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

solgel matrix protects beta-glucosidase against thermal and chemical 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 antibodycatalysed 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, 245250
109 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 atrazine chlorohydrolase 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
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
Campostrini, R. et al. (1996) Immobilization of plant cells in hybrid
solgel materials. J. Solgel Sci. Technol. 7, 8797
Carturan, G. et al. (1999) Production of valuable drugs from plant
cells immobilized by hybrid solgel SiO2. J. Solgel Sci. Technol.
13, 273276
Reetz, M.T. (1997) Entrapment of biocatalysts in hydrophobic
solgel materials for use in organic chemistry. Adv. Mater. 9,
Pope, E.J.A. et al. (1995) Encapsulation of living tissue cells in an
organosilicon. J. Solgel Sci. Technol. 55, 3349
McFarland, E.W. and Weinberg, W.H. (1999) Combinatorial
approaches to materials discovery. Trends Biotechnol. 17, 107115
Wang, J. et al. (1998) Self-assembled silica gel networks. J. Am.
Chem. Soc. 120, 58525853
Xia, Y.N. and Whitesides, G.M. (1998) Soft lithography. Angew.
Chem., Int. Ed. Engl. 37, 550575

Biochemical engineering approaches to the

challenges 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 plasmids 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.

he direct application of genes as vaccines and for

medical therapy shows great promise1,2. Having
reviewed the potential of DNA vaccines, the
American Academy of Microbiology concluded that
recent results obtained in animals indicate that this
new technology might revolutionize the vaccination
of humans3 (http://www.asmusa.org/acasrc/pdfs/
dnareprt.pdf ). Further, there are currently greater than
390 active clinical trials of gene therapy worldwide,
involving approximately 3500 patients (http://www.
wiley.co.uk/genmed/clinical). The main options for
gene therapy are to use disabled viruses or plasmidbased genes complexed with agents such as lipids or
polypeptides, although each system has its advantages
and disadvantages4. This article will focus on plasmid
DNA, which has potential for both vaccines and
medical therapy.

M.S. Levy, R.D. OKennedy, P. Ayazi-Shamlou (p.shamlou@ucl.ac.uk)

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


At present, the size of the plasmid DNA being used

in clinical trials is at the lower end of the possible range,
typically ,10 kb. However, there are strong indications
that the size of the plasmid DNA used will grow. Thus,
for DNA vaccines, their effectiveness might be increased
by, for example, incorporating genes for signalling molecules (eg. cytokines) into antigen-carrying plasmids5.
For gene therapy using either plasmid DNA or disabled
viruses, there is increasing concern that regulation of
the functioning of the gene is ensured once it is in place.
This, together with the expectation of the progression
of gene therapy to metabolic and other multigene diseases6,7, will lead to an increase in vector size. Although
multiple plasmids represent an alternative method in
some cases, their separate processing and blending will
not be easy. Artificial chromosomes are beginning to
excite interest as a method of achieving reliable longterm action8,9, and these chromosomes will range up
to mega-base size.
In the laboratory, plasmid DNA is typically isolated
from a suitable recombinant Escherichia coli strain.
Recovery usually begins with a chemical lysis step using

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

TIBTECH JULY 2000 (Vol. 18)


an alkaline solution of, for example, sodium dodecyl

sulphate (SDS) followed by neutralization with an agent
such as concentrated potassium acetate10. Although the
alkaline SDS mixture causes irreversible denaturation
of the chromosomal DNA and protein, providing the
pH value is not too high, denaturation is reversible for
the plasmid DNA; the optimum pH value varies with
the nature of the plasmid11. Plasmids are therefore soluble in the neutralized solution whereas most chromosomal DNA and cellular proteins form an insoluble
precipitate (a floc). In the laboratory, centrifugation
removes the floc before stages that commonly use
reagents, such as flammable alcohols, which are unsuitable on a large scale. Similarly, the use of caesium-chloride gradients for purification is not easy to scale up,
although chromatographic columns offer an alternative.
The quantity of plasmid DNA required will differ
between therapies, and each disease is likely to have
specific needs. However, there are some general indications suggesting that the dose will range from micrograms to milligrams12. International regulatory agencies are likely to set stringent specifications with regard
to the level of endotoxins, RNA, protein, bacterial host
DNA, quantities of linear and supercoiled DNA in the
preparation and presence of toxic chemicals13. Contaminant levels will have to comply with the maximum
allowable level per administered dose and therefore
specifications are likely to be strongly dependent on the
dose given. For example, release specifications for bulk
plasmid DNA used in a cancer clinical trial14 were:
RNA, non-visualized on a 0.8% agarose gel; E. coli
DNA, ,0.01 mg mg21 plasmid DNA; protein, ,1 mg
mg21 plasmid DNA; endotoxin, ,0.1 endotoxin units
(EU) mg21 plasmid DNA and circular DNA, .95%.
For process monitoring, quality control and validation,
it is of major importance to develop sensitive and quantitative analytical methods. Examples of such methods
include recent studies by Lahijani et al.15 on the sensitive quantitation of chromosomal DNA contamination,
by Noites et al.16 on the rapid quantitation of plasmid
DNA in solution and by Levy et al.17 on the rapid
monitoring of supercoiled DNA content in solution.
This review does not set out to cover all aspects of manufacture, but the removal and testing of contaminants
such as endotoxins is certainly critical.
Central biochemical engineering issues
As Table 1 indicates, the apparent range of operations
available from the literature (most concerned with laboratory-scale studies) is large. However, the particular
properties of plasmid DNA and its contaminants can
set 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 coded
by three larger nucleotides, the plasmid gene required
to specify a given protein is much larger (relatively). As
indicated, it is likely that the size of plasmids to be
processed will increase and a size of 50 kb might not
be unusual. In addition, the purification of plasmid
DNA involves the removal of large quantities of
chromosomal DNA, which has a much larger size,
typically 40004500 kb for E. coli.
The size of these key components means that their
susceptibility to fluid mechanical forces during processing is probably the most critical issue in the large-scale
TIBTECH JULY 2000 (Vol. 18)

