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The direct application of genes as vaccines and formedical therapy shows great promise1,2. Havingreviewed the potential of DNA vaccines, the

American Academy of Microbiology concluded thatrecent results obtained in animals indicate that thisnew technology might revolutionize the vaccinationof humans3 (http://www.asmusa.org/acasrc/pdfs/dnareprt.pdf ). Further, there are currently greater than390 active clinical trials of gene therapy worldwide,involving approximately 3500 patients (http://www.wiley.co.uk/genmed/clinical). The main options forgene therapy are to use disabled viruses or plasmid-based genes complexed with agents such as lipids orpolypeptides, although each system has its advantagesand disadvantages4. This article will focus on plasmidDNA, which has potential for both vaccines andmedical therapy.

At present, the size of the plasmid DNA being usedin clinical tr ials is at the lower end of the possible range,typically10 kb. However, there are strong indicationsthat the size of the plasmid DNA used will grow. Thus,for DNA vaccines, their effectiveness might be increasedby, for example, incorporating genes for signalling mol-ecules (eg. cytokines) into antigen-carrying plasmids5.For gene therapy using either plasmid DNA or disabledviruses, there is increasing concern that regulation ofthe functioning of the gene is ensured once it is in place.This, together with the expectation of the progressionof gene therapy to metabolic and other multigene dis-eases6,7, will lead to an increase in vector size. Althoughmultiple plasmids represent an alternative method insome cases, their separate processing and blending willnot be easy. Artificial chromosomes are beginning toexcite interest as a method of achieving reliable long-term action8,9, and these chromosomes will range upto mega-base size.

In the laboratory, plasmid DNA is typically isolatedfrom a suitable recombinant Escherichia coli strain.Recovery usually begins with a chemical lysis step using

106 Heichal-Segal, O. et al. (1995) Immobilization in alginate-silicate

solgel matrix protects beta-glucosidase against thermal and chemi-

cal denaturation: enzyme stabilization for use in e.g. wine aroma

improvement. Biotechnology 13, 798800

107 Shabat, D. et al. (1997) An efficient solgel reactor for antibody-

catalysed transformations. Chem. Mater. 9, 22582260

108 Hsu, A.F. et al. (1999) Immobilized lipoxygenase in a packed-bed

column bioreactor: continuous oxygenation of linolenic acid.

Biotechnol. Appl. Biochem. 30, 245250109 Wu, S. et al. (1994) Oxidation of dibenzothiophene catalysed by

heme-containing enzymes encapsulated in solgel glass: a new

form of biocatalysts.Appl. Biochem. Biotechnol. 47, 1120

110 Obert, R. and Dave, B.C. (1999) Enzymatic conversion of

carbon dioxide to methanol: enhanced methanol production in

silica solgel matrices.J. Am. Chem. Soc. 121, 1219212193

111 Kaufmann, C.G. and Mandelbaum, R.T. (1996) Entrapment of atra-

zinechlorohydrolase in solgel glass matrix.J. Biotechnol.51, 219225

112 Rietti-Shati, M. et al. (1996) Atrazine degradation by Pseudomonas

strain ADP entrapped in solgel glass.J. Solgel Sci. Technol. 7, 7779

113 Inama, L. et al. (1993) Entrapment of viable microorganisms by

silicon dioxide solgel layers on glass surfaces: trapping, catalytic

performance and immobilization durability of Saccharomyces

cerevisiae.J. Biotechnol. 30, 197210

114 Armon, R. et al. (1996) Denitrification by a mixture of bacterial

strains derived from an upflow sludge blanket reactor, following

entrapment in solgel glass.J. Biotechnol. 51, 279285

115 Campostrini, R. et al. (1996) Immobilization of plant cells in hybrid

solgel materials.J. Solgel Sci. Technol. 7, 8797

116 Carturan, G. et al. (1999) Production of valuable drugs from plant

cells immobilized by hybrid solgel SiO2.J. Solgel Sci. Technol.13, 273276

117 Reetz, M.T. (1997) Entrapment of biocatalysts in hydrophobic

solgel materials for use in organic chemistry. Adv. Mater. 9,

943954

118 Pope, E.J.A. et al. (1995) Encapsulation of living tissue cells in an

organosilicon.J. Solgel Sci. Technol. 55, 3349

119 McFarland, E.W. and Weinberg, W.H. (1999) Combinatorial

approaches to materials discovery. Trends Biotechnol. 17, 107115

120 Wang, J. et al. (1998) Self-assembled silica gel networks. J. Am.

Chem. Soc. 120, 58525853

121 Xia, Y.N. and Whitesides, G.M. (1998) Soft lithography.Angew.

Chem., Int. Ed. Engl. 37, 550575

296 0167-7799/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01446-3 TIBTECH JULY 2000 (Vol. 18)

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Biochemical engineering approaches to thechallenges of producing pure plasmid DNA

M. Susana Levy, Ronan D. OKennedy, Parviz Ayazi-Shamlou and Peter Dunnill

Plasmid-based genes offer promise for a new generation of vaccines and for gene therapy, but the size and character of plas-

mids pose new challenges to biochemical engineers. By acknowledging these and using bioprocess-design information based

on fundamental studies of the systems properties, it will be possible to create efficient and consistent processes for these

materials. This review addresses the purity required, the key issue of the sensitivity of the chromosomal DNA contaminant

and larger plasmids to hydrodynamic forces, and the impact of this and other characteristics of plasmids on the recovery

and purification of DNA for pharmaceutical purposes.

