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

Enzyme and Microbial Technology 33 (2003) 865883

Review

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

Kristala Jones Prather, Sangeetha Sagar, Jason Murphy, Michel ChartrainMerck Research Laboratories, Department of Bioprocess R&D, RY80Y-105, Rahway, NJ 07065, USA

Received 9 January 2003; received in revised form 16 June 2003; accepted 24 June 2003

Abstract

The past several years have witnessed a rapidly increasing number of reports on utilizing plasmid DNA as a vector for the introductionof genes into mammalian cells for use in both gene therapy and vaccine applications. Naked DNA vaccines allow the foreign genes to betransiently expressed in transfected cells, mimicking intracellular pathogenic infection and triggering both the humoral and cellular immuneresponses. While considerable attention has been paid to the potential of such vaccines to mitigate a number of infections, substantially lessconsideration has been given to the practical challenges of producing large amounts of plasmid DNA for therapeutic use in humans, forboth clinical studies and, ultimately, full-scale manufacturing. Doses of naked DNA vaccines are on the order of milligrams, while typicalsmall-scale Escherichia coli fermentations may routinely yield only a few mg/l of plasmid DNA. There have been many investigationstowards optimizing production of heterologous proteins over the past three decades, but in these cases, the plasmid DNA was not the finalproduct of interest. This review addresses the current state-of-the-art means for the production of plasmid DNA at large scale in compliancewith 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 ofplasmid DNA are also discussed. 2003 Elsevier Inc. All rights reserved.

Keywords: Plasmid DNA; Gene therapy; Vaccine; DNA vaccine

1. Plasmid DNA in vaccines and gene therapy

The use of plasmid DNA as vector for gene therapy or es-pecially vaccination has gained considerable interest duringthe 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 oftransgenes inserted on a plasmid injected into the leg muscleof mice by Wolf et al. [2] generated tremendous interest. Theexpressed heterologous proteins were detectable for up to 60days after injection, indicating that the genes were expressedin vivo over an extended period of time, thus suggesting po-tential therapeutic applications [2,3]. Since then, intramus-cular injection of plasmid DNA has been extensively usedin gene therapy and in the design of novel DNA-based vac-cines. To date, well over 600 plasmid DNA-based gene ther-apy, cancer vaccine, and therapeutic vaccine clinical trialshave been initiated [48]. Notably in gene therapy, the use ofa 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).

factor that has been shown to promote revascularization inischemic limbs and hearts of patients, has generated exten-sive interest and publicity [5,9,10]. With regard to vaccines,several investigators have demonstrated that the injection ofplasmid DNA containing selected genes from pathogens canelicit a protective immune response [1113]. Subsequently,plasmid DNA vaccines have yielded very encouraging re-sults in the fights against malaria [14] and AIDS [1518].Currently the use of plasmid DNA vaccines is being investi-gated against many other infectious diseases including hep-atitis B and C, and tuberculosis [1,7,19].

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

0141-0229/$ see front matter 2003 Elsevier Inc. All rights reserved.doi:10.1016/S0141-0229(03)00205-9

866 K.J. Prather et al. / Enzyme and Microbial Technology 33 (2003) 865883

fibroblasts

MUSCLE

VACCINATION WITH PLASMIDS

APC

Internal synthesis of

Release of

Stimulation of naive B cells

Internal synthesis of

Presentation of foreign

Stimulation of naive CD 8+ cells

HUMORAL IMMUNE CELLULAR IMMUNE

foreign peptide/protein foreign peptide/protein

foreign peptide/proteinforeign peptide/protein

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 histocompatibilitycomplex 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 draininglymph nodes where they elicit a cellular immune response by triggering the maturation and proliferation of CD8+ lymphocytes or cytotoxic T-lymphocytes(CTL). The foreign antigen can also be released in the extra-cellular environment, via either shedding from the MHC-I sites or from the lysis of myocytes.Once in the extracellular milieu, the solubilized antigen comes in contact with CD4 T cells and nave B-cells, and initiates a classical humoral immuneresponse cascade.

[3,21]. Although the lack of genetic integration may appearas a drawback for gene therapy applications, plasmids canbe maintained episomally and transcribed for extended pe-riods of time prior to being eliminated. This property makestheir use very attractive where transient trans gene expres-sion is desired such as for example, cardiac revasculariza-tion [9,10,22]. In addition, unlike live attenuated vaccines,plasmid DNA vaccines do not carry the hypothetical risk ofreversion and causing illness [1].

On a small scale, plasmids are viewed as relatively easy toproduce and purify. Plasmid DNA also offers extended sta-bility, thus presenting an invaluable advantage in vaccinatingpopulations in areas where sophisticated storage technologymay not be available. On the other hand, while this new vac-cine technology is very attractive, injection of naked DNAusually leads to a weak immune response unless relatively

large amounts are administered (up to several milligrams perdose in humans) [20].

However, plasmid production under non-optimized lab-oratory conditions invariably leads to very low (540 mg/l)volumetric titers. Additionally, the utilization of laboratory-scale purification methods at larger scales leads to pooryields. For vaccination of a potentially large population, andassuming dosages ranging from one to a few milligrams,it is readily apparent that these un-optimized processes andlow productivities are inadequate at the industrial scale. Inaddition, commercial scale processes will have to supportnot only the economics but also the regulatory standardsrequired for commercial DNA vaccine production. In par-ticular, during plasmid design and the development of aproduction process, it is crucial that the regulatory en-vironment be constantly considered. DNA vaccines are

K.J. Prather et al. / Enzyme and Microbial Technology 33 (2003) 865883 867

considered by the Food and Drug Administration (FDA)as biological products and are therefore regulated underthe well-established vaccine review process controlled bythe 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 re-spect to DNA vaccines, both the FDA and the WHO haveissued points to consider guidelines and a guideline forassuring the quality of DNA vaccines, respectively. Bothpublications specifically and extensively address the pro-duction and testing of experimental DNA vaccines [23,24].

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

In addition, several publications offer comments and adviceon the interpretation of these guidelines [2330].