Table 1. Potential unit operations and process options for the

downstream recovery and purification of plasmid DNA
Unit operation

Process options



Solids removal

Membrane separation

Intermediate purification

Fractional precipitation
Membrane fractionation

High-resolution purification

Reversed phase
Gel filtration



preparation of plasmid DNA. For proteins, after much

initial concern18, there was clear evidence that they are
not easily damaged by shear fields usually encountered
in process equipment19. By contrast, the high mixing
fields associated with such shear fields are capable of
causing severe damage if they bring a protein into contact with a gasliquid interface20. The lack of direct
shear-induced damage is explicable for proteins in
terms of their size and shape. Because globular proteins
typically have an equivalent sphere diameter of 310
nm (Ref. 21), they are situated within the individual
fluid packages defined by the Kolmogorov microscale
of turbulence, which are unaffected by the geometry
of the external source providing the motion22. The size
and particularly the extended shape of plasmids (Fig. 1),
with an average hydrodynamic diameter of 150250 nm


1.24 M 0

500 nm
trends in Biotechnology

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 3 500 nm area. (Images kindly provided by Dr Y. Lyubchenko.)



13 kb
20 kb


29 kb
76 kb



SC DNA (%)

Supercoiled plasmid DNA (%)




89 kb


40 kb


50 kb
76 kb
89 kb
Time of exposure to shear (sec)


Time of exposure to shear (sec)
trends in Biotechnology

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 3 106 s21 using a rotating disk
device 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 damage 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.

Supercoiled plasmid DNA (%)


Experimental condition

trends in Biotechnology

Figure 3
Supercoiled plasmid-DNA content after shearing in the absence or presence of air
liquid interfaces for a 20 kb plasmid under different DNA concentration and ionicstrength conditions: (1) clarified alkaline lysate (20 mg ml21); (2) ultrapure plasmid in
TE buffer (20 mg ml21); (3) ultrapure plasmid in TE buffer (5 mg ml21); (4) ultrapure
plasmid in TE buffer (0.2 mg ml21); (5) ultrapure plasmid in 150 mM NaCl, TE buffer
(0.2 mg ml21). 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.)

for 510 kb plasmids23 and potentially .1 mm for

larger plasmids, means that this complete protection is
not available. For chromosomal DNA, compaction in
the native state can be protective but bacterial material,
especially that denatured in the chemical lysis procedure


for plasmid DNA isolation, will be acutely sensitive to

fluid mechanical force. Any significant degradation will
reduce chromosomal DNA to the same order of magnitude as plasmid DNA, making separation very difficult.
The danger of shear-induced degradation of chromosomal DNA is more serious in process terms because
the large size of both plasmid and degraded chromosomal DNA makes chromatographic procedures relatively inefficient. Conventional macromolecular, chromatographic media were developed for the separation
of proteins that are both smaller and more compact.
This limitation in capacity when using high-resolution
chromatographic separation means that every gain that
can be made upstream in the process is especially critical.
Characterizing the effect of shear and
associated factors
In principle, all the operations ranging from cell lysis
to vialing can be influenced by shear effects; a fundamental understanding of the extent of these effects is
therefore essential. Shear forces can convert the desirable supercoiled, circular form of the plasmid DNA
into undesirable forms, such as open circular and linear DNA. Sensitivity to shear forces has been found to
be critically dependent on plasmid size and the ionic
strength of the environment. Levy et al.24 have shown
that plasmids >20 kb are sensitive to shear rates greater
than 1 3 106 s21 (Fig. 2), a level that can be encountered in industrial-bioprocess equipment. Studies investigating increasing shear rate indicate that there is a
critical value, above which damage is severe. A firstorder-reaction kinetic model describes the decrease in
the concentration of supercoiled plasmid content as a
function 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 equipment, in combination with shear have been shown to
increase damage to purified plasmids resuspended in
low-ionic-strength buffer (Fig. 3). Material in a highionic-strength environment, characteristic of an early
stage of the process, is not as sensitive to these effects.
The impact of physicochemical conditions on product
stability parallels findings with proteins wherein, for
example, fermentation media can protect secreted
proteins in the sheared and aerated environment25.
Lysis and the removal of solids
Cell lysis is the operation in which shear effects are
perhaps the most critical. It is complex in terms of biochemical engineering; rapid mixing of the alkaline
detergent and cells is essential, but once the chromosomal DNA is exposed, it is acutely sensitive to
mechanical damage. Before a large-scale operation is
designed, basic information on rheology, mixing and
the effects of shear must be obtained. The rheology of
alkaline lysis has been studied using a co-axial cylinder
rheometer26; this was used to continuously record
changes in viscosity during the lysis of two strains of
plasmid-bearing Escherichia coli. Figure 4 shows a typical viscositytime profile during the lysis of a cell suspension containing a 76 kb plasmid26. The apparent
viscosity increased rapidly for 30s following the addition of a NaOH/SDS solution to the cell suspension.
The change in viscosity reached a plateau after ~25s
TIBTECH JULY 2000 (Vol. 18)


Intact cells (%)


Viscosity (mPas)

before rising again to a peak value between 80120s.

