Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PURIFICATION OR/AND CONCENTRATION OF DNA BY CROSS-FLOW
FILTRATION, SEPARATION OF ENDOTOXINS FROM A NUCLEIC ACID
PREPARATION
Description
The present invention concerns a method for purifying
or/and concentrating nucleic acids in a solution, a
method for separating endotoxins from a nucleic acid
preparation and the use of a cross-flow filtration
system to purify or/and concentrate nucleic acids in a
solution and to separate endotoxins from a nucleic acid
preparation.
Nucleic acid purification methods are common methods in
the field of molecular biology. In methods known from
the prior art the isolated biological material, such as
E. coli bacterial cells is for example centrifuged after
they have been lysed (usually lysis with lysozyme or
ultrasound) and the supernatant is shaken out with
phenol. Subsequently an ultracentrifugation is carried
out on a caesium chloride gradient (Birnboim & Doly,
Nucl.Acid Res. 7 (1979) 1513-1523; Garger et al.,
Biochem.Biophys.Res.Comm. 117 (1983) 835-842). However,
such preparations usually contain bacterial endotoxins,
phenol, caesium chloride and/or ethidium bromide as
impurities.
Endotoxins are bacterial toxins that are found in all
enterobacterioceae e.g. Salmonella, Shigella and E.
coli. In mammals endotoxins act as a pyrogen and, in
addition to fever, have numerous other pathological
effects. The component lipid A is especially responsible
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t.for the toxic effect of endotoxins.
Another method for purifying nucleic acids is described
in the QIAGEN~ Plasmid Handbook (Qiagen Inc.,
Chatsworth, USA) and in EP-B 0 268 946. According to
this the cell lysate obtained by the usual lysis is
chromatographed on a QIAGEN~ TIP which contains QIAGEN~
resin (a support material based on silica gel). A
disadvantage of this method is that DNA binding proteins
are not completely detached from the DNA so that the
plasmid preparation that is obtained contains a
considerable amount of proteins and in particular
endotoxins (e. g. from the membrane of the E. coli cell).
In another nucleic acid purification method after
alkaline lysis of the biological material for example E.
coli cells the centrifugation supernatant is
chromatographed according to Birnboim % Doly under high
salt conditions over anion exchange columns (e. g. Mono-
Q, Source-Q from Pharmacia, Macroprep-Q from Biorad,
Poros-Q from Perspective Biosystems or HyperD-Q from
Biosepra, cf. Chandra et al., Analyt. Biochem. 203
(1992) 169-172; Dion et al., J.Chrom. 535 (1990) 127-
147) . Even after this purification step the plasmid
preparation still contains impurities such as proteins
and especially a considerable amount of endotoxins.
In yet another method for purifying nucleic acids a
chromatography is carried out by gel filtration after
alkaline lysis and subsequent phenol-chloroform
extraction (McClung & Gonzales, Anal.Biochem. 177 (1989)
378-382; Raymond et al., Analyt.Biochem. 173 (1988) 124-
133). This purification method is also not able to
completely remove the impurities from the plasmid
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preparation.
The said purification methods all have a final desalting
and concentration step. This usually involves an
isopropanol/ethanol precipitation of the nucleic acid
with subsequent centrifugation and resuspension of the
nucleic acid pellet in buffer (cf. e.g. Sambrook J. et
al. (1989), Molecular Cloning; A Laboratory Manual, 2nd
Ed., Cold Spring Harbor Laboratory Press). In this
process a DNA solution is for example admixed with 1/10
volumes 4 M LiCl and subsequent with 0.7 volumes
isopropanol at room temperature. Subsequently the
precipitate that forms of the nucleic acid is
centrifuged and the supernatant is discarded. The pellet
which contains the nucleic acid is taken up in 70
ethanol in a subsequent step, centrifuged again, the
supernatant is discarded and, after drying the pellet,
it is resuspended in a desired buffer. However, the
isopropanol/ethanol precipitation method is only
practical for applications in a laboratory where
relatively small volumes are used.