The understanding, optimization, and validation ofsteps, from plasmid design and host strain selection tomass-cultivation and purification, are crucial if this novelvaccine technology is to be commercially successful. Todate, while there are not yet any commercial applicationsof DNA vaccines, several clinical trials are advancing andare accompanied by research and development activitiesoccurring in both industry and academia. The flow diagrampresented in Fig. 2 outlines the steps involved in the devel-


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

2. The choice of a replicon for DNA vaccine or genetherapy constructs

The choice of a replicon for plasmid DNA constructs isan important one, as it provides an appropriate framework inwhich production process development decisions should oc-cur. Most researchers have settled on the ColE1-type vectorsas the basis for DNA vaccine or gene therapy constructs tobe propagated in the well-studied Gram-negative bacteriumEscherichia coli [3133]. The reasons for this selection arethree-fold. First, ColE1-derivatives have been most com-monly employed as vectors useful for recombinant proteinexpression and are widely available. Second, they have suc-cessfully been modified to replicate at high copy numbers,thereby increasing the overall theoretical yield from a plas-mid DNA production process. Last, production processesand scale-up conditions have been extensively studied usingE. coli as host for recombinant protein production.

An extensive amount of literature exists detailing the se-lection and optimization of plasmid DNA vectors for re-combinant protein expression (see the following reviews[3436]). This literature can be very useful in choosing areplicon as the foundation of a DNA vaccine candidate,though the desire to produce plasmid DNA as the ultimateproduct of interest necessitates that a different set of cri-teria guide the final selection. Many possibilities exist forthis choice; however, most naturally occurring replicons ex-ist at low to moderate copy numbers (Table 1). Such lowcopy numbers were a limitation for the production of largeamounts of recombinant protein, as the gene dosage becamethe limiting factor in maximizing expression. Likewise, theuse of a low-copy number vector is undesirable for plasmidDNA vaccine production since the product yields will beunfavorably low. One solution to this potential problem canbe found in the form of the multi-copy derivatives of theColE1-type plasmid pBR322, which is derived from pMB1[37,38].

ColE1-type plasmids require only host-encoded proteinsfor replication [3941]. The necessary plasmid-based func-

Table 1Replicons commonly used in Escherichia coli

Representativeplasmid

Replicon(incompatibility group)

Copy number(copies per cell)

pML31 F plasmid (FI) 14P1 prophage P1 (Y) 14pSC101 R6 (FII) 6pMMB66 RSF1010 (P4/Q) 1213pFF1 RK2 (P1) 1015pACYC184 p15A 1822pBR322 pMB1 (ColE1) 4055

tions exist in the form of two RNA transcripts, known asRNAI and RNAII. The RNAII transcript is the pre-primerfor DNA replication. It binds to the complementary plas-mid 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 encodesfor an anti-sense strand complementary to a small regionof RNAII, and the RNAI:RNAII hybrid prevents forma-tion of the stable DNA:RNAII hybrid necessary for RNaseH cleavage and the formation of the active primer. Theinteraction between RNAI and RNAII is enhanced by aplasmid-encoded protein, called Rop or Rom (for repressorof primer and RNA one modulator, respectively). Fig. 3 de-picts these various effectors that lead to the control of plas-mid copy number.

The first elevated copy number derivatives of pBR322were found to exclude the Rop/Rom protein [4244].These plasmids (e.g. pAT153) exhibited an approximatelythree-fold improved copy number of 150 per cell overpBR322. The pUC vectors, including the still commonlyused plasmids pUC18 and pUC19 [45], were also con-structed without a functional rop gene; however, thesevectors were found to behave differently than other ropderivatives with respect to copy number. Minton [46] re-ported an increase in recombinant protein activity whenexpressed from a pUC8-derived vector that was ultimatelycorrelated to an additional 34-fold increase in plasmidcopy number (500700 copies per cell) over pAT153-likeplasmids [47]. This additional increase in copy number wasattributed to a G A point mutation in the pUC vectorsrelative to pBR322 that was not identified in the originallypublished sequence of pUC19 [48]. The point mutationmaps to the RNAII sequence, impacts binding affinity ofthe RNAI:RNAII complex, and affects copy number ina temperature-dependent manner [49]. The copy numbereffects 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 numberpBR322-derived constructs has been successfully employedto improve the yield of plasmid DNA for gene therapypurposes [31].

Because of the high copy numbers achievable with thepUC-like versions of ColE1-type plasmids, they have longbeen the replicons of choice for recombinant protein expres-sion in cases where toxicity due to high expression is not anissue. As a result, much work has been done to create a vari-ety of useful expression vectors, many of which are commer-cially available. The high copy numbers and wide availabil-ity have also made these vectors very attractive as the basisfor DNA vaccine constructs, especially since other methodsto increase yields of lower copy vectors, such as chloram-phenicol or rifampicin addition are not viable for pharma-ceutical products. Recently, a new class of high-copy plas-mids comparable to pUC and derived from the ColE1-likeand -compatible RSF1030 replicon has been described that


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

RNA II

Rop/Rom

RNA I

ori

(a)RNA II

RNA II

RNaseHcleavage

(1) degradation of DNA:RNAII hybrid to ori by RNaseH

(2) elongation by DNA polymerase

RNA Iori

(b)

(c)

ori

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:RNAIIhybrid formation, enhanced by the Rop/Rom protein (illustration adapted from [114,154]).