Finally, the apparent viscosity decreased to a steady
value after ~200400s. The absolute magnitude of the
apparent viscosities at various points during the lysis
operation was shear-dependent. However, the shape of
the viscositytime profiles and the position of the
various peaks on these profiles were unaffected by shear
Before neutralization, measurements revealed that
the cell suspension and the NaOH/SDS solution were
both Newtonian with viscosities comparable to water.
However, the reaction between the cell suspension and
the NaOH/SDS solution produced a liquor exhibiting
strong non-Newtonian pseudoplastic properties with a
flow-behaviour index of approximately 0.3 at shear
rates ,370 s21. The development of non-Newtonian
behaviour during lysis will have major consequences
on the mixing pattern in the reactor, thus affecting the
quality of the product27. The cell-lysis reaction was
subsequently stopped at predetermined times by the
rapid addition of the neutralizing agent, and total cell
counts were carried out to determine the number of
intact cells present in the lysate. Figure 4 (inset) shows
total counts obtained from the initial phase of the lysis
reaction. This indicates that the time taken to reach the
initial transient peak is necessary in order to solubilize
the cell-wall material sufficiently to start the release of
intracellular contents.
Alternative methods of lysis have also been examined.
The use of mechanical disruption equipment was associated with substantial damage to plasmid DNA28. An
interesting approach29 used a continuous-flow heat
step, typically at a temperature of 702778C and with a
residence time of ~35s. In another procedure, a static
mixer was used30 to carry out both the alkaline cell lysis
and the neutralization steps. Pressure drops across
static mixers are significantly larger than those across an
empty pipe of equal diameter. Because shear damage is
directly related to pressure drop, the flocculated
chromosomal DNA will be likely to suffer significant
damage owing to the shear generated by flow over the
static mixers. The extent of such damage will depend
on the pressure drop which, in turn, is determined by
flow rate and mixer design. In studies aimed at assessing the shear susceptibility of the flocculated material,
samples of flocculated material from the neutralization
step were subjected to a small-amplitude sinusoidal
strain of fixed frequency in a rheometer with a coneand-plate attachment27,31. The measurements were
obtained using a flat plate and a convex cone with a





Time (sec)






Time (sec)



trends in Biotechnology

Figure 4
Viscosity profile as a function of time after adding NaOH/sodium dodecyl sulphate
(SDS) to a suspension of Escherichia coli cells containing a 76 kb plasmid. The inset
shows the percentage of intact cells for the initial period of the reaction. The reaction was performed inside a co-axial cylinder viscometer operated at a shear rate of
231 s21. (Adapted from Ref. 26.)

shallow angle of 48. A small quantity of the flocculated

material was placed on the bottom plate and the cone
was positioned such that its apex touched the centre of
the plate thus trapping the material in the region
between the centre and the outer rim. Small-amplitude
sinusoidal strain (or stress) with a fixed frequency was
applied to the system and the stress (or strain) response
was measured simultaneously.
The data were obtained using a modern computercontrolled rheometer, which permitted automatic plotting of the results. Measurements of the response of stress
to such oscillations (Table 2) indicated that the floc had
both strong elastic and viscous properties, typical of
viscoelastic materials. In Table 2, this is reflected by the
values of the storage modulus and the loss modulus.
The loss modulus gives a measure of the energy that is
stored elastically during deformation and is normally
fully recoverable; the storage modulus is a measure of
the viscous (non-recoverable) energy dissipation.
When a perfectly elastic material is subjected to such
deformation, the sinusoidal strain and stress are fully in

Table 2. Viscoelastic properties of the insoluble floca

10%, 0.02

100%, 0.2


angle d

modulus G9

modulus G0


angle d

modulus G9

modulus G0












compiled largely from Ref. 27.

TIBTECH JULY 2000 (Vol. 18)









Relative clarification (%)

Supercoiled plasmid DNA (%)

phase and the phase angle is 908. By contrast, for a

perfectly viscous material, the phase angle is zero. As
shown in Table 2, the phase angle of the floc has a value
of ~138 for low strains and 22288 for high strains,
indicating considerable viscoelastic behaviour. The
change in phase angle indicates that the two moduli
have a different dependence on the imposed strain. This
is confirmed by the change in the dynamic viscosity of
the material in Table 2, which is defined by the two
moduli. According to the data in Table 2, the dynamic
viscosity appears to decrease with increasing strain.
This, together with the observed changes in phase
angle, are indicative of a degree of breakdown of the
three-dimensional structure of the floc as the imposed
strain is increased. Given the susceptibility of chromosomal DNA to damage by fluid mechanical forces and
the requirement for minimum contamination of the
liquor by cellular material, the method of separation will
be 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 neutralization, it is especially critical that the effect is not compromised in the subsequent removal of the flocculated
debris. Continuous-flow industrial centrifuges apply
strong shear fields to an entrant stream32,33 and although
new designs are addressing this, the problem is substantial when dealing with sensitive materials such as
flocs. Studies with a tubular-bowl centrifuge (M.S. Levy,
unpublished) indicate that lower speeds are tolerated by
a 20 kb plasmid, but not higher speeds (Fig. 5). This
has implications for both throughput and the level of
entrained liquid in the solids discharge. Filtration in
membrane-based systems must involve some shear in
order to move the retentate so that dead-end filtration
is a more likely option. However, because of the shear
sensitivity of the denatured chromosomal DNA, damage in the pores is an issue. Theodossiou et al.34 established that a variety of filtration media, with and