M.S. Levy, R.D. OKennedy, P. Ayazi-Shamlou ([email protected])

and P. Dunnill are at The Advanced Centre for Biochemical Engineering,Department of Biochemical Engineering, University College London,London, UK WC1E 7JE.


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TIBTECH JULY 2000 (Vol. 18) 297

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an alkaline solution of, for example, sodium dodecylsulphate (SDS) followed by neutralization with an agentsuch as concentrated potassium acetate10. Although thealkaline SDS mixture causes irreversible denaturationof the chromosomal DNA and protein, providing thepH value is not too high, denaturation is reversible forthe plasmid DNA; the optimum pH value varies with

the nature of the plasmid11. Plasmids are therefore sol-uble in the neutralized solution whereas most chromo-somal DNA and cellular proteins form an insolubleprecipitate (a floc). In the laboratory, centrifugationremoves the floc before stages that commonly usereagents, such as flammable alcohols, which are unsuit-able on a large scale. Similarly, the use of caesium-chlo-ride gradients for purification is not easy to scale up,although chromatographic columns offer an alternative.

The quantity of plasmid DNA required will differbetween therapies, and each disease is likely to havespecific needs. However, there are some general indi-cations suggesting that the dose will range from micro-grams to milligrams12. International regulatory agen-

cies are likely to set stringent specifications with regardto the level of endotoxins, RNA, protein, bacterial hostDNA, quantities of linear and supercoiled DNA in thepreparation and presence of toxic chemicals13. Con-taminant levels will have to comply with the maximumallowable level per administered dose and thereforespecifications are likely to be strongly dependent on thedose given. For example, release specifications for bulkplasmid DNA used in a cancer clinical trial14 were:RNA, non-visualized on a 0.8% agarose gel; E. coliDNA,0.01 g mg1 plasmid DNA; protein,1 gmg1 plasmid DNA; endotoxin,0.1 endotoxin units(EU) g1 plasmid DNA and circular DNA, 95%.For process monitoring, quality control and validation,

it is of major importance to develop sensitive and quan-titative analytical methods. Examples of such methodsinclude recent studies by Lahijani et al.15 on the sensi-tive quantitation of chromosomal DNA contamination,by Noites et al.16 on the rapid quantitation of plasmidDNA in solution and by Levy et al.17 on the rapidmonitoring of supercoiled DNA content in solution.This review does not set out to cover all aspects of manu-facture, but the removal and testing of contaminantssuch as endotoxins is certainly critical.

Central biochemical engineering issuesAs Table 1 indicates, the apparent range of operations

available from the literature (most concerned with lab-oratory-scale studies) is large. However, the particularproperties of plasmid DNA and its contaminants canset limits on the type of operation that is appropriate.The key feature of plasmids is their size and shape(Fig. 1); because each amino acid of a protein is codedby three larger nucleotides, the plasmid gene requiredto specify a given protein is much larger (relatively). Asindicated, it is likely that the size of plasmids to beprocessed will increase and a size of 50 kb might notbe unusual. In addition, the purification of plasmidDNA involves the removal of large quantities ofchromosomal DNA, which has a much larger size,typically 40004500 kb for E. coli.

The size of these key components means that theirsusceptibility to fluid mechanical forces during process-ing is probably the most critical issue in the large-scale

preparation of plasmid DNA. For proteins, after muchinitial concern18, there was clear evidence that they arenot easily damaged by shear fields usually encounteredin process equipment19. By contrast, the high mixingfields associated with such shear fields are capable ofcausing severe damage if they bring a protein into con-tact with a gasliquid interface20. The lack of directshear-induced damage is explicable for proteins interms of their size and shape. Because globular proteins

typically have an equivalent sphere diameter of 310nm (Ref. 21), they are situated within the individualfluid packages defined by the Kolmogorov microscaleof turbulence, which are unaffected by the geometryof the external source providing the motion22. The sizeand particularly the extended shape of plasmids (Fig. 1),with an average hydrodynamic diameter of 150250 nm

Table 1. Potential unit operations and process options for thedownstream recovery and purification of plasmid DNA

Unit operation Process options

Lysis ChemicalThermalMechanical

Solids removal CentrifugationFiltrationMembrane separationFlotation

Intermediate purification Fractional precipitationMembrane fractionationAdsorption

High-resolution purification Chromatography:Ion-exchangeReversed phaseGel filtrationAffinity

Finishing DryingFormulationVialing

Figure 1

Visualization of supercoiled plasmid DNA with atomic force microscopy: (a) a 6 kb

plasmid deposited onto mica functionalized with 3-aminopropyltriethoxy silane

(APTES, AP-mica) from TE buffer (20 mM Tris HCl, pH 7.6, 1 mM EDTA) with 100 mM

NaCl; (b) a high-resolution image of one molecule obtained by rescanning over a

500 500 nm area. (Images kindly provided by Dr Y. Lyubchenko.)

trends in Biotechnology

0 1.24 M0 500 nm

(a) (b)


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for 510 kb plasmids23 and potentially 1 m forlarger plasmids, means that this complete protection isnot available. For chromosomal DNA, compaction inthe native state can be protective but bacterial material,especially that denatured in the chemical lysis procedure

for plasmid DNA isolation, will be acutely sensitive tofluid mechanical force. Any significant degradation willreduce chromosomal DNA to the same order of magni-tude as plasmid DNA, making separation very difficult.