Apart from the limitation to small volumes, the
described isopropanol/ethanol precipitation method has
other serious disadvantages. Thus for reasons of
operational safety and environmental protection it is
very unfavourable to use the required isopropanol/
ethanol volumes on an industrial process scale.
Furthermore, the isopropanol/ethanol precipitation
method does not fulfil the technical requirements for
providing nucleic acids that can be used therapeutically
since endotoxins contained in the nucleic acid solution
cannot be completely removed by this method.
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method for isolating and purifying nucleic acids for
use in gene therapy is described in WO 95/21177 in which
the purification is essentially by centrifugation,
filtration, an affinity chromatography or a
chromatography on an inorganic chromatographic material
and a chromatography on an ion exchanger. In order to
remove endotoxins the nucleic acid is treated with an
endotoxin removal buffer which contains 10 % Triton~
X100 and 40 mmol/1 MOPS buffer (3-morpholino-1-propane
sulfonate). A disadvantage of this method is that the
nucleic acid purified in this manner is contaminated
with the pharmacologically unsafe substances Triton~ and
MOPS. In addition, although it is possible to deplete
endotoxins to a content of ca. 100 EU/mg DNA (QIAGEN
News 1-96, 3-5), a more extensive removal of endotoxins
is not possible. However, nucleic acid preparations with
an even higher purity are required for a therapeutic
application, like for example a gene therapy, which are
as free as possible of all impurities (in particular
substantially free of endotoxins). Above all the
endotoxin content of plasmid DNA preparations has been a
hitherto unsolved problem as for example described by
Cotten et al., Gene Therapy 1 (1994) 239 - 246.
K.-G.Wahlund and A. Litzen (Journal of Chromatography,
461 (1989), 73-87; 476 (1989), 413-421) describe a
method named "field flow fractionation (FFF)" suitable
for analytical and micropreparative applications for
separating protein mixtures and plasmids according to
their respective molecular weights. As in a cross-flow
filtration the approach flow on the ultrafiltration
membrane is tangential but, in contrast to cross-flow
filtration, the separation is based on the different
migration of the molecules in the stream of carrier
fluid. Hence the elution of the molecules to be
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separated depends on their molecular size and the
correlating diffusion coefficients. The separation is
not continuous i.e. the molecules to be separated flow
over the membrane only once during the separation
process.
F.M. Fernandez, J.M. Romano and M.A. Otero (Acta
Biotechnol. 12 (1992) 1, 49-56) describe the
concentration of RNA in solution in a cross-flow
filtration method with hollow fibre membranes. However,
the purification of DNA or plasmid DNA is neither
described nor made obvious.
G.W. Rembhotkar and G.S. Khatri (Analytical Biochemistry,
176, 337-374 (1989)) describe the purification of a
phage lysate by means of tangential flow filtration. A
subsequent ~ phage DNA preparation is carried out with
common methods using chloroform, phenol-chloroform
treatment and ethanol precipitation.
A method for the isolation and purification of plasmid
DNA from microorganisms is described in WO 96/36706 and
96/02658 A1 which were produced on a large scale. The
cells are lysed by adding a lysis solution and heating to
70°C to 100°C in a flow heat exchanger and subsequently a
clear supernatant is obtained by batch-wise
centrifugation and diafiltration. The diafiltration is
carried out in the dead-end modus but not by tangential
overflow of the membrane according to a cross-flow
fraction method. Afterwards a further purification is
carried out by anion exchange chromatography and reversed
phase HPLC. In this method only the cell lysis is carried
out in a continuous process step, the further
purification of the plasmid DNA is carried out in a batch
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a manner in common centrifuges and diafiltration devices.