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

Although pUC-based plasmids are in widespread use aspotential DNA vaccines, the runaway-replication plas-mids derived from some low-copy repliconsso calledbecause of the loss of control of replication usually inducedby a temperature shiftwould also seem an attractive op-tion from a process development perspective. The loss ofreplication control results in a large accumulation of plas-mid DNA within the host cell, up to copy numbers near1000 [51,52]. Such vectors have been used successfully toachieve inducible high gene dosages for the production ofrecombinant proteins [53,54]. These vectors have causedsome concerns due to the large metabolic burden typicallyencountered with recombinant gene expression from veryhigh-copy number plasmids [5558]. However, in the casewhere plasmid DNA is the ultimate product of interestand expression of the transgene in the host cell is minimalor non-existent, concerns about transcriptional and trans-lational limitations typically associated with recombinantprotein expression are no longer relevant. In spite of thisapparent advantage of using runaway-replication vectors toproduce high titers of plasmid DNA, these authors did notlocate any examples of these vectors being used as the basisfor DNA vaccines in the literature. It is not entirely clearwhy such vectors have not been exploited; however, one

may speculate that the primary reason lies in the traditionalseparation between basic research and process developmentactivities. Proof of concept is readily obtained with smallquantities 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 controlregion to modify plasmid copy number is not possible sincethis results in the creation of a new molecule, requiringsuch 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 therapyconstruct is conceptually straightforward [32]. The plas-mid should contain elements required for maintenanceand propagation in the bacterial host and for expressionof the transgene in the human host (Fig. 4). The primaryelements required for bacterial propagation include the ori-gin of replication and any plasmid-encoded functions (e.g.RNAI/RNAII sequences in ColE1-type replicons) requiredfor replication. These are determined by the initial choiceof the replicon upon which the vector will be based. Aselectable marker should be included as well to allow for se-lection of successful transformants upon initial introduction


promoter/enhancer

selection mechanism

pathogen gene

bacterial replication elements

transciption terminator/(e.g., CMVIE, SV40,

EF-1, UbC, native)

(e.g., antibiotic resistance)(e.g., modified ColE1-type)

human -globin) polyadenylation signal

(e.g., BGH, SV40,

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

of the plasmid into the bacterial host and to create selectivepressure against a predominance of plasmid-free segre-gants arising in the population upon subsequent growth ofclones. The choices that exist for the selectable marker canbe grouped into two categories: (i) those that require bothplasmid- 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 thatcan 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 genesthat can be complemented or repressed, respectively, byplasmid-based genes at permissive temperatures. Recently,a selectable strain-plasmid system was described in whichthe host genome contained a kanamycin resistance geneunder the control of the lac operator and promoter and theplasmid contained lacO [59]. In the presence of kanamycin,the transformed strains could only grow if host-encodedLacI repressor molecules were successfully titrated byplasmid-based lacO, creating selective pressure for plasmidpresence. In each of these cases, the options for growthmedia may be limited by the nature of the marker, and theconstruction of special hosts is required, limiting the abil-ity to rapidly evaluate several strains to determine the besthost for propagation of the desired construct. Additionally,plasmid-encoded gene products may accumulate in the cyto-plasm, allowing plasmid-free segregants to continue growthfor a finite number of generations in the absence of themarker gene [35]. Most researchers have therefore settled onmarkers that only require plasmid-based sequences, the mostcommon of these conferring antibiotic resistance. As dis-cussed in the following sections, the choice of antibiotic andthe decision to include this component in the final produc-tion vessel is governed in part by regulatory considerations.

The expression elements should be designed to maximizetransient production of the transgene upon entry into thehuman host. These elements include a eukaryotic promoter,a transcription termination/polyadenylation signal, and thetransgene of interest. Promoters available for recombinant

gene expression in mammalian cells include those fromhuman cytomegalovirus/immediate-early (CMVIE), simianvirus/early (SV40), human elongation factor-1 (EF-1),and human ubiquitin C (UbC). The CMVIE promoter hasbeen shown to be more effective than both SV40 [60,61]and UbC [62], and inclusion of intron A can further improveCMVIE-directed expression [61]. A survey of commercialsuppliers of expression vectors will show that those con-taining the CMVIE promoter/enhancer are most widelyavailable, further increasing the likelihood that this pro-moter will be used for commercial development of plasmidDNA vaccines. Promoters derived from the antigen sourcemay also be used for plasmid-based expression, though thisapproach would limit the ability to construct a single plas-mid backbone for the expression of multiple antigens [63].Transcription terminators/polyadenylation signals that arecommonly used include those from bovine growth hormone(BGH), SV40, and human -globin.

In addition to the basic elements of a DNA vaccine con-struct, it may be possible to further optimize the plasmidbackbone by exploiting the immunostimulatory effects ofthe bacterial DNA. In particular, it has been observed thatbacterial CpG dinucleotide motifs can enhance the immuno-genicity of plasmid DNA vaccines [32,6466]. These dinu-cleotides occur in bacterial DNA at an expected frequencyand are generally un-methylated. Conversely, mammalianDNA contains CpG motifs at a suppressed frequency, some1020-fold lower than expected, in a largely methylated-Cform. It is believed that this difference in frequency andmethylation patterns results in an enhanced immune re-sponse following injection of plasmid DNA vaccines with abacterial backbone. The magnitude of the immune responseis also affected by the identity of the flanking dinucleotides[64]. Accordingly, it may be possible to optimize codon usein the plasmid vector to increase the occurrence of stimula-tory CpG motifs or the nature of the surrounding nucleotidesand thereby improve the natural adjuvant properties of theplasmid DNA.

During selection and construction of a plasmid vectorsuitable for vaccine usage, the regulatory environment mustbe closely considered. A major regulatory concern, poten-tial DNA integration, should be addressed during the de-sign of the plasmid. Care should be taken in the choice ofthe insert, and in avoiding homologous sequences betweenthe human genome and the plasmid DNA. Promoters andterminator regions should also be carefully selected to re-strict their biological activities on the sequences inserted onthe plasmid. When selecting an antibiotic resistance marker,one should avoid the use of beta-lactams and rather selectthe aminoglycosides neomycin or kanamycin [28]. Both ofthese aminoglycoside antibiotics are appropriate as they areseldom used in clinics and have a low incidence of adverseeffects such as ototoxicity and nephrotoxicity. In addition,all steps that lead to the construction of the plasmid mustbe well recorded, with detailed descriptions of cell lines, in-termediates manipulation, and substrates used. Finally, the


complete sequence of the plasmid must be provided priorto the initiation of phase I clinical studies. Host selectionrequires that the source of the microbial strain be character-ized and free of any adventitious agents, and that the strainbe genotypically and phenotypically well characterized. Itis also important that the methods leading to the selectionof the clone be well documented [2330].