The danger of shear-induced degradation of chromo-somal DNA is more serious in process terms becausethe large size of both plasmid and degraded chromo-

somal DNA makes chromatographic procedures rela-tively inefficient. Conventional macromolecular, chro-matographic media were developed for the separationof proteins that are both smaller and more compact.This limitation in capacity when using high-resolutionchromatographic separation means that every gain thatcan be made upstream in the process is especially critical.

Characterizing the effect of shear andassociated factors

In principle, all the operations ranging from cell lysisto vialing can be influenced by shear effects; a funda-mental understanding of the extent of these effects istherefore essential. Shear forces can convert the desir-

able supercoiled, circular form of the plasmid DNAinto undesirable forms, such as open circular and lin-ear DNA. Sensitivity to shear forces has been found tobe critically dependent on plasmid size and the ionicstrength of the environment. Levy et al.24 have shownthat plasmids20 kb are sensitive to shear rates greaterthan 1 106 s1 (Fig. 2), a level that can be encoun-tered in industrial-bioprocess equipment. Studies inves-tigating increasing shear rate indicate that there is acritical value, above which damage is severe. A first-order-reaction kinetic model describes the decrease inthe concentration of supercoiled plasmid content as afunction of the duration of exposure to shear. Figure 2(inset) indicates that it is possible from the model to

predict the shear sensitivity of much larger plasmids.Airliquid interfaces, often present in large-scale equip-ment, in combination with shear have been shown toincrease damage to purified plasmids resuspended inlow-ionic-strength buffer (Fig. 3). Material in a high-ionic-strength environment, characteristic of an earlystage of the process, is not as sensitive to these effects.The impact of physicochemical conditions on productstability parallels findings with proteins wherein, forexample, fermentation media can protect secretedproteins in the sheared and aerated environment25.

Lysis and the removal of solidsCell lysis is the operation in which shear effects are

perhaps the most critical. It is complex in terms of bio-chemical engineering; rapid mixing of the alkalinedetergent and cells is essential, but once the chromo-somal DNA is exposed, it is acutely sensitive tomechanical damage. Before a large-scale operation isdesigned, basic information on rheology, mixing andthe effects of shear must be obtained. The rheology ofalkaline lysis has been studied using a co-axial cylinderrheometer26; this was used to continuously recordchanges in viscosity during the lysis of two strains ofplasmid-bearing Escherichia coli. Figure 4 shows a typi-cal viscositytime profile during the lysis of a cell sus-pension containing a 76 kb plasmid26. The apparentviscosity increased rapidly for 30s following the addi-tion of a NaOH/SDS solution to the cell suspension.The change in viscosity reached a plateau after ~25s

298 TIBTECH JULY 2000 (Vol. 18)

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Figure 2

Supercoiled plasmid-DNA content as a function of exposure to shear and plasmid size.

Clarified lysates containing plasmids of 13 kb (solid triangle), 20 kb (solid square) or

29 kb (solid circle) were subjected to shear rates of 1 106 s1 using a rotating diskdevice operated at 27 700 rpm. Experimental conditions were as described in Ref. 24.

The curves in the inset are the predictions based on a first-order-kinetic model of dam-

age to plasmid DNA; the data points in the inset were obtained by the authors for

clarified lysates containing plasmids of 76 kb (inverted solid triangle) or 89 kb (solid

diamond). Abbreviation: SC, supercoiled circular.


0.0 0.5 1.0 1.5 2.0

Time of exposure to shear (sec)

89 kb76 kb 50 kb

40 kb

SCDNA(%)

0 5 10 15 20

Superco

ile

dplasm

idDNA(%)

Time of exposure to shear (sec)

20 kb

29 kb

13 kb

76 kb

89 kb

0

20

40

60

80

100

0

20

40

60

80

100

Figure 3

Supercoiled plasmid-DNA content after shearing in the absence or presence of airliquid interfaces for a 20 kb plasmid under different DNA concentration and ionic-

strength conditions: (1) clarified alkaline lysate (20 g ml1); (2) ultrapure plasmid in

TE buffer (20 g ml1); (3) ultrapure plasmid in TE buffer (5 g ml1); (4) ultrapure

plasmid in TE buffer (0.2 g ml1); (5) ultrapure plasmid in 150 mM NaCl, TE buffer

(0.2 g ml1). The solutions were stirred for 5s at 27 700 rpm in the absence of

airliquid interfaces (filled bar) or at 26 650 rpm in the presence of airliquid interfaces

(open bar). Data shown are the average of two independent experiments, error bars

reflect standard deviation. Experimental conditions were as described in Ref. 24.

Abbreviations: TE buffer, 10 mM TrisCl, pH 8, 1 mM EDTA. (Adapted from Ref. 24.)