Speed (rpm)

trends in Biotechnology

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 monitored by optical density at 600 nm (OD), relative clarification was calculated by normalizing OD values to those obtained for the lysate centrifuged at 15 000 rpm. Supercoiled plasmid content was quantified in solution using a fluorescence-based method17.
Abbreviation: SC, supercoiled circular.


without filter aids, always entail some loss of plasmid

and some passage of chromosomal DNA.
The use of more-refined and inert filter aids did not
alleviate these problems35. The finest filter aid gave
the best compromise of filter clarity (solids content of
0.05 g l21 from 100 g l21) and plasmid purity (71%).
Many reports have discussed the addition of diatomaceous earth, glass and other adsorbents to the original
lysis mixture3537. A more radical possibility that avoids
shear emerged when it was observed that the floc of
denatured cellular solids would float leaving a relatively
clear liquid beneath. At a scale of 15 litres, the drained
liquor contained ~80% of the plasmid with a solids
content of 0.2 g l21. Although flotation allowed an
increased flux (~2.7 fold), elevated levels of solids,
chromosomal DNA and protein were observed in the
filtrate35. This is probably because the floc itself previously acted as a pre-filter and emphasizes the importance of lysis and the design of neutralization-stage
mixing in order to reduce this problem.
Rheological information has been used to define a
scaleable reactor design with the capacity to process
large quantities of cell suspension27. Thus, the rapid rise
in the viscosity of the lysate shown in Fig. 4 is indicative of a fast reaction between the cell suspension and
the alkaline solution. For such a system, rapid and
intense mixing is critical. However, after the two components are mixed, agitation intensity must be reduced
in order to minimize damage to chromosomal DNA.
The neutralization step does require a degree of mixing but it must be of a lower intensity and once the
flocculated material appears, the mixing intensity must
again be further reduced in order to avoid damage to
flocs. These process objectives can be met by separating the initial lysis step from the neutralization reaction.
The former process can be carried out using twoimpinging-jet mixing27. In such a device, intense mixing energy is dissipated over a small volume and the
time of mixing is extremely short. Next, the lysate mixture is collected in a reactor in which the neutralization
step is carried out by air-assisted injection of cold potassium acetate from the base of the reactor. The volume
of potassium acetate required for complete neutralization is jetted rapidly into the reactor and this ensures
that mixing of the reactants is completed before flocculation commences. Additional air sparged at the base
of the reactor provides low shear mixing throughout
the neutralization and enhances the up-flow of flocs to
the surface27.
Intermediate purification
After clarification with respect to cell debris has been
achieved, the shear-related issues that remain will focus
on the essentially soluble components: the plasmid
DNA and any soluble or colloidal chromosomal DNA
that has escaped the clarification step. If the level of
soluble or colloidal chromosomal material is high, it
can be a significant constraint. For example, it can limit
the velocity of retentate recirculation during use of
membrane-based separation and will constrain the level
of mixing during any fractional precipitation. Bussey et
al.38 reported the use of tangential-flow ultrafiltration
for plasmid-DNA purification. They described the use
of membranes with typical cut-off points of 300500 kDa.
Membranes of 500 kDa cut-off were used for plasmids
TIBTECH JULY 2000 (Vol. 18)





trends in Biotechnology

Figure 6
Plasmid purification by retention of contaminant Escherichia coli chromosomal DNA on nitrocellulose: (a) agarose gel electrophoresis and
(b) Southern blot analysis using a sequence specific for the E. coli chromosome 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.)

ranging from 15 kb to 50 kb. Contaminating proteins,

carbohydrates and nucleotides passed through the
membrane. The patent application describes a requirement typically of five square feet of membrane for
400600 mg of nucleic acid. It noted that a gel layer
helps to reduce product loss into the permeate. Their
comment on the need to manage shear-induced damage suggest that this will be an issue, especially for larger
plasmids and where prior removal of chromosomal
DNA has been inadequate. Rotating disc dynamic
membrane systems are being examined because they
enable the separation of the factors of pressure drop
across the membrane from the level of shear on the
retentate side (M.S. Levy et al., unpublished). If precipitation is used, solidliquid separation once again
raises the possibility of shear-induced damage during
centrifugation and while re-dissolving precipitates. Redissolving precipitates can pose concern particularly if
a high-molecular-weight agent is used that inhibits
rapid dissolution.
An alternative approach based on the selective retention of contaminating nucleic acids (chromosomal
DNA and RNA) using a nitrocellulose membrane has
been described39. Most of the plasmid-DNA macromolecules pass through the membrane and are recovered in the filtrate. The denatured single-stranded
chromosomal DNA and RNA, by contrast, are adsorbed
on the membrane. Figure 6 shows the selective removal
of denatured E. coli chromosomal DNA from a clarified
lysate by filtration through a 0.45 mm nitrocellulose
TIBTECH JULY 2000 (Vol. 18)

There have been several reports on the use of fractional precipitation to purify plasmid DNA. Some of
these use divalent and trivalent metal salts and combinations of polyethylene glycol (PEG) and salts as well
as PEG alone40; there are also some earlier published
reports of PEG precipitation of DNA41,42. Chen and
Ruffner43 reported the use of 1 M magnesium chloride
and 3.3% (w/v) PEG to remove debris, chromosomal
DNA, RNA and protein followed by 1.5 M salt and 5%
(w/v) PEG to precipitate plasmid DNA. The basis of
selective precipitation of RNA and DNA might partly
be the differences in charge density between the plasmid DNA and other nucleic-acid contaminants and
partly the higher regional phosphate-charge density of
inter-helix junctions found in transfer RNAs and
recombinant RNAs compared with DNA44. Alternative approaches using ammonium sulphate45, spermidine46 and spermine46 fractional precipitation have also
been described.
High-resolution separation
It is unlikely that plasmid DNA of the quality required
for clinical purposes can be produced without the use
of chromatography; this has been the subject of several
studies13,14,47,48. In this article, the only element that will
be addressed in particular is the impact of the size of
the macromolecules and the way this affects available
chromatographic media. Plasmid DNA is large, relative
to the proteins for which most macromolecule-directed
chromatographic media were created; the effect of this
is illustrated in Fig. 7, which relates to experiments with