4. Plasmid production

Economic large-scale plasmid production from E. colirequires the concomitant optimization of plasmid copy num-ber (specific yield) and of biomass concentration. Achievingelevated specific yields is particularly important as it alsopositively impacts downstream processing and ultimatelypurification yields. Fermentation methods for the produc-tion of recombinant protein by E. coli have been extensivelystudied, successfully optimized, and implemented at thecommercial scale [67,68]. It is highly probable that a sub-stantial fraction of the knowledge gained by experimentalistsduring studies directed at optimizing recombinant proteinproduction will be translatable to processes aimed at theover-production of plasmid. However, conditions leading tothe optimization of protein expression may differ somewhatfrom the circumstances necessary for achieving high plas-mid copy number. For example, it is well established that themetabolic burden of high plasmid copy number and elevatedforeign protein synthesis is far greater than that of the pres-ence of a high copy number plasmid only [69]. It is thereforelikely that achieving optimal plasmid production will requireseveral paradigm shifts to occur in the cultivation methods.

4.1. Medium formulation

The cultivation medium formulation can dramatically in-fluence the performance of microbial processes. E. coli is anon-fastidious microorganism that grows in both rich com-plex organic media as well as in salt-based chemically de-fined media providing that a source of organic carbon isprovided. Through the type and concentration of ingredientsused, cultivation medium composition directly dictates theamount of biomass produced, and is therefore likely to in-fluence plasmid volumetric yield. In addition, it is probablethat medium composition will directly bear on the physi-ology of the microorganisms by influencing their intricateregulatory systems, and therefore will control plasmid copynumber or specific yield. It is also generally understood thatplasmid copy number of ColE1-type plasmids in E. colivaries inversely with growth rate in batch culture. Lin-Chaoand Bremer [70] observed that the copy number of pBR322changes from 15 to 23 plasmids per genome as the growthrate decreases from 1.7 to 0.4 h1 as a result of growth incomplex Luria Bertani (LB) medium versus glycerol min-imal medium. These data were subsequently confirmed bydetermining both plasmid copy number and the expression

of plasmid-encoded cat as a function of growth rate in LBand minimal media [71]. A similar trend was observed withColE1-type plasmids of altered copy number [72]. This ex-periment also suggested that there was an upper limit toplasmid content per cell, as the growth rate was further sup-pressed to 0.25 h1 by adding glucose uptake inhibitors tominimal medium.

Cultivation media suitable for E. coli can be purchased ortheir formulations can be obtained from the literature. Com-positions of both complex and chemically defined media areabundantly discussed in many publications [7377]. Due tothe simplicity of use associated with off-the-shelf cultivationmedia, a number of laboratory-scale DNA vaccine produc-tion schemes rely on the use of un-optimized small-scaleprocesses employing commercially available complex for-mulations [7880]. Generally, these simple processes arebased on the growth of E. coli in either large shake flasksor small laboratory fermentors, usually employing simplemedia formulations such as Luria Bertrani (LB), or brainheart infusion (BHI). These processes yield low cell mass,ranging from 1 to 7 g dry cell weight/l, which in turn supportmodest volumetric plasmid yields (Table 2). Such processesare, however, sufficient for the production of amounts ofplasmids needed for studies employing a limited number ofsmall animals. Interestingly, the data presented in Table 2show that although employing different E. coli strains andplasmids, the specific yields achieved by several researchgroups when using LB broth are all remarkably similar, withall reaching about 46g of plasmid/mg dry cell weight.

Modification of pre-existing media, leading to a bettercontrol of the physiology of the host cells, may result inenhanced process yields. When E. coli DH5 harboring theplasmid pSV was cultivated in a semi-defined medium,a higher plasmid specific yield of 9.12g plasmid/mg drycell weight, was achieved over the standard complex LBformulation which only supported 6.0g plasmid/mg drycell weight [81]. Increasing medium strength, by eitheradding additional nutrients (LB plus glucose) or by em-ploying richer formulations (BHI, Terrific Broth), supportshigher volumetric yields without compromising specificyields (Table 2). In these specific examples, the benefits ob-served can be entirely attributed to the increase in biomasscaused by employing a richer cultivation medium. Whenmodifying a semi-defined cultivation medium, OKennedyet al. [81] demonstrated the existence of an optimum car-bon/nitrogen (C:N) ratio. They found that a C:N ratio of2.78:1 was optimal for plasmid production. Higher ratios ofup to 12:1, which are more in line with standard media for-mulations, yielded lower plasmid specific yields. Becauseno differences were observed in the growth kinetics andfinal biomass production when testing variable C:N ratiomedia, the authors speculate that the changes in C:N ratiomay have influenced the physiology of the microbes in afashion 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 30g/ml)

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

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

OD600 = 14 174 mg/4.5-l or 38.66 mg/l [79]5.52g/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 50g/ml of kanamycin) OD600 = 30 4 mg/l [95]

0.26g/mg

10-l fermentor (complex medium with 59g/ml kanamycin) NA 7.5 mg/l [31]Temperature shift 3742 C 5.1 g/l 34.1 mg/l (6.7g/mg) "Temperature shift 3745 C 4.8 g/l 49.3 mg/l (10.3g/mg) "Fed-batch (with step increase) and 3742 C shift NA 220 mg/l "

Fermentor fed-batch 55 g/l 225 mg/l (4.1g/mg) [103]Fermentor 2.5 l batch

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

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

Fermentor (7- and 80-l scale) semi definedFed-batch (manual) 1012 g/l 58 mg/l (0.7g/mg) [104]Fed-batch (DO-stat) 60 g/l 100 mg/l (1.7g/mg)

Fermentor (10-l working volume) (LB broth with 100 g/mlampicilin 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[73,8285]. Employing this approach, Wang et al. [86] re-ported the rational design of a defined medium optimized forplasmid production by E. coli strain JM109 harboring thepcDNA3S plasmid. After identifying six key amino acids(aspartate, glutamine, glycine, histidine, leucine and trypto-phan) to add to a glucose-basal salt medium, the designedformulation supported higher plasmid specific yield whencompared with those routinely achieved in the complex LBmedium (7.5 versus 5.0g 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 17.1g plasmid/mgdry cell weight.

