtration. Anal Biochem1991;198(1):30–5.

[128] Murphy J, Wibbenmeyer J, Fox G, Willson R. Purification of plasmid DNA using selective precipitation by compaction agents. Nat Biotechnol 1999;17(8):822–3.

[129] Yeung M, Lau A. Fast and economical large-scale preparation of high-quality plasmid DNA. Biotechniques 1993;15(3):381–2.

[130] Freitag R, Costioli M, Garret F. Stimulus-responsive polymers for bioseparation. Chimia 2001;55(3):196–200.

[131] Tebbe H, Lutkemeyer D, Gudermann F, Heidemann R, Lehmann J.Lysis-free separation of hybridoma cells by continuous disc stack centrifugation. Cytotechnology 1996;22(1–3):119–27.

[132] Horn N, Marquet M, Meek J, Budahazi G. Process for reducing RNA concentration in a mixture of biological material using diatomaceous earth. Vical Inc., US Patent 5,576,196.

[133] Kahn D, Butler M, Cohen D, Gordon M, Kahn J, Winkler M.Purification of plasmid DNA by tangential flow filtration. BiotechnolBioeng 2000;69(1):101–6.

[134] Chandra G, Patel P, Kost T, Gray J. Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi- Load Q Sepharose column. Anal Biochem 1992;203(1):169–72.

[135] Murphy J, Jewell D, White K, Fox G, Willson R. Nucleic acid separations utilizing immobilized metal affinity chromatography. Biotechnol Prog 2003;19:982–6.

[136] Ellegren H, Laas T. Size-exclusion chromatography of DNA restriction fragments: fragment length determinations and a comparison with the behavior of proteins in size-exclusion chromatography. J Chromatogr 1989;467(1):217–26.

[137] Mueller W. New ion exchangers for the chromatography of biopolymers. J Chromatogr 1990;510:133–40.

[138] Murphy J, Fox G, Willson R. Enhancement of anion-exchange chromatography of DNA using compaction agents. J Chromatogr.2003;984:215–21.


[139] Ferreira G, Cabral J, Prazeres D. Anion exchange purification of plasmid DNA using expanded bed adsorption. Bioseparation2000;9(1):1–6.

[140] Varley D, Hitchcock A, Weiss A, Horler W, Cowell R, Peddie L, et al. Production of plasmid DNA for human gene therapy using modified alkaline cell lysis and expanded bed anion exchange chromatography. Bioseparation 1999;8(1–5):209–17.

[141] Theodossiou I, Sondergaard M, Thomas O. Design of expanded bed supports for the recovery of plasmid DNA by anion exchange adsorption. Bioseparation 2001;10(1–3):31–44.

[142] Winters M, Richter J, Sagar S, Lee A, Lander R. Plasmid DNA purification by selective calcium silicate adsorption of closely related impurities. Biotechnol Prog 2003;19:440–7.

[143] Grunwald A, Shields M. Plasmid purification using membrane-based anion-exchange chromatography. Anal Biochem 2001;296(1):138–41.

[144] Levy M, Collins I, Tsai J, Shamlou P, Ward J, Dunnill P.

Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing. J Biotechnol2000;76(2/3):197–205.

[145] Horn N, Marquet M, Meek J. Special issues for DNA vaccines.

In: Sofer G, Zabriskie DW, editors. Biopharmaceutical process validation. New York: Marcel Dekker; 2000. p. 329–45.

[146] Luo D, Saltzman W. Synthetic DNA delivery systems. NatBiotechnol 2000;18(1):33–7.

[147] Lian T, Ho R. Trends and developments in liposome drug delivery systems. J Pharm Sci 2001;90(6):667–80.

[148] Hobart P, Parker S, Khatibi S. Cancer treatment method utilizing plasmids suitable for IL-2 expression. USA: Vical Incorporated;2002.

[149] Nabel G, Marquet M. Plasmids suitable for gene therapy. In: PCT application. Vical Incorporated and Regents of the University of Michigan: WO application 94/29469; 1994.

[150] Butler M, Cohen D, Kahn D, Winkler M. Purification of plasmidDNA. Genentech, Inc., US Patent 6,313,285; 2001.

[151] Marquet M, Horn N, Meek J, Budahazi G. Production of pharmaceutical-grade plasmid DNA. 1996, Vical Inc., US Patent5,561,064.

[152] McNeilly, D. Method for purifying plasmid DNA and plasmidDNA substantially free of genomic DNA. Genzyme Inc., US Patent6,214,586; 2001.

[153] Turnes C, Antonio J, Alexio G, Monteiro A, Dellagostin O. DNA inoculation with a plasmid vector carrying the faeG adhesin gene of Escherichia coli K88ab induced immune responses in mice and pigs. Vaccine 1999;17(15/16):2089–95.

[154] Jung YH, Lee Y. RNases in ColE1 DNA metabolism. Mol BiolReports 1996;22:195–200.