In WO 97/29113 the purification of nucleic acid
preparations by an anion exchanger using a high salt
gradient is suggested in order to obtain nucleic acid
solutions which have a protein content of less than
0.1 %, are free of impurities such as ethidium bromide,
phenol, caesium chloride and detergents and are
substantially free of endotoxins.
Hence in the prior art neither methods are known which
would enable large amounts of nucleic acids to be
purified or concentrated nor are methods known which are
suitable for preparing nucleic acids in large amounts and
in a highly purified form which are in particular free of
endotoxins that would be suitable for a therapeutic
application.
One of the objects of the present invention was
therefore to provide a purification and concentration
method which enables nucleic acids to be purified in
large amounts in a simple manner. An additional object
of the present invention was to reduce the concentration
of endotoxins in a nucleic acid preparation to such an
extent that it is suitable for a therapeutic
application.
A first aspect of the present invention concerns a method
for purifying or/and concentrating nucleic acids in a
solution which is characterized in that the solution
containing the nucleic acid is guided tangentially past
one or several semi-permeable membranes such that the
nucleic acid molecules are retained by the membranes and
substances with a lower molecular weight can pass through
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.the membranes so that a purified or/and concentrated
nucleic acid solution is obtained.
A further aspect of the present invention concerns a
method for separating endotoxins from a nucleic acid
preparation which is characterized in that the
preparation containing nucleic acid is guided
tangentially past one or several semi-permeable membranes
such that the nucleic acid molecules are retained by the
membranes and substances with a lower molecular weight
can pass through the membranes or/and are adsorbed by the
membrane to obtain an essentially endotoxin-free nucleic
acid solution.
It has now been found that nucleic acid solutions can be
purified and concentrated with the method of the present
invention using a cross-flow filtration system. In this
connection a surprising and new feature is that the
nucleic acids are not damaged by the cross-flow
filtration (CFF). Previously it has always been assumed
that the shear forces occurring in the CFF would lead to
damage of nucleic acids in particular to strand breaks.
Therefore CFF was previously only used to concentrate and
diafiltrate proteins. In addition the method of the
invention not only enables nucleic acids to be obtained
in large amounts and of a desired purity, but the method
of the present invention also avoids the use of organic
solvents which is a major advantage toxicologically as
well as with regard to safety and environmental aspects.
Surprisingly it was also found that nucleic acid
preparations can also be obtained by the method of the
present invention which are substantially free of
impurities and in particular of endotoxins so that they
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can be used in therapeutic methods or applications. Thus
the present invention solves the problem that has been
created by the demand for strongly increasing amounts of
nucleic acids that can be used therapeutically and in
particular of DNA that can be used therapeutically which
can be expected to increase further in the future.
The method of the present invention is used to purify
or/and concentrate linear or circular nucleic acids,
preferably plasmid DNA and most preferably circular
plasmid DNA. In this connection the size of the nucleic
acid is preferably in the range of > 150 base pairs,
particularly preferably in the range of 1 kbp - 200 kbp.
In the method according to the invention the nucleic acid
can be purified or/and concentrated batch-wise but the
method is preferably carried out continuously. Any volume
size can be processed but it is preferable to process a
solution with a volume of 1 to 10,000 1, particularly
preferably of 1 - 100 1. The solution containing the
nucleic acids is guided past the membrane or membranes
under suitable pressure conditions whereby the cross-flow
pressure is preferably larger than the transmembrane
pressure. It is particularly preferable to operate at a
transmembrane pressure of 0.2 to 3.0 bar and most
preferably of 0.8 - 1.5 bar in which case the cross-flow
pressure is larger than the transmembrane pressure. The
retentate flow rate (RF) can be varied over a wide range
and it is preferably to operate with an RF of 100 1/h~m2
to 4,000 1/h~m2. The process can also be carried out at
varying temperatures and it is preferable to work in a
temperature range of 4°C - 25°C.