The effects of medium composition on plasmid produc-tion are closely intertwined with those affecting plasmidsegregational stability. An important factor contributing toplasmid stability is the presence of an antibiotic, selectingfor cells containing the desired plasmid, in the cultivationmedium. Antibiotic presence in the cultivation mediumis not problematic since its clearance, or clearance of itsinactivated form, can be readily achieved during the down-

stream steps [68]. However, the mechanisms of resistanceencoded by the plasmid usually lead to either deactivationor degradation of the antibiotic. In liquid culture, selectivepressure may diminish with time as deactivation of the an-tibiotic occurs [87,88]. Therefore, a potential for plasmidloss exists due to inherent instability, even when antibi-otics are added to the cultivation medium. The recombinantprotein over-production literature, with the caveat that ex-periments were performed in the context of concomitantprotein synthesis, has reported extensively on the effectof nutrition on plasmid stability. The mechanisms of plas-mid instability observed, dimer formation, segregationalinstability, and degradation, probably remain valid [8991].Using frequent serial transfers, OKennedy et al. [81] eval-uated plasmid stability when employing several media(complex LB versus semi-defined formulations) and foundthat a semi-defined medium supported the highest plasmidstability, while the complex LB medium offered the low-est plasmid stability. Media that supported similar growthrates between plasmid-bearing and plasmid-free cells alsosupported high plasmid stability. The authors concludedthat higher plasmid instability resulted from a wider growthrate 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 twocomplex richer media, plasmid stability was higher, by upto 63%, when employing defined media. These authors alsospeculate that higher stability in the defined media may re-sult 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 suc-cessfully employed for plasmid over-production by E. coli[9294]. Batch cultivation, although logistically simple, isseverely limited with respect to achieving elevated biomass.

DNA production processes that employ simple batchcultivation methodology yield relatively low biomass, withcell mass values ranging from 1 to 8 g/l, and correlativelysupport low plasmid volumetric yields (Table 2). For ex-ample, Horn et al. [95] report reaching an optical density,measured at 600 nm (OD600) of 30 when employing a richcomplex cultivation medium (TB medium). The harvestedcells, in late log phase, yielded about 4 mg of plasmid perliter. In another example, a volumetric yield of 38 mg/l wasachieved by Diogo et al. [79] when cultivating E. coli in arich complex medium and harvesting the cells in late logphase at an OD600 of about 14. Generally, volumetric yieldsachieved in batch cultivation tend to be relatively low, withvalues ranging from 4 to 40 mg/l (Table 2). When employingrich cultivation media, the fermentor oxygen transfer ca-pacity is eventually exceeded, resulting in the creation of anoxygen-limited environment. This lack of oxygen triggersthe fermentative metabolism of E. coli, rapidly leading tothe production of toxic by-products, mostly acetic acid, thatseverely limit growth and even lead to cell death [9698].

Fed-batch technology delivers nutrients over an extendedperiod of time, thereby achieving the control of nutrientavailability to a level compatible with the oxygen transfercapacities of the bioreactor, offering a reasonable solutionthat has been amply explored. The feeding of nutrients,usually glucose, has been extensively researched, and in-corporates a range of approaches that span from simpleto very elaborate, with each presenting its own advantagesand disadvantages. Feeding regimens based on feed rateincrease, either simple or following sophisticated algo-rithms aimed at maintaining a more constant environmentthus maintaining a desired growth-rate, have been success-fully implemented. In general, these strategies lead to theaccumulation of biomass in the 60120 g dry cell/l range,with each recombinant construct presenting its own limita-tions. These limitations are probably the result of the extrametabolic burden caused by the over-expression of plasmidand recombinant product [9294].

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

growth rate by changing medium composition affects plas-mid copy number, several investigators have clearly demon-strated that plasmid content (yield) is directly affected by thedilution rate in continuous culture. Using continuous culture,Seo and Bailey [99] established that fast growing cultures(0.60.8 h1) contain less plasmid than those growing atmuch slower rates (0.30.4 h1). However, their data suggestthat maintaining lower dilution rates (below 0.2 h1) wouldresult in lower plasmid copy number. Reinikainen and Virka-jrvi [100] reported similar trends, employing a differentplasmid. These low plasmid copy numbers may be reflectiveof elevated stress caused by excessive nutritional limitations.In another study, Reinikainen et al. [101] demonstrated thatpH and temperature greatly influenced plasmid copy num-ber. The authors speculate that these environmental factors(elevated pH and reduced temperature), positively influ-enced plasmid copy number by down-regulating the growthrate. Several investigators have observed that plasmid copynumber increases during both the late exponential and earlystationary phases of growth [101,102]. This phenomenonhas been coined plasmid amplification in these publicationsand these observations also support a strong correlation be-tween growth rate and plasmid accumulation. Higher plas-mid content achieved under reduced growth rate conditionsis attributed to both higher plasmid stability (due to effectiveequivalent growth rates of plasmid-free and plasmid-bearingcells) and to privileged plasmid synthesis over other bio-chemical pathways. The following examples show that thesefindings in conjunction with fed-batch technology have beensuccessfully applied for the production of plasmid DNA.

Schmidt et al. [103] used a feeding strategy that main-tained a slow growth rate (about 0.15 h1) for the most partof the cultivation (1030 h). Plasmid concentration contin-ued to increase during the first 5 h of stationary phase afterwhich it reached a plateau. This strategy allowed the authorsto achieve a biomass of about 50 g/l of dry cell weight anda final plasmid yield of about 225 mg/l.