1 2 3 4 5

Superco

ile

dp

lasm

idDNA(%)

Experimental condition

0

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before rising again to a peak value between 80120s.Finally, the apparent viscosity decreased to a steadyvalue after ~200400s. The absolute magnitude of theapparent viscosities at various points during the lysisoperation was shear-dependent. However, the shape ofthe viscositytime profiles and the position of thevarious peaks on these profiles were unaffected by shear

rate.Before neutralization, measurements revealed that

the cell suspension and the NaOH/SDS solution wereboth Newtonian with viscosities comparable to water.However, the reaction between the cell suspension andthe NaOH/SDS solution produced a liquor exhibitingstrong non-Newtonian pseudoplastic properties with aflow-behaviour index of approximately 0.3 at shearrates 370 s1. The development of non-Newtonianbehaviour during lysis will have major consequenceson the mixing pattern in the reactor, thus affecting thequality of the product27. The cell-lysis reaction wassubsequently stopped at predetermined times by therapid addition of the neutralizing agent, and total cell

counts were carried out to determine the number ofintact cells present in the lysate. Figure 4 (inset) showstotal counts obtained from the initial phase of the lysisreaction. This indicates that the time taken to reach theinitial transient peak is necessary in order to solubilizethe cell-wall material sufficiently to start the release ofintracellular contents.

Alternative methods of lysis have also been examined.The use of mechanical disruption equipment was asso-ciated with substantial damage to plasmid DNA28. Aninteresting approach29 used a continuous-flow heatstep, typically at a temperature of 7077C and with aresidence time of ~35s. In another procedure, a staticmixer was used30 to carry out both the alkaline cell lysis

and the neutralization steps. Pressure drops acrossstatic mixers are significantly larger than those across anempty pipe of equal diameter. Because shear damage isdirectly related to pressure drop, the flocculatedchromosomal DNA will be likely to suffer significantdamage owing to the shear generated by flow over thestatic mixers. The extent of such damage will dependon the pressure drop which, in turn, is determined byflow rate and mixer design. In studies aimed at assess-ing the shear susceptibility of the flocculated material,samples of flocculated material from the neutralizationstep were subjected to a small-amplitude sinusoidalstrain of fixed frequency in a rheometer with a cone-and-plate attachment27,31. The measurements wereobtained using a flat plate and a convex cone with a

shallow angle of 4. A small quantity of the flocculatedmaterial was placed on the bottom plate and the conewas positioned such that its apex touched the centre ofthe plate thus trapping the material in the regionbetween the centre and the outer rim. Small-amplitudesinusoidal strain (or stress) with a fixed frequency was

applied to the system and the stress (or strain) responsewas measured simultaneously.

The data were obtained using a modern computer-controlled rheometer, which permitted automatic plot-ting of the results. Measurements of the response of stressto such oscillations (Table 2) indicated that the floc hadboth strong elastic and viscous properties, typical ofviscoelastic materials. In Table 2, this is reflected by thevalues of the storage modulus and the loss modulus.The loss modulus gives a measure of the energy that isstored elastically during deformation and is normallyfully recoverable; the storage modulus is a measure ofthe viscous (non-recoverable) energy dissipation.When a perfectly elastic material is subjected to suchdeformation, the sinusoidal strain and stress are fully in


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Figure 4

Viscosity profile as a function of time after adding NaOH/sodium dodecyl sulphate

(SDS) to a suspension of Escherichia colicells containing a 76 kb plasmid. The inset

shows the percentage of intact cells for the initial period of the reaction. The reac-

tion was performed inside a co-axial cylinder viscometer operated at a shear rate of

231 s1. (Adapted from Ref. 26.)


0 100 200 300 400

Time (sec)

Viscos

ity

(mPa

s)

0 10 20 30 40

Time (sec)

Intac

tce

lls

(%)

0

10

20

30

40

50

0.1

1

10

100

Table 2. Viscoelastic properties of the insoluble floca

Strain Strain10%, 0.02 100%, 0.2

Frequency Phase Storage Loss Dynamic Phase Storage Loss Dynamic(Hz) angle modulus G modulus G viscosity angle modulus G modulus G viscosity

(Pa) (Pa) (Pas) (Pa) (Pa) (Pas)

1 13.2 142.0 33.4 5.3 22.1 69.3 28.2 4.55 13.4 471.0 113.0 3.6 27.9 172.0 90.9 2.9

aTable compiled largely from Ref. 27.


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phase and the phase angle is 90. By contrast, for aperfectly viscous material, the phase angle is zero. Asshown in Table 2, the phase angle of the floc has a valueof ~13 for low strains and 2228 for high strains,indicating considerable viscoelastic behaviour. Thechange in phase angle indicates that the two modulihave a different dependence on the imposed strain. This

is confirmed by the change in the dynamic viscosity ofthe material in Table 2, which is defined by the twomoduli. According to the data in Table 2, the dynamicviscosity appears to decrease with increasing strain.This, together with the observed changes in phaseangle, are indicative of a degree of breakdown of thethree-dimensional structure of the floc as the imposedstrain is increased. Given the susceptibility of chromo-somal DNA to damage by fluid mechanical forces andthe requirement for minimum contamination of theliquor by cellular material, the method of separation willbe governed largely by the shear properties of the floc.