In order to separate the solution containing nucleic
acids from low molecular impurities and in particular
from endotoxins, common membranes are used such as
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membranes made of polyether sulfone (PES), modified PES,
polyvinylidene difluoride (PVDF), cellulose triacetate or
regenerated cellulose. Hollow fibre coil modules are also
suitable for the method according to the invention.
Membranes with an exclusion size of 1 - 1000 kilodalton
(kD) are preferably used, 10 - 300 kD is more preferred
and 10 - 100 kD is most preferred. The endotoxin
depletion factor (ratio of endotoxin content of the
nucleic acid preparation before cross-flow filtration to
the endotoxin content of the nucleic acid solution after
cross-flow filtration) that is achieved in the present
invention is at least 10 . 1, preferably at least 200 .
1. The endotoxin content of the solution is very low
after cross-flow filtration and is preferably < 0.1 EU/mg
nucleic acid. The nucleic acids obtained in the present
invention are essentially undamaged and essentially have
no single-strand or double-strand breaks.
In particular a plasmid DNA purified according to the
invention exhibits only~one dominant band after gel
electrophoretic separation which corresponds to the
"covalently closed circle" conformation. Furthermore,
apart from small amounts of the open circle and
linearized circle conformations, no other bands are
present.
A further aspect of the present invention concerns the
use of a cross-flow filtration system to purify or/and to
concentrate nucleic acids in a solution.
Yet a further aspect of the present invention concerns
the use of a cross-flow filtration system to separate
endotoxins from a nucleic acid preparation.
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Yet a further aspect of the previous invention concerns
the use of the nucleic acids purified or/and concentrated
by cross-flow filtration for cloning, transformation,
transfection, microinjection into cells, for use in
methods of gene therapy, DNA vaccination or/and for the
polymerase chain reaction (PCR).
Examples
In the described experiments membranes of the OMEGA type
made of modified polyether sulfone from the Filtron
Company (order No. 8S 100C01 exclusion size 100 kD),
membranes made of cellulose acetate from the Sartorius
Company or PVDF membranes from the Millipore Company were
used. A membrane with an exclusion limit of 100 kilodalton
was used in particular for endotoxin separation. In order
to check the separation of endotoxins, E-toxate~ from the
Sigma Company (order No. 210) was used as a spiking
solution. The endotoxins were tested by the solid gel
method in which the addition of a solution containing
endotoxin to a limulus amoebocyte lysate solution (LAL
solution) leads to a gel formation of the mixture. The gel
formation is due to a coagulation cascade that occurs in
several steps.
1. Cross-flow filtration (CFF) of a plasmid DNA solution
In order to examine CFF as a method for concentrating
plasmid DNA, a production preparation of 2000 g E. coli
biomass is lysed by alkali lysis, processed by Q-Sepharose*
and hydroxylapatite chromatography and the plasmid DNA
solution that is obtained is used as a starting solution
in the CFF.
*Trademark
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a.1.1 Production of a starting solution
2000 g wet E. coli biomass from the fermenter is filled
into depyrogenized centrifuge beakers. 22.5 1
resuspension buffer (50 mmol/1 Tris-HC1, 10 mmol/1 EDTA-
Na2, pH 8 + 0.2) is added and slowly stirred (ca. 35 rpm)
for at least 24 hours at 5 + 4°C until the biomass is
completely suspended. In this process the temperature of
the suspension is slowly increased to 25°C.
22.5 1 0.2 mol/1 NaOH, 1 % SDS is added to the suspension
while stirring at ca. 80 rpm and incubated for 5 minutes
at 25°C. 22.5 1 potassium acetate buffer (3 mol/1
potassium acetate buffer pH 5.5) is added while stirring
and the temperature of the biomass is reduced as rapidly
as possible to 4°C. The biomass is centrifuged for 30
minutes at 26,000 x g and 4°C. The supernatant which
contains the plasmid DNA is isolated and filtered clear
over a 5 ~Cm candle filter.