Binding capacity (mg ml1)






1 2

Reciprocal of mean particle radius (mm1)


trends in Biotechnology

Figure 7
Binding capacity as a function of particle diameter of resin for a 6.9 kb plasmid binding 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 mm);
(2) DEAE Sepharose FF (93 mm); (3) Q Sepharose FF (93 mm); (4) DE52 Cellulose
(75 mm), the particle is a rod 120 mm length and 40 mm diameter; (5) Source 30Q
(30 mm); (6) Source 15 Q (15 mm); (7) Q Sepharose XL (93 mm); (8) Q Hyper D (M) 65
(65 mm); (9) Fractogel EMD DMAE 650 (M) (65 mm); and (10) POROS 50 DEAE (50 mm).

classical anion-exchange matrices and several novel

types of anion exchange matrices (O.R.T. Thomas et
al., unpublished). As Fig. 7 indicates, the capacities of
traditional materials are typically 1000-fold lower than
those reported for proteins. This could be attributed to
the greater size of a plasmid-DNA molecule compared
with proteins and their correspondingly larger hydrodynamic volume. The capacity of the matrices for
resuspended PEG fractionally precipitated DNA was
double that seen for the clarified lysate, and this could
be a result of the removal of contaminants present in
the lysate during the precipitation process (e.g. 95% of
the protein) and possibly also to DNA compaction.
The addition of salt to the equilibration buffers and
the high ionic strength of the lysate prevented the binding of any residual proteins to the matrix. The use of
the clarified lysate in breakthrough studies on newer
matrices generally resulted in capacities higher than
those seen with the Sepharose FF media, although the
recoveries tended to be lower. The low capacity for
DNA from the clarified lysate suggests that the contaminants present in the lysate are successfully competing for the charged groups present on the surface of
the matrix. The different matrices were seen to form
into groups depending on their physical composition.
These results are consistent with a recent study using
confocal-scanning laser microscopy49, which indicated
adsorption in the outer layer of the media. These make
the studies with nitrocellulose membranes notable in
that hydrodynamic methods are available to bring the
single-stranded chromosomal DNA to the adsorbent
surface. However, as with all cross-flow membrane


systems, it is necessary to examine the level of shear

that is allowed before damage to the product occurs.
As is so often the case, nature suggests alternative
approaches to the plasmid-DNA size problem. In higher
organisms, DNA is formed into structures, such as
chromatin, which allows it to be maintained in a physically 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 compacting agents can be expected to interact heavily with
contaminants. The agent must not be too expensive
and should not interfere with selective separation by
techniques such as chromatography. Horn et al.50
described the use of concentrations of PEG (typically
,1% ) as an agent to be used before anion-exchange
chromatography. It is worth noting that chromatography, especially size exclusion, is associated with large
reductions in endotoxin levels14,51.
At formulation stage, the plasmid DNA has its greatest value, thus damage is particularly undesirable. Damage can occur in any step that introduces the product
into a high-velocity stream, such as those entailed in
drying and injection into vials. For plasmid DNA to be
effective, processing must include the formation of an
agent for its delivery. For vaccines, this can be small particles52; for gene therapy, DNA complexing with lipids
or peptides is most common53,54. The theme of size,
shape and shear sensitivity persists. For some time, it
has been known (in semi-quantitative terms) that spraying aerosols of plasmids into the airways of an animal
or human can lead to a substantial loss of activity55, and
this has been associated with a decrease in the supercoiled plasmid content. Plasmid damage is presumably
caused by shear, interfacial effects or a combination of
both. Mixing plasmid DNA and complexing agents,
such as lipids and polypeptides, to assist transfection56
can yield compact and shear-resistant particles. For
example, an examination of complexes of plasmid
DNA and polylysine and the subsequent application of
shear (Fig. 8) indicated that compaction does indeed
reduce damage. In terms of more-radical approaches to
the downstream processing of compacted DNA, phages
have been used as a vehicle for carrying DNA into
cells57,58. Phages are not completely insensitive to shear
themselves but their distinctive physical properties
allow a different approach to processing.
The sensitivity of macromolecules, such as proteins
and plasmid DNA, to combinations of shear and
gasliquid interfaces can pose problems if the plasmid
DNA is to be dried. Freeze drying can avoid such problems but the production of particles of a broad size
range might require subsequent milling that can cause
damage. The method of solution-enhanced dispersion
by supercritical fluids (SEDS) can overcome these difficulties. Studies with a 6.9 kb plasmid co-formulated
with mannitol showed no shear-related damage,
although it was necessary to counter the effective
acidity of the aqueous CO2 (Ref. 59). If plasmid DNA
is processed as a liquid, it is still possible to suffer losses
of active material if, for example, the fluid is injected
into vials from fine orifices. The levels of shear tolerated by flow from such small pipes have been defined
by Levy et al.24.
TIBTECH JULY 2000 (Vol. 18)