If the shear levels have been held close to a minimum,consistent with effective mixing in lysis and neutraliz-

ation, it is especially critical that the effect is not com-promised in the subsequent removal of the flocculateddebris. Continuous-flow industrial centrifuges applystrong shear fields to an entrant stream32,33 and althoughnew designs are addressing this, the problem is sub-stantial when dealing with sensitive materials such asflocs. Studies with a tubular-bowl centrifuge (M.S. Levy,unpublished) indicate that lower speeds are tolerated bya 20 kb plasmid, but not higher speeds (Fig. 5). Thishas implications for both throughput and the level ofentrained liquid in the solids discharge. Filtration inmembrane-based systems must involve some shear inorder to move the retentate so that dead-end filtrationis a more likely option. However, because of the shear

sensitivity of the denatured chromosomal DNA, dam-age in the pores is an issue. Theodossiou et al.34 estab-lished that a variety of filtration media, with and

without filter aids, always entail some loss of plasmidand some passage of chromosomal DNA.

The use of more-refined and inert filter aids did notalleviate these problems35. The finest filter aid gavethe best compromise of filter clarity (solids content of0.05 g l1 from 100 g l1) and plasmid purity (71%).Many reports have discussed the addition of diatoma-

ceous earth, glass and other adsorbents to the originallysis mixture3537. A more radical possibility that avoidsshear emerged when it was observed that the floc ofdenatured cellular solids would float leaving a relativelyclear liquid beneath. At a scale of 15 litres, the drainedliquor contained ~80% of the plasmid with a solidscontent of 0.2 g l1. Although flotation allowed anincreased flux (~2.7 fold), elevated levels of solids,chromosomal DNA and protein were observed in thefiltrate35. This is probably because the floc itself previ-ously acted as a pre-filter and emphasizes the impor-tance of lysis and the design of neutralization-stagemixing in order to reduce this problem.

Rheological information has been used to define a

scaleable reactor design with the capacity to processlarge quantities of cell suspension27. Thus, the rapid risein the viscosity of the lysate shown in Fig. 4 is indica-tive of a fast reaction between the cell suspension andthe alkaline solution. For such a system, rapid andintense mixing is critical. However, after the two com-ponents are mixed, agitation intensity must be reducedin order to minimize damage to chromosomal DNA.The neutralization step does require a degree of mix-ing but it must be of a lower intensity and once theflocculated material appears, the mixing intensity mustagain be further reduced in order to avoid damage toflocs. These process objectives can be met by separat-ing the initial lysis step from the neutralization reaction.

The former process can be carried out using two-impinging-jet mixing27. In such a device, intense mix-ing energy is dissipated over a small volume and thetime of mixing is extremely short. Next, the lysate mix-ture is collected in a reactor in which the neutralizationstep is carried out by air-assisted injection of cold potas-sium acetate from the base of the reactor. The volumeof potassium acetate required for complete neutraliza-tion is jetted rapidly into the reactor and this ensuresthat mixing of the reactants is completed before floc-culation commences. Additional air sparged at the baseof the reactor provides low shear mixing throughoutthe neutralization and enhances the up-flow of flocs tothe surface27.

Intermediate purificationAfter clarification with respect to cell debris has been

achieved, the shear-related issues that remain will focuson the essentially soluble components: the plasmidDNA and any soluble or colloidal chromosomal DNAthat has escaped the clarification step. If the level ofsoluble or colloidal chromosomal material is high, itcan be a significant constraint. For example, it can limitthe velocity of retentate recirculation during use ofmembrane-based separation and will constrain the levelof mixing during any fractional precipitation. Bussey etal.38 reported the use of tangential-flow ultrafiltrationfor plasmid-DNA purification. They described the useof membranes with typical cut-off points of 300500kDa.Membranes of 500 kDa cut-off were used for plasmids


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Figure 5

Clarification and supercoiled plasmid-DNA content in supernatants as a function of

centrifugal force. A lysate containing a 20 kb plasmid was filtered through muslin cloth

before centrifugation in a continuous-flow tubular centrifuge. Clarification was moni-

tored by optical density at 600 nm (OD), relative clarification was calculated by nor-

malizing OD values to those obtained for the lysate centrifuged at 15 000 rpm. Super-

coiled plasmid content was quantified in solution using a fluorescence-based method17.

Abbreviation: SC, supercoiled circular.


0 5000 10000 15000

Speed (rpm)

Superco

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id

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ranging from 15 kb to 50 kb. Contaminating proteins,

carbohydrates and nucleotides passed through themembrane. The patent application describes a require-ment typically of five square feet of membrane for400600 mg of nucleic acid. It noted that a gel layerhelps to reduce product loss into the permeate. Theircomment on the need to manage shear-induced dam-age suggest that this will be an issue, especially for largerplasmids and where prior removal of chromosomalDNA has been inadequate. Rotating disc dynamicmembrane systems are being examined because theyenable the separation of the factors of pressure dropacross the membrane from the level of shear on theretentate side (M.S. Levy et al., unpublished). If pre-cipitation is used, solidliquid separation once againraises the possibility of shear-induced damage duringcentrifugation and while re-dissolving precipitates. Re-dissolving precipitates can pose concern particularly ifa high-molecular-weight agent is used that inhibitsrapid dissolution.

An alternative approach based on the selective reten-tion of contaminating nucleic acids (chromosomalDNA and RNA) using a nitrocellulose membrane hasbeen described39. Most of the plasmid-DNA macro-molecules pass through the membrane and are recov-ered in the filtrate. The denatured single-strandedchromosomal DNA and RNA, by contrast, are adsorbedon the membrane. Figure 6 shows the selective removalof denatured E. colichromosomal DNA from a clarifiedlysate by filtration through a 0.45 m nitrocellulosemembrane.