Chen et al. [104] report the use of several fed-batch strate-gies, employing a semi-defined medium, used for the culti-vation of E coli DH10B carrying a plasmid coding for HIVpEnv. When glucose was fed, employing a predeterminedfeeding algorithm designed to match consumption rate, ahigh specific growth rate of about 0.7 h1 was maintainedfor about 8 h. However, during the course of experiment,the feed rate of glucose exceeded the anticipated consump-tion rate and caused glucose accumulation and oxygen lim-itation which triggered the accumulation of acetic acid. Alimited biomass of about 1012 g/l was achieved and plas-mid volumetric yield reached a value of 810 mg/l. To avoidthe accumulation of glucose, a feeding strategy based onfeed-back control using the input from the pH and DO con-trol loops was designed. The nutrient solution, a mixture ofglucose and yeast extract, was fed when either the DO roseabove 50% (indicative of a lack of nutrient) or the pH roseabove 7.2 (caused by the consumption of alternative carbonsources such as fatty acids). Both conditions were believed


to accurately reflect a lack of glucose in the medium. Thisfed-batch strategy was initiated after about 8 h of cultivation.During the initial batch portion a growth rate of 0.69 h1 wasachieved while a lower specific growth rate of 0.13 h1 wasmaintained during the fed-batch portion. Glucose accumu-lation was never observed, and final biomass concentrationsranging from 70 to 100 g dry cell/l were achieved. Plasmidvolumetric and specific yields were improved over those ob-tained with the glucose fed-batch strategy (7090 mg plas-mid/l versus 810 mg plasmid/l and 1.7g plasmid/mg drycell weight versus 0.7g plasmid/mg dry cell weight, re-spectively). The authors clearly attribute the specific yieldimprovements to achieving a slower growth rate, conduciveto plasmid amplification and higher stability, during the sec-ond portion of the fermentation process. The scale-up of thisstrategy was demonstrated from 7 to 80 l.

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

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

a semi-defined medium. When the temperature was shiftedin mid-log phase (OD of 12) from 37 C to either 42 or45 C, plasmid volumetric yields of 34 and 49 mg/l wererespectively achieved. These volumetric yields comparedvery favorably with the 7.5 mg/l achieved without temper-ature shift. A fed-batch approach employing the feeding ofa mixed nutrient solution (glucose, yeast extract, MgSO4,and l-leucine) coupled with a pre-determined program thatkept the culture growing with a specific growth rate of0.25 h1, was implemented in conjunction with the tempera-ture shift strategy. At mid log phase (OD 56) the temperaturewas shifted to 45 C and the batch continued until the ODvalue dropped. A volumetric plasmid yield of 220 mg/l wasachieved under these conditions. Close examination of thedata show that the specific yield was comparable betweenthe fed-batch and batch cultivation.

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


Rapid Growth

Time

Slow Growth

Fed-Batch Cultivation

Bio

mas

s (

)

Plas

mid

DN

A (

)

Fig. 6. Representative plot of cell growth and plasmid accumulation in a typical fed-batch process. Commonly used units for biomass are either opticaldensity 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). Thecultivation of the microbial strain should be performed un-der controlled conditions as to reproducibly yield a productof high quality [2330]. It should be well understood thatwhile it is acceptable to use an antibiotic for the selection ofplasmid-bearing cells, the antibiotic should not be used tomaintain the culture axenic in replacement of proper steriletechniques [106].

Medium composition and cultivation conditions play animportant role by controlling plasmid copy number, stabilityand the amount of biomass produced. Important intertwin-ing inputs that lead to an efficient plasmid DNA productionprocess are depicted in Fig. 5. It is apparent from the exam-ples reviewed here that media formulations specifically de-signed for the over-production of plasmid DNA can supporthigher yields and increase plasmid stability. While perhapsmore difficult and time consuming to initially complete, thedesign and development of an optimized cultivation mediumcan provide an extensive pay-off in the long-term. Coupledwith the powerful tool of statistical experimental design,the design of chemically defined cultivation medium willlikely yield highly optimized media formulations [107,108].Chemically defined formulations offer the possibility to per-form extensive analytical investigations, which in turn cansupport metabolic studies and quality control purposes. Inaddition, the use of chemically defined media eliminatesmost of the uncertainty facing the origin and composition

of the raw complex ingredients. In the context of develop-ing processes for the commercial production of vaccines, theuse of chemically defined cultivation media will also helpin achieving a better position with respect to the regulatoryenvironment by supporting safety and reproducibility claims[109]. Although the available information is limited in com-parison with the extensive recombinant protein productionliterature, some general rules and methodologies pertainingto the production of plasmid DNA by cultivation of E. coliare beginning to emerge. As anticipated, while many of theconditions that lead to elevated recombinant protein produc-tion can be directly translated, several modifications are nec-essary in order to achieve maximum yields. Productive plas-mid DNA processes tend to employ a two-phase strategy.Namely, a phase of biomass buildup where the cells growexponentially followed by a phase of slow growth, achievedvia fed-batch technology, where plasmid amplification takesplace (Fig. 6). Additional studies in this field should revolvearound the optimization of these steps and will bring muchneeded additional information.

5. Plasmid DNA purification

When a molecular biologist thinks of large-scale plas-mid DNA production, the range of 10100 mg of DNAusually comes to mind. However, at a pharmaceutical


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

production-scale, plasmid DNA requirements may exceed50 g per batch. In extreme cases, many kilograms of plasmidDNA per year will be needed to fill the ultimate market-ing demand for DNA vaccines currently in clinical trials.The following section is to be considered in conjunctionwith several reviews recently published [110112]. Here,we specifically emphasize the practicality and potentialscale-up issues facing existing plasmid DNA purificationtechnologies.

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

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

Historically, plasmid DNA purification is accomplishedthrough the use of cesium chloride/ethidium bromide(CsCl/EtBr) buoyant density gradient separation [113,114].This method allows the separation of plasmid DNA bybuoyant density into purified bands of different forms (e.g.supercoiled, open circular, linear, multimeric) when theCsCl/EtBr/plasmid DNA mixture is subjected to ultracen-trifugation. The covalently closed plasmid DNA band isthen withdrawn from the ultracentrifuge bottle(s) and fur-ther extracted and alcohol precipitated to remove both CsCland EtBr. While it yields highly purified plasmid, this ap-proach is not scaleable because of personnel safety issuesand the hazardous waste considerations associated with theuse of cesium chloride and ethidium bromide. In addition,the use of ultracentrifugation is also a major impedimentto the scale-up of this technology. However, this procedureis still considered the purity benchmark against which allother plasmid DNA purification techniques are evaluated.