In the next step a chromatography on Q-Sepharose* is
carried out. The decanted centrifuge supernatant is
adjusted to a conductivity of 49 - 50 mS/cm by addition
of TE buffer (10 mmol/1 Tris-HC1, 1 mmol/1 EDTA pH 8.5 +
0.2) and cooled tp 5 + 4°C. The entire chromatography is
carried out at this temperature. The centrifugation
supernatant is absorbed to the equilibrated column.
Subsequently the column is washed with ca. 8 CV 10 mmol/1
Tris-HCl, 1 mmol/1 EDTA, 0.65 mol/1 NaCl pH 8.5 + 0.2.
For the elution a gradient (5 CV buffer A (10 mmol/1
Tris-HC1, 1 mmol/1 EDTA, 0.65 mmol/1 NaCl, pH 8.0 + 2),
CV buffer B (10 mmol/1 Tris-HC1, 1 mmol/1 EDTA,
0.85 mol/1 NaCl pH 8.0 ~ 0.2)) is applied to the column
*Trademark
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~ and the eluate is fractionated at a flow rate of 5 to 8
CV/h, the detection is carried out at 254 nm. The prepeak
(impurities) is separated from the main peak (plasmid
DNA) by collecting the main peak in a separate vessel
starting from the ascending flank.
Subsequently a chromatography on hydroxylapatite (HA
ceramic) is carried out at 5 + 4°C.
Equilibration buffer: 0.1 mol/1 potassium phosphate,
6 mol/1 urea pH 7.0 + 0.2.
Wash buffer 1: 0.15 mol/1 potassium. phosphate, 6 mol/1
urea pH 7.0 ~ 0.2.
Wash buffer 2: 0.02 mol/1 potassium phosphate buffer
pH 7.0 ~ 0.2.
Elution buffer: 0.5 mol/1 potassium phosphate pH 7.0 +
0.2.
The detection is carried out at 254 nm using a W
detector/recorder unit. A 1 % product solution (plasmid
DNA) is used as a calibration solution that was measured
with a calibrated photometer.
The Q-Sepharose* pool is adjusted to a final concentration
of 1.1 mmol/1 calcium chloride and absorbed onto the
equilibrated column.
Then the column is successively washed at a flow rate of
5-8 CV/h with:
*Trademark
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1. 0.1 mol/1 potassium phosphate, 6 mol/1 urea pH 7.0
~ 0.2 until an absorbance is no longer detectable
at the detector.
2. 2-4 CV, 0.15 mol/1 potassium phosphate, 6 mol/1
urea pH 7.0 ~ 0.2
3. 5 CV, 0.02 mol/1 potassium phosphate pH 7.0 + 0.2.
It is eluted with 0.5 mol/1 potassium phosphate buffer pH
7.0 + 0.1 after the wash steps. The peak is collected and
used as a plasmid DNA starting solution in the CFF.
1.2 Cross-flow filtration
The plasmid DNA starting solution has a plasmid DNA
concentration of ca. 200 ug/ml and a volume of ca.
3750 ml. The CFF is carried out at a retentate flow rate
of 100-200 1/h~m2, a transmembrane pressure of ca. 0.8
bar and a cross-flow pressure of ca. 1.2 bar. The volume
is concentrated to ca. 50 ml with the aid of the CFF and
retentate is subsequently diafiltered against TE buffer
(10 mmol/1 Tris-HC1, 1 mmol/1 EDTA, pH 8.0) until the
values for pH and conductivity of the retentate and TE
buffer agree. After completion of the diafiltration
process the retentate is adjusted to a plasmid DNA
concentration of 1 mg/ml by dilution with diafiltration
buffer. A sample of the prepared plasmid DNA solution is
taken and this is analysed as described under item 3.1.