Sheared DNA

trends in Biotechnology

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 3 106 s21 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 and
sheared 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)

Biological approaches to enhance purification

As in other processes involving recombinant
materials, any biological solutions that can be applied
before downstream processing will be valuable. For
example, it is common in the processing of plasmids to
use ribonucleases to degrade RNA rather than removing it by conventional fractionation. A new approach60
allows recombinant bovine ribonuclease to be induced
in the plasmid-bearing strain. Maximizing plasmid
DNA levels and minimizing chromosomal DNA levels during and after cell growth will be useful. Enhancing the segregational stability of a plasmid ensures that
a high proportion of biomass contains plasmid DNA.
Plasmid-free cells will only contribute to contamination, which, as noted, is difficult to remove downstream.
The addition of antibiotics to select for plasmid-containing cells can alleviate plasmid instability and recent
studies on plasmid autoselection systems show that this
dependence on antibiotics can be removed61,62.
The plasmid-DNA production rate can also be
increased by using several plasmid-amplification strategies. Many recombinant plasmids replicate in a relaxed
mode, in which their replication can occur in the
absence of protein synthesis; by contrast, chromosomal
DNA replication is stringent, requiring protein
synthesis to occur. Plasmid DNA can be amplified by
either starving certain mutant host strains of amino
acids63 (and thereby stalling protein synthesis) or by
halting protein synthesis completely using antibiotics
such as chloroamphenicol63,64. However, antibiotic
clearance from the process stream might be a problem
from a regulatory point of view. Certain temperaturesensitive mutations, when introduced into the plasmidreplication control systems, can result in large increases
in plasmid-copy number upon undergoing a temperature shift during culture growth65. Runaway replication
vectors can be induced to amplify plasmid levels up to
1000 copies per cell66.
TIBTECH JULY 2000 (Vol. 18)

As mentioned previously, final-product-release specifications are likely to require .90% of the plasmid
DNA molecules to be in the supercoiled form14,67. These
forms are produced within the cell by the action of DNAsupercoiling enzymes, such as DNA topoisomerases,
which introduce negative superhelical twists into plasmid-DNA molecules. OKennedy et al.68 have shown
that the proportion of supercoiled forms can depend
strongly on the type of growth medium used. This
suggests that the proportion of supercoiled forms can
be modulated using different growth conditions and
could therefore be a possible target for recombinant
genetic modification. Supercoiled plasmids exhibit a
distribution with respect to the number of superhelical
twists that have been introduced, and this supercoiling
density can affect the transcription efficiency of plasmid-encoded genes. This is known to depend on factors
that include growth temperature and exposure to cold
or heat shocks69.
The biochemical engineering studies reviewed here
have created the foundation for the efficient isolation
of plasmid DNA. At present, scales and efficiencies are
modest; however, if DNA vaccines prove to be effective, the ultimate scale will be large. With tuberculosis,
often in forms that are resistant to drugs, causing three
million deaths per year worldwide, malaria (two
million deaths per year) and AIDS (one million deaths
per year), there is a great need for stable new vaccines;
an influenza epidemic could demand much-greater
quantities quickly3. Recent reports on the use of DNA
vaccines in animal studies are promising70, although the
situation with gene therapy is less clear at present.
However, the potential of gene therapy and DNA
vaccines to address currently incurable diseases indicates
a large possible demand. Given these two broad categories of potential use, the establishment of strong



biochemical engineering foundations will be important as a general guide to future processing. As indicated
in this review, the interactions between particular stages
are especially pronounced, a whole bioprocess approach
will therefore be even more important for DNA than
for proteins.
We 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 and
comments. We are also grateful for the support of
the Biotechnology and Biological Sciences Research
1 Liljeqvist, S. and Stahl, S. (1999) Production of recombinant subunit vaccines: protein immunogens, live delivery and nucleic acid
vaccines. J. Biotechnol. 73, 133
2 Friedman, T. (1997) The road toward human gene therapy a
25-year perspective. Ann. Med. 29, 575577
3 Robinson, H.L. et al. (1997) The scientific future of DNA for
immunization. Academy Colloquia Report The American Society for
Microbiology, Washington, DC, USA
4 Mountain, A. (2000) Gene therapy: the first decade. Trends Biotechnol.
18, 119128
5 Cohen, A.D. et al. (1998) Modulating the immune response to
genetic immunization. FASEB J. 12, 16111626
6 Newgard, C.B. (1992) Cellular engineering for the treatment of
metabolic disorders: prospects for therapy in diabetes. Biotechnology
10, 11121120
7 Yarmush, M.L. and Berthiaume, F. (1997) Metabolic engineering
and human disease. Nat. Biotechnol. 15, 525528
8 Westphal, E.M. et al. (1998) A system for shuttling 200-kb BAC/PAC
clones into human cells: stable extrachromosomal persistence and
long-term ectopic gene activation. Hum. Gene Ther. 9, 18631873
9 Willard, H.F. (1998) Human artificial chromosomes coming into
focus. Nat. Biotechnol. 16, 415416
10 Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res.
7, 15131523
11 Thatcher, D.R. et al. (1997) Method of plasmid DNA production
and purification. International Patent Application WO 97/29190
12 Nabel, G.J. et al. (1993) Direct gene transfer with DNAliposome
complexes in melanoma: expression, biologic activity, and lack of
toxicity in humans. Proc. Natl. Acad. Sci. U. S. A. 90, 1130711311
13 Schleef, M. (1999) Issues for large-scale plasmid DNA manufacturing. In Biotechnology: a Multi-Volume Comprehensive Treatise. Recombinant Proteins, Monoclonal Antibodies, and Therapeutic Genes (Vol. 5a)
(Rehm, H.J. and Reed, G., eds), pp. 443470, Wiley-VCH
14 Horn, N.A. et al. (1995) Cancer gene therapy using plasmid DNA:
purification of DNA for human clinical trials. Hum. Gene Ther. 6,
15 Lahijani, R. et al. (1998) Quantitation of host cell DNA contaminate in pharmaceutical-grade plasmid DNA using competitive
polymerase chain reaction and enzyme-linked immunosorbent assay.
Hum. Gene Ther. 9, 11731180
16 Noites, I.S. et al. (1999) Rapid quantitation and monitoring of
plasmid DNA using an ultrasensitive DNA-binding dye. Biotechnol.
Bioeng. 66, 195201
17 Levy, M.S. et al. Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method. Nucleic Acids
Res. (in press)
18 Charm, S.E. and Wong, B.L. (1981) Shear effects on enzymes.
Enzyme Microb. Technol. 3, 111118
19 Thomas, C.R. and Dunnill, P. (1979) Action of shear on enzymes:
studies with catalase and urease. Biotechnol. Bioeng. 21, 22792302
20 Virkar, P.D. et al. (1981) Studies of the effects of shear on globular
proteins: extension to high shear fields and to pumps. Biotechnol.
Bioeng. 23, 425429