Current bench-scale purification methods employ kitsmarketed by commercial companies that take advantage ofseveral of the techniques reviewed below, including alkalinelysis and the use of disposable chromatography columns.However, these purification conditions do not lend them-selves to scale up because they may employ hazardouschemicals, such as phenol and chloroform, require the useof large amounts of chromatographic resin per milligram ofplasmid purified, or necessitate the use of relatively expen-sive enzymes in order to degrade contaminants (e.g. RNaseand protease K). Finally, the use of fairly dilute solutionswould lead to extremely large process equipment on the>50 g plasmid DNA production scale and would translateinto considerable capital investment. Due to their simplic-ity, these purification methods are however the method ofchoice for the purification of small amounts of plasmidDNA (


5.2. Cell lysis

Most current bacterial cell lysis techniques (homogeniza-tion, freeze/thaw, detergent-based extraction, etc.) have beenoptimized for the purification of recombinant proteins andmost are not applicable to plasmid DNA purification. Sincelarge-scale plasmid DNA production is in its infancy, cur-rently only a handful of lysis techniques are used. By far, themost common technique used in the production of plasmidDNA is that of alkaline lysis [117]. This procedure relies onthe use of sodium hydroxide and the detergent sodium dode-cyl sulfate (SDS) whose combined effects result in the dis-ruption of the cell membrane, leading to lysis and release ofthe intracellular components. Subsequent neutralization withsodium acetate precipitates protein and genomic DNA. Uponneutralization, supercoiled plasmid DNA anneals from itspH-denatured state while large molecular weight (200 kb)genomic DNA cannot diffuse and anneal properly, resultingin single-stranded genomic DNA precipitation. In addition,at high pH, RNA degrades, which benefits downstream sep-aration steps. However, this lysis is rather delicate as plas-mid DNA can be degraded at high pH if high shear con-ditions are produced during this procedure [118]. Since theDNA does denature during this lysis, some of the super-coiled DNA is converted to alternative forms (e.g. denaturedsupercoiled, multimeric, open circle, and linear). Also, alka-line lysis can cause fragmentation of genomic DNA, whichcan make downstream removal more difficult.

Another method of lysis used in the production of plas-mid DNA is that of boiling cell lysis first introduced byHolmes and Quigley [119]. This lysis method utilizes alysozyme digestion to break down the peptidoglycan in cellwalls (through hydrolysis of glycosidic linkages) followedby heating to the point that the membranes disassociate,leading to the release of plasmid DNA from its cytosoliclocation. In addition, undesirable DNAses are inactivatedand proteins are denatured and precipitated during this heatstep. Scale-up issues with heat lysis include heat exchangerdesign and cleaning. The prime scale-up example of this ly-sis technique utilizes a continuous heat exchanger with rapidheating and cooling [120]. This lysis has been carried out atthe multiple-gram scale and is highly scaleable.

In addition, lysozyme alone can be used to lyse cells inthe presence of a detergent (e.g. Triton X-100) [114]. How-ever, it should be noted that lysozyme is traditionally pro-duced from hen-egg whites and is not a desirable raw mate-rial in the cGMP production of plasmid DNA due to the riskof contamination from this avian-derived material. An alter-native to hen egg lysozyme, although slightly more costly,has been the use of a recombinant lysozyme, purified fromE. coli [121,122]. Scale-up challenges include minimizingthe use of relatively high cost lysozyme; however, the ly-sis is very simple and can be done in a single process tankwith very high plasmid DNA recoveries. A major advantageof a lysozyme-based lysis, beside simplicity, is that prod-uct 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 pro-cedure is that lysozyme lysis releases cellular contents anddoes not aid in purification.

The use of mechanical lysis is a fairly new developmentin the field of nucleic acid separation. A common problem,and an advantage of mechanical lysis for protein purifica-tion, is that high shear degrades nucleic acids [123]. It hasbeen shown that relatively low shear induced by a bead millcan release plasmid DNA from E. coli while keeping it upto 90% intact [124]. Currently, the only published work uti-lizing mechanical lysis in plasmid DNA purification is ata relatively small scale; however, mechanical lysis is usedin large-scale protein manufacture and equipment is readilyavailable.

5.3. Affinity precipitation of plasmid DNA

Most large-scale production processes utilize an upstreamaffinity precipitation of plasmid DNA. Alcohols, namelyethanol and isopropanol, are well-known DNA precipitationagents. In addition, polyethylene glycol [125], cationic de-tergents, for example CTAB, [122,126], certain salts such asLiCl and CaCl2 [127], polyamines such as spermidine [128],and polyethylene imine [129], are used to remove the ma-jority of contaminants in a large-scale preparation withoutresorting to costly chromatographic separations.

A practical example of affinity precipitation is the useof CTAB. When employing this technology, plasmid DNAyields are normally greater than 90% and this precipitationmethod has been used on the multiple gram scale by Landeret al. [122]. An additional example of affinity precipitationin practice is the use of polyamines, specifically spermidine.Again yields are commonly greater than 90% and Murphyet al. [128] have applied this purification strategy at thegram scale.

In addition, DNA-binding ligands (triplex interaction)affixed to temperature sensitive polymers have been usedin the purification of plasmid DNA. These polymers aresoluble at low temperature but are insoluble at higher temper-atures resulting in a temperature controllable affinity precip-itation agent [130]. Most recently, the company Dr. MullerAG has applied this approach and is marketing a large-scaleplasmid DNA separation strategy based on triplex affinityligands attached to a stimulus responsive polymer.

Scaleability issues with affinity precipitations mainly in-volve proper mixing, separation of the precipitants, washingof the precipitants, and the method of addition of the affin-ity precipitation agent. Temperature, pH, degree of mixing,and the mixing time can affect the precipitant particle sizeand the extent of any precipitation. The mixing and methodof addition of the affinity precipitation agent are also im-portant because high local concentration of affinity agentscan cause coprecipitation of impurities. Additionally, thequantity of residual liquid remaining prior to re-suspensioncan greatly affect the levels of impurities.


5.4. Solidliquid separation in plasmid DNA production

The separation of solids from liquids is a necessity in theproduction of plasmid DNA. With plasmid DNA doses onthe order of milligrams per dose and the solubility limit ofcrude DNA-containing lysates at 12 mg/ml, extremely largesolution volumes with moderate levels of solids volume needto be processed. The most economical means of clearingsolids is by a centrifugation-based separation; however, thismethod of solidliquid separation cannot produce a highlyclarified solution without substantial residence times, partlydue to the high viscosity of plasmid DNA in solution. Largeresidence times require either extremely large centrifugesor long processing times, neither of which is optimal in amanufacturing setting. In addition, cleaning concerns withcurrent industrial centrifuges have been reported [131].