There have been several reports on the use of frac-

tional precipitation to purify plasmid DNA. Some ofthese use divalent and trivalent metal salts and combi-nations of polyethylene glycol (PEG) and salts as wellas PEG alone40; there are also some earlier publishedreports of PEG precipitation of DNA41,42. Chen andRuffner43 reported the use of 1 M magnesium chlorideand 3.3% (w/v) PEG to remove debris, chromosomalDNA, RNA and protein followed by 1.5 M salt and 5%(w/v) PEG to precipitate plasmid DNA. The basis ofselective precipitation of RNA and DNA might partlybe the differences in charge density between the plas-mid DNA and other nucleic-acid contaminants andpartly the higher regional phosphate-charge density ofinter-helix junctions found in transfer RNAs andrecombinant RNAs compared with DNA44. Alterna-tive approaches using ammonium sulphate45, spermi-dine46 and spermine46 fractional precipitation have alsobeen described.

High-resolution separationIt is unlikely that plasmid DNA of the quality required

for clinical purposes can be produced without the useof chromatography; this has been the subject of severalstudies13,14,47,48. In this article, the only element that willbe addressed in particular is the impact of the size ofthe macromolecules and the way this affects availablechromatographic media. Plasmid DNA is large, relativeto the proteins for which most macromolecule-directedchromatographic media were created; the effect of thisis illustrated in Fig. 7, which relates to experiments with


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Figure 6

Plasmid purification by retention of contaminant Escherichia colichromosomal DNA on nitrocellulose: (a) agarose gel electrophoresis and

(b) Southern blot analysis using a sequence specific for the E. colichromosome as a probe. Abbreviations: M, supercoiled DNA ladder

(216 kb); (1) plasmid DNA (500 ng) purified by isopropanol precipitation; (2) same as 1 followed by anion-exchange chromatography; (3)

same as 2 but filtered through nitrocellulose before anion-exchange chromatography; (4) material retained by the nitrocellulose membrane

(DNA amount loaded was not determined). Densitometric scanning of the Southern blot gave relative signal values of 1:0.24:0.07 for lanes

1, 2 and 3, respectively. Abbreviations: CHR, chromosomal DNA; OC, open circular plasmid DNA; SC, supercoiled circular plasmid DNA.

(Reprinted from Levy et al.39 with permission.)


(a) (b)M 2 3 41M 2 3 41

SC

OC + CHR

CHR

CHR


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classical anion-exchange matrices and several noveltypes of anion exchange matrices (O.R.T. Thomas et

al., unpublished). As Fig. 7 indicates, the capacities oftraditional materials are typically 1000-fold lower thanthose reported for proteins. This could be attributed tothe greater size of a plasmid-DNA molecule comparedwith proteins and their correspondingly larger hydro-dynamic volume. The capacity of the matrices forresuspended PEG fractionally precipitated DNA wasdouble that seen for the clarified lysate, and this couldbe a result of the removal of contaminants present inthe lysate during the precipitation process (e.g. 95% ofthe protein) and possibly also to DNA compaction.

The addition of salt to the equilibration buffers andthe high ionic strength of the lysate prevented the bind-ing of any residual proteins to the matrix. The use ofthe clarified lysate in breakthrough studies on newermatrices generally resulted in capacities higher thanthose seen with the Sepharose FF media, although therecoveries tended to be lower. The low capacity forDNA from the clarified lysate suggests that the con-taminants present in the lysate are successfully compet-ing for the charged groups present on the surface ofthe matrix. The different matrices were seen to forminto groups depending on their physical composition.These results are consistent with a recent study usingconfocal-scanning laser microscopy49, which indicatedadsorption in the outer layer of the media. These makethe studies with nitrocellulose membranes notable inthat hydrodynamic methods are available to bring thesingle-stranded chromosomal DNA to the adsorbentsurface. However, as with all cross-flow membrane

systems, it is necessary to examine the level of shearthat is allowed before damage to the product occurs.

As is so often the case, nature suggests alternativeapproaches to the plasmid-DNA size problem. In higherorganisms, DNA is formed into structures, such aschromatin, which allows it to be maintained in a phys-ically stable and highly compact state. In principle, this

is an approach that can be adopted during processing.The constraint at early stages is that potential com-pacting agents can be expected to interact heavily withcontaminants. The agent must not be too expensiveand should not interfere with selective separation bytechniques such as chromatography. Horn et al .50

described the use of concentrations of PEG (typically1% ) as an agent to be used before anion-exchangechromatography. It is worth noting that chromatogra-phy, especially size exclusion, is associated with largereductions in endotoxin levels14,51.