21 Smith, E.L. et al. (1978) Principles of Biochemistry: General Aspects,

22 Ayazi Shamlou, P. and Titchener-Hooker, N. (1993) Turbulent
aggregation and breakup of particles in stirred vessels. In Processing of
SolidLiquid Suspensions (Ayazi Shamlou, P., ed.), pp. 124, Butterworth Heinemann
23 Ledley, F.D. (1996) Pharmaceutical approach to somatic gene therapy.
Pharm. Res. 13, 15951614
24 Levy, M.S. et al. (1999) Effect of shear on plasmid DNA in solution.
Bioprocess Eng. 20, 713
25 Hoare, M. et al. (1993) Interfacial damage of proteins during intensive mixing in fermentation and downstream processing. In Proceedings
of Stability and Stabilization of Enzymes (Van den Tweel, W.J. et al.,
eds), pp.2127, Elsevier Science
26 Ciccolini, L.A.S. et al. (1998) Time course of SDS-alkaline lysis of
recombinant bacterial cells for plasmid release. Biotechnol. Bioeng. 60,
27 Ciccolini, L.A.S. et al. (1999) Rheological properties of chromosomal and plasmid DNA during alkaline lysis reaction. Bioprocess Eng.
21, 231237
28 Carlson, A. et al. (1995) Mechanical disruption of Escherichia coli for
plasmid recovery. Biotechnol. Bioeng. 48, 303315
29 Lee, A.L. and Sagar, S. (1996) A method for large scale plasmid
purification. US Patent Application PCT/US95/08749
30 Wan, N.C. et al. (1997) Method for lysing cells. International Patent
Application WO 97/23601
31 Levy, M.S. et al. (1999) The effects of material properties and fluid
flow intensity on plasmid DNA recovery during cell lysis. Chem.
Eng. Sci. 54, 31713178
32 Bell, D.J. and Dunnill, P. (1982) The influence of precipitation
reactor configuration on the centrifugal recovery of isoelectric soya
protein precipitate. Biotechnol. Bioeng. 24, 23192336
33 Maybury, J. et al. (1998) The performance of a scaled down industrial disc stack centrifuge with a reduced feed material requirement.
Bioprocess Eng. 18, 231237
34 Theodossiou, I. et al. (1997) The processing of a plasmid-based gene
from Escherichia coli. Primary recovery by filtration. Bioprocess Eng.
16, 175183
35 Theodossiou, I. et al. (1999) Methods of enhancing the recovery of
plasmid genes from neutralised cell lysate. Bioprocess Eng. 20, 147156
36 Hayashizaki, Y. (1997) Method for the purification of DNA.
European Patent Application EP/0814156A2
37 Horn, N. et al. (1996) Process for reducing RNA concentration in
a mixture of biological material using diatomaceous earth. International Patent Application WO 96/21729
38 Bussey, L. et al. (1998) Methods for purifying nucleic acids. International Patent Application WO 98/05673
39 Levy, M.S. et al. (2000) Removal of contaminant nucleic acids by
nitrocellulose filtration during pharmaceutical plasmid DNA processing. J. Biotechnol. 76, 197205
40 Marquet, M. et al. (1995) Production of pharmaceutical-grade
plasmid DNA. International Patent Application WO 95/21250
41 Lis, J.T. (1980) Fractionation of DNA fragments by polyethylene
glycol induced precipitation. Methods Enzymol. 65, 347353
42 Lis, J.T. and Schleif, R. (1975) Size fractionation of double stranded
DNA by precipitation with polyethylene glycol. Nucleic Acids Res.
2, 383389
43 Chen, Z. and Ruffner, D. (1998) Compositions and methods for
rapid isolation of plasmid DNA. International Patent WO 98/16653
44 Olmsted, M.C. and Hagerman, P.J. (1994) Excess counterion accumulation around branched nucleic acids. J. Mol. Biol. 243, 919929
45 McNeilly, D. (1999) Method for purifying plasmid DNA and plasmid DNA substantially free of genomic DNA. International Patent
WO 99/29832
46 Murphy, J.C. et al. (1999) Purification of plasmid DNA using selective precipitation by compaction agents. Nat. Biotechnol. 17, 822823
47 Wils, P. et al. (1997) Efficient purification of plasmid DNA for gene
transfer using triple-helix affinity chromatography. Gene Ther. 4,
48 Prazeres, D.M.F. et al. (1998) Preparative purification of supercoiled
plasmid DNA using anion exchange chromatography. J. Chromatogr.
A 806, 3145
TIBTECH JULY 2000 (Vol. 18)