The most attractive filtration-based solidliquid separa-tion for plasmid DNA large-scale processing is that of abody feed filtration (e.g. addition of a filter aid such as di-atomaceous earth (DE)). This cake-building filtration de-creases the amount of filter fouling as the filtration surface iscontinuously regenerated as filter aid is deposited, allowingfor the removal of the large amounts of cell waste resultingfrom E. coli lysis. In addition, DE is known to bind RNA[132], aiding downstream purification steps. However, a ma-jor 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-con-taining alkaline lysates has been demonstrated as a feasi-ble purification unit operation. TFF can be accomplished forbuffer exchange and small ion removal. However, at a highloading, large bio-molecules cannot be adequately removedduring TFF without use of expensive nucleases, long termexposure to high pH, and costly proteases due to the forma-tion of a DNA gel layer [133]. In addition, upon scale up,the elevated liquid flow rates achieved necessitate the use ofhigh shear pumps that can degrade the shear-sensitive plas-mid DNA and the necessary membrane area can becomeexcessive.

5.5. Adsorbent-based plasmid DNA separation

Chromatography, a hallmark of bioseparation, turns outto be a very tedious step in the purification of plasmid DNA.Common chromatography resins used in the separation ofnucleic acids are anion-exchange (e.g. functional groupsof quaternary amine (Q) and diethyl aminoethyl (DEAE)),reverse phase, hydrophobic interaction, hydroxyapatite, sizeexclusion, and immobilized metal affinity chromatography[78,95,134,135]. Most chromatographic separation mediacurrently produced are optimized for the production of pro-teins. The average pore size of common chromatographicsupports is smaller than or approximately the same size asthe radius of gyration of a plasmid DNA molecule [136].

Thus, plasmid DNA cannot access pores in standard chro-matographic media and can only bind to the outer surfaceof the particles; hence, only approximately 0.22 g plas-mid DNA bind per liter of resin in contrast to proteinswhere the loading can range from 10 to 100 g per liter ofresin. This means a substantial amount of chromatographymedia is needed in a large-scale plasmid DNA separation(5002000 l of resin/kg plasmid DNA processed). Sagaret al. [121] goes into further detail about the application ofchromatographic techniques to the purification of plasmidDNA.

Immobilized Metal Affinity Chromatography has recentlybeen applied to plasmid DNA purification. For example,an iminodoacetic acid (IDA) resin charged with Cu(II) wasfound to bind free purine bases in solution. Thus, damagedDNA and RNA bind to an IMAC resin in flow-through modewhile plasmid DNA is not retained, thereby allowing down-stream polishing to handle >100 g of plasmid DNA per literof IMAC resin (for a feed with 2% (w/w) RNA contamina-tion) [135]. IMAC has only been applied to plasmid DNA atthe 10 mg-scale to this point; however, due to the scaleabil-ity of flow-through chromatography (no gradients or steps),scale-up issues should be minimal.

Perfusion chromatography offers higher flow rates andmoderately higher binding capacities of plasmid DNA [121].The typical perfusion media for example has 4000 con-vective pores along with the standard 1001000 poresof a typical media. The convective flow through the media(typically 5% of the overall fluid flow) decreases pressuredrop and the larger convective pores can allow some plas-mid DNA to enter the chromatography media, increasingplasmid DNA-binding capacity. In addition, linear velocitiesof 10005000 cm/h are achievable using a perfusion chro-matography media (as opposed to 50400 cm/h for conven-tional chromatography media).

Tentacle chromatographic supports have also been usedin the large-scale production of plasmid DNA. These resinscontain long poly-electrolyte chains with large linkers thatextend from the surface of the chromatographic resin. Thisincreases the number of charged sites on the resin, and pos-sibly the loading capacity. To this point, however, there havebeen only limited publications detailing the use of tentacleresins for plasmid DNA purification [137].

Studies have been completed using additives to in-crease the loading of plasmid DNA on columns by re-ducing the effective size of plasmid DNA molecules insolution [121,138]. However, even with condensed par-ticles, the binding of DNA to chromatography media isquite low (typically less than 5 mg plasmid DNA/ml ofresin).

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


lowing for plates of separation to still exist. Also, this typeof chromatographic separation allows lysates to be purifiedwith limited particulates and allows for linear velocities inexpanded bed mode of 300 cm/h. The major drawbackof expanded bed adsorbents for plasmid DNA purificationis 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 particlesizes, loading capacities are typically much lower that thatof classic packed bed chromatography columns. To com-bat this inherent problem with expanded bed adsorbents,prototype adsorbents with smaller particle sizes and chro-matographic ligands based on polyethylene imine (that actas a tentacle ligand) have been utilized with some success[141].

With the inherent issues with chromatographic purifica-tion of plasmid DNA, other forms of adsorptive separationhave been attempted. To this end, batch adsorption of pro-cess contaminants can be performed as opposed to columnchromatography, when a low cost adsorbent is utilized. Oneexample of this approach was demonstrated with the use ofa hydrated calcium silicate-based batch adsorption of DNAand other contaminants [142]. In this approach hydrated cal-cium silicate is used to bind genomic DNA, open circleplasmid, endotoxin, proteins, and residual detergents leav-ing supercoiled plasmid in solution.

Adsorptive membrane separations also show somepromise 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 ad-dition, the adsorption of E. coli genomic DNA on nitrocel-lulose based filters has been discussed but the low loadingcapacity of genomic DNA on nitrocellulose makes themimpractical at the large scale [144].

5.6. Buffer exchange/concentration

Membrane-based TFF can be used for buffer exchangeand small ion removal; however, at a high loading, largebiomolecules cannot be permeated due to the formation ofa DNA gel layer. Possible scale-up issues facing this tech-nology include both the requirements for large membranesurface area and high fluid flow rates in order to keep mem-branes from fouling prematurely.

Buffer exchange and concentration can also be accom-plished by using an alcohol-based precipitation. This allowsfor 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 90%

Percentage E. coli genomic DNA (qPCR)


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