FormulationAt formulation stage, the plasmid DNA has its great-

est value, thus damage is particularly undesirable. Dam-age can occur in any step that introduces the productinto a high-velocity stream, such as those entailed indrying and injection into vials. For plasmid DNA to beeffective, processing must include the formation of anagent for its delivery. For vaccines, this can be small par-ticles52; for gene therapy, DNA complexing with lipidsor peptides is most common53,54. The theme of size,shape and shear sensitivity persists. For some time, ithas been known (in semi-quantitative terms) that spray-ing aerosols of plasmids into the airways of an animalor human can lead to a substantial loss of activity55, andthis has been associated with a decrease in the super-coiled plasmid content. Plasmid damage is presumably

caused by shear, interfacial effects or a combination ofboth. Mixing plasmid DNA and complexing agents,such as lipids and polypeptides, to assist transfection56

can yield compact and shear-resistant particles. Forexample, an examination of complexes of plasmidDNA and polylysine and the subsequent application ofshear (Fig. 8) indicated that compaction does indeedreduce damage. In terms of more-radical approaches tothe downstream processing of compacted DNA, phageshave been used as a vehicle for carrying DNA intocells57,58. Phages are not completely insensitive to shearthemselves but their distinctive physical propertiesallow a different approach to processing.

The sensitivity of macromolecules, such as proteinsand plasmid DNA, to combinations of shear andgasliquid interfaces can pose problems if the plasmidDNA is to be dried. Freeze drying can avoid such prob-lems but the production of particles of a broad sizerange might require subsequent milling that can causedamage. The method of solution-enhanced dispersionby supercritical fluids (SEDS) can overcome these dif-ficulties. Studies with a 6.9 kb plasmid co-formulatedwith mannitol showed no shear-related damage,although it was necessary to counter the effectiveacidity of the aqueous CO2 (Ref. 59). If plasmid DNAis processed as a liquid, it is still possible to suffer lossesof active material if, for example, the fluid is injectedinto vials from fine orifices. The levels of shear toler-ated by flow from such small pipes have been definedby Levy et al.24.


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Figure 7

Binding capacity as a function of particle diameter of resin for a 6.9 kb plasmid bind-

ing to commercial anion-exchange adsorbents. Feedstock and column equilibration

conditions: feedstock, resuspended PEG precipitated material supplemented with

0.30.5 M NaCl/10 mM Tris-HCl pH 8; column equilibration 1 mM EDTA containing

0.30.5 M NaCl. Chromatographic media tested: (1) STREAMLINE DEAE (186 m);

(2) DEAE Sepharose FF (93 m); (3) Q Sepharose FF (93 m); (4) DE52 Cellulose

(75 m), the particle is a rod 120 mm length and 40 mm diameter; (5) Source 30Q

(30 m); (6) Source 15 Q (15 m); (7) Q Sepharose XL (93 m); (8) Q Hyper D (M) 65

(65m); (9) Fractogel EMD DMAE 650 (M) (65m); and (10) POROS 50 DEAE (50 m).


0 20 40 60 80 100 120 140

Reciprocal of mean particle radius (mm1)

Bindingcapac

ity(mgm

l1)

1 23 4

5

6

7

8

9

10

0

1

2

3

4

5

6


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Biological approaches to enhance purificationAs in other processes involving recombinant

materials, any biological solutions that can be appliedbefore downstream processing will be valuable. Forexample, it is common in the processing of plasmids touse ribonucleases to degrade RNA rather than remov-ing it by conventional fractionation. A new approach60

allows recombinant bovine ribonuclease to be induced

in the plasmid-bearing strain. Maximizing plasmidDNA levels and minimizing chromosomal DNA lev-els during and after cell growth will be useful. Enhanc-ing the segregational stability of a plasmid ensures thata high proportion of biomass contains plasmid DNA.Plasmid-free cells will only contribute to contami-nation, which, as noted, is difficult to remove downstream.The addition of antibiotics to select for plasmid-con-taining cells can alleviate plasmid instability and recentstudies on plasmid autoselection systems show that thisdependence on antibiotics can be removed61,62.

The plasmid-DNA production rate can also beincreased by using several plasmid-amplification strat-egies. Many recombinant plasmids replicate in a relaxedmode, in which their replication can occur in theabsence of protein synthesis; by contrast, chromosomalDNA replication is stringent, requiring proteinsynthesis to occur. Plasmid DNA can be amplified byeither starving certain mutant host strains of aminoacids63 (and thereby stalling protein synthesis) or byhalting protein synthesis completely using antibioticssuch as chloroamphenicol63,64. However, antibioticclearance from the process stream might be a problemfrom a regulatory point of view. Certain temperature-sensitive mutations, when introduced into the plasmid-replication control systems, can result in large increasesin plasmid-copy number upon undergoing a tempera-ture shift during culture growth65. Runaway replicationvectors can be induced to amplify plasmid levels up to1000 copies per cell66.

As mentioned previously, final-product-release speci-fications are likely to require 90% of the plasmidDNA molecules to be in the supercoiled form14,67. Theseforms are produced within the cell by the action of DNA-supercoiling enzymes, such as DNA topoisomerases,which introduce negative superhelical twists into plas-mid-DNA molecules. OKennedy et al.68 have shownthat the proportion of supercoiled forms can depend

strongly on the type of growth medium used. Thissuggests that the proportion of supercoiled forms canbe modulated using different growth conditions andcould therefore be a possible target for recombinantgenetic modification. Supercoiled plasmids exhibit adistribution with respect to the number of superhelicaltwists that have been introduced, and this supercoilingdensity can affect the transcription efficiency of plas-mid-encoded genes. This is known to depend on factorsthat include growth temperature and exposure to coldor heat shocks69.