49 Ljunglof, A. et al. (1999) Direct visualisation of plasmid DNA in
individual chromatography adsorbent particles by confocal scanning
laser microscopy. J. Chromatogr. A 844, 129135
50 Horn, N. et al. (1998) Purification of plasmid DNA during column
chromatography. US Patent US/5707812
51 Durland, R.H. and Eastman, E.M. (1998) Manufacturing and quality
control of plasmid-based gene expression systems. Adv. Drug Del.
Rev. 30, 3348
52 McCabe, D.E. (1999) Gas driven gene delivery instrument. US
Patent US/5865796
53 Chonn, A. and Cullis, P.R. (1995) Recent advances in liposomal
drug-delivery systems. Curr. Opin. Biotechnol. 6, 698708
54 Yanez, R.J. and Porter, A.C.G. (1998) Therapeutic gene targeting.
Gene Ther. 5, 149159
55 Crook, K. et al. (1996) Plasmid DNA molecules complexed with
cationic liposomes are protected from degradation by nucleases and
shearing by aerosolisation. Hum. Gene Ther. 3, 834839
56 Tsai, J.T. et al. (1999) Characterisation of plasmid DNA conjugates
as a basis for their processing. Bioprocess Eng. 21, 279286
57 Larocca, D. et al. (1998) Targeting bacteriophage to mammalian cell
surface receptors for gene delivery. Hum. Gene Ther. 9, 23982399
58 Perham, R.N. et al. (1997) Engineered bacteriophages and the
vaccines containing them. European Patent EP/0552267 B1
59 Tservistas, M. et al. (1999) Supercritical fluids for dry powder formulation of plasmid DNA. Proceedings of Ninth European Congress of




Biotechnology (ECB9) (Hofman, M., ed.) Branche Belge de la Societe

de Chimie Industrielle
Hanak, J.A.J. et al. (1999) Purification of cellular components that
are substantially RNA free. International Patent WO/9953018
Sherratt, D.J. et al. (1999) Plasmid stabilization. US Patent US/5972708
Reinikainen, P. et al. (1989) Escherichia coli plasmid production in a
fermenter. Biotechnol. Bioeng. 33, 386393
Hecker, M. et al. (1983) Replication of pBR322 DNA in stringent
and relaxed strains of Escherichia coli. Mol. Gen. Genet. 190, 355357
Clewell, D.B. (1972) Nature of ColE1-plasmid replication in Escherichia
coli in the presence of chloroamphenicol. J. Bacteriol. 110, 667676
Lahijani, R. et al. (1996) High-yield production of pBR322-derived
plasmids intended for human gene therapy by employing a temperature-controllable point mutation. Hum. Gene Ther. 7, 19711980
Leipold, R.J. et al. (1994) Mathematical model for temperaturesensitive plasmid replication. Plasmid 32, 131167
Middaugh, C.R. et al. (1998) Analysis of plasmid DNA from a pharmaceutical perspective. J. Pharm. Sci. 87, 131146
OKennedy, R.D. et al. (2000) Effects of growth medium selection
on plasmid DNA production and initial processing step. J. Biotechnol.
76, 175183
Mizushima, T. et al. (1997) Increase in negative supercoiling of plasmid
DNA in Escherichia coli exposed to cold shock. Mol. Microbiol. 23, 381386
Lowrie, D.B. et al. (1999) Therapy of tuberculosis in mice by DNA
vaccination. Nature 400, 269271

Chitin deacetylases: new, versatile tools in

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.

hitin, a homopolymer comprising b-(1-4)-linked

N-acetyl-D-glucosamine residues is one of the
most abundant, easily obtained and renewable
natural polymers, second only to cellulose. It is commonly found in the exoskeletons or cuticles of many
invertebrates and in the cell walls of most fungi1.
Because of its high crystallinity, chitin is insoluble in
aqueous solutions and organic solvents2.
Chitosan is a polycationic biopolymer that occurs
naturally or is obtained by the N-deacetylation of
chitin; its name does not refer to a uniquely defined
compound but rather to a family of copolymers with

I. Tsigos, A. Martinou and D. Kafetzopoulos are at the Institute of

Molecular Biology and Biotechnology, Foundation of Research and
Technology, Crete, Greece. V. Bouriotis (bouriotis@imbb.forth.gr) is
at the Division of Applied Biology and Biotechnology, Department of
Biology, University of Crete, Crete, Greece.
TIBTECH JULY 2000 (Vol. 18)

various fractions of acetylated units. It is biodegradable,

non-toxic to animals (in mice, the LD50 was .16 g kg21),
soluble in acidic solutions, available in various physical
forms and much more tractable than chitin2,3. Thus,
chitosan offers properties with great potential for many
industrial applications.
Today, several companies are producing chitin and
chitosan products on a commercial scale; the majority
are located in Japan, where .100 billion tons of
chitosan are manufactured each year from the shells of
crabs and shrimps, an amount that accounts for ~90%
of the global chitosan market (approximately four trillion yen). The major areas of application include water
treatment, biomedical applications (including wound
dressing and artificial skin) and personal-care products213.
In addition, oligomers of chitin and chitosan have
also attracted considerable attention because they have
been reported to exhibit certain interesting physiological

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