ConclusionThe biochemical engineering studies reviewed here

have created the foundation for the efficient isolationof plasmid DNA. At present, scales and efficiencies aremodest; however, if DNA vaccines prove to be effec-tive, the ultimate scale will be large. With tuberculosis,often in forms that are resistant to drugs, causing threemillion deaths per year worldwide, malaria (twomillion deaths per year) and AIDS (one million deathsper year), there is a great need for stable new vaccines;an influenza epidemic could demand much-greaterquantities quickly3. Recent reports on the use of DNAvaccines in animal studies are promising70, although thesituation with gene therapy is less clear at present.However, the potential of gene therapy and DNAvaccines to address currently incurable diseases indicatesa large possible demand. Given these two broad cat-egories of potential use, the establishment of strong


REVIEWS

Figure 8

Effect of shear on plasmid-DNApolylysine complexes. The complexes were mixed at a charge ratio of 1.5 using a semi-automated syringe

pump and sheared using a rotating disk device operated at a shear rate of 1 106 s1 for 5s using the rotating disk device (Ref. 31). The

sheared complexes together with unsheared controls were treated with trypsin before agarose gel electrophoresis analysis. Lanes: (1,2)

unsheared and sheared plasmid (29 kb) in TE buffer; (3,4) unsheared and sheared plasmid in TE buffer/150 mM NaCl; (5, 6) unsheared andsheared plasmid-polylysine (26 kDa) in TE buffer; (7,8) unsheared and sheared plasmid-polylysine (26 kDa) in TE buffer/150 mM NaCl; and

(9,10) unsheared and sheared plasmid-polylysine (100 kDa) in TE buffer. Abbreviations: SC, supercoiled circular plasmid DNA; TE buffer,

10 mM TrisCl, pH 8, 1mM EDTA. (Adapted from J.T. Tsai, PhD thesis, University of London, London, UK, 1999)


Sheared DNA

SC

2 3 41 65 87 109


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biochemical engineering foundations will be impor-tant as a general guide to future processing. As indicatedin this review, the interactions between particular stagesare especially pronounced, a whole bioprocess approachwill therefore be even more important for DNA thanfor proteins.

AcknowledgmentsWe are very grateful to our colleagues (D. Cooke,M. Hoare, D. Kendall, L. Lee, G. Lye, P. McHugh,M. Tservistas and J. Ward) for valuable advice andcomments. We are also grateful for the support ofthe Biotechnology and Biological Sciences ResearchCouncil.

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Chitin, a homopolymer comprising -(1-4)-linkedN-acetyl-D-glucosamine residues is one of themost abundant, easily obtained and renewable

natural polymers, second only to cellulose. It is com-monly found in the exoskeletons or cuticles of manyinvertebrates and in the cell walls of most fungi1.Because of its high crystallinity, chitin is insoluble inaqueous solutions and organic solvents2.

Chitosan is a polycationic biopolymer that occursnaturally or is obtained by the N-deacetylation ofchitin; its name does not refer to a uniquely definedcompound but rather to a family of copolymers with

various fractions of acetylated units. It is biodegradable,non-toxic to animals (in mice, the LD

50

was16gkg1),soluble in acidic solutions, available in various physicalforms and much more tractable than chitin2,3. Thus,chitosan offers properties with great potential for manyindustrial applications.

Today, several companies are producing chitin andchitosan products on a commercial scale; the majorityare located in Japan, where 100 billion tons ofchitosan are manufactured each year from the shells ofcrabs and shrimps, an amount that accounts for ~90%of the global chitosan market (approximately four tril-lion yen). The major areas of application include watertreatment, biomedical applications (including wounddressing and artificial skin) and personal-care products213.

In addition, oligomers of chitin and chitosan havealso attracted considerable attention because they havebeen reported to exhibit certain interesting physiological

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and relaxed strains of Escherichia coli. Mol. Gen. Genet. 190, 35535764 Clewell, D.B. (1972) Nature of ColE1-plasmid replication inEscherichia

coliin the presence of chloroamphenicol.J. Bacteriol. 110, 667676

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plasmids intended for human gene therapy by employing a tempera-

ture-controllable point mutation. Hum. Gene Ther. 7, 19711980

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sensitive plasmid replication. Plasmid32, 131167

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maceutical perspective.J. Pharm. Sci. 87, 131146

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76, 175183

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vaccination. Nature400, 269271

TIBTECH JULY 2000 (Vol. 18) 0167-7799/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(00)01462-1 305

REVIEWS

Chitin deacetylases: new, versatile tools inbiotechnology

Iason Tsigos, Aggeliki Martinou, Dimitris Kafetzopoulos and Vassilis Bouriotis

Chitin deacetylases have been identified in several fungi and insects. They catalyse the hydrolysis of N-acetamido bonds of chitin,

converting it to chitosan. Chitosans, which are produced by a harsh thermochemical procedure, have several applications

in areas such as biomedicine, food ingredients, cosmetics and pharmaceuticals. The use of chitin deacetylases for the

conversion of chitin to chitosan, in contrast to the presently used chemical procedure, offers the possibility of a controlled,

non-degradable process, resulting in the production of novel, well-defined chitosan oligomers and polymers.

I. Tsigos, A. Martinou and D. Kafetzopoulos are at the Institute ofMolecular Biology and Biotechnology, Foundation of Research and

Technology, Crete, Greece. V. Bouriotis ([email protected]) isat the Division of Applied Biology and Biotechnology, Department ofBiology, University of Crete, Crete, Greece.

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