2. Measurement of the endotoxin depletion by CFF
A plasmid DNA solution with a volume of 100 ml is
supplemented with 1000 EU of the E-toxate~ endotoxin
standard solution to 10 EU/ml and then used as a
starting solution in the experiment. The solution is
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diluted to 1000 ml with TE buffer and then again
concentrated with the aid of CFF to its initial volume
of 100 ml (concentrate 1). The dilution and
concentration step is repeated four times in succession.
After each concentration step a sample is taken from
each concentrate (samples: concentrate 2, 3, 4, 5) and
the endotoxin concentration of the sample is analysed
with the limulus-amoebocyte lysate method.
3. Results
3.1 CFF of the plasmid DNA solution
The plasmid DNA solution can be concentrated and
diafiltered without difficulty using the CFF. The
results of the examination are summarized in the
following table.
Parameter PLASMID DNA PLASMID DNA Diafiltration
initial solutionafter CFF buf f er ( TE
(HA pool)
10 mmol/l Tris-HCI,
1 mmol/I EDTA,
pH 8.0
Volume (ml) 3750 505 -
OD260/280 1. 89 1. 90 -
Conduct ivity2 6 . 5 1. 11 1. 11
(mS/cm)
pH 6.99 7.93 7.98
Yield (mg) 763 666 -
An aliquot of the plasmid DNA after completion of the
CFF is applied at various concentrations to an agarose
gel. The agarose gel that is shown shows the DNA length
standard No. II (fragment sizes: 125, 564, 2027, 2322,
4361, 6557, 9416, 23130 bp) in lanes 1 and 10 and the
DNA length standard No. III (fragment sizes: 125, 564,
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1831, 647, 1375, 1584, 1904, 2027, 3530, 4268, 4973,
5148, 21226 bp) in lanes 2 and 9. pBR322 (4162 bp) is
applied as a reference plasmid in lane 3 which was
purified by a conventional caesium chloride gradient
method. It is known that plasmid DNA purified by this
method essentially contains plasmid DNA which
corresponds to the covalently closed circle conformation
(dominant supercoiled band). The plasmid DNA (pCMV-CAT)
purified by the method according to the invention is
applied in different amounts in lanes 4, 5 and 6. The
plasmid DNA purified according to the invention, like
the reference plasmid DNA (lane 3), essentially shows a
dominant band. This plasmid DNA band corresponds to the
covalently closed circle conformation (dominant
supercoiled band). This shows that the plasmid DNA
isolated according to the invention is not damaged and
retains its original conformation. This therefore rules
out the possibility that the plasmid DNA is fragmented
during the CFF or is converted into an undesired plasmid
DNA conformation.
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{ Legend
1% agarose gel
Lane 1: DNA length standard II (Boehringer Mannheim GmbH;
Cat. No. 236250)
Lane 2: DNA length standard III (Boehringer Mannheim GmbH,
Cat. No. 528552).
Lane 3: pBR322 (Boehringer Mannheim GmbH, Cat. No. 481238)
(0.4 ~cg)
Lane 4: pCMV-CAT after CFF, 0.19 ~,g (bulk active substance
solution)
Lane 5: pCMV-CAT after CFF, 0.45 ~Cg (bulk active substance
solution)
Lane 6: pCMV-CAT after CFF, 0.71 ~Cg (bulk active substance
solution)
Lane 7: TE buffer
Lane 8: pBR322 (Boehringer Mannheim GmbH, Cat. No. 481238)
(0.4 ~cg)
Lane 9: DNA length standard III (Boehringer Mannheim GmbH;
Cat. No. 528552)
Lane 10: DNA length standard II (Boehringer Mannheim GmbH,
Cat. No. 236250) .
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3.2 Endotoxin depletion by CFF
The following table shows that the endotoxins are
already substantially removed after the first
concentration step. The additional CFF reduces the
endotoxin concentration down to the detection limit of
the test method.
Sample Retentate volume Measured endotoxin
[ml] concentration in
the retentate
[EU/ml]
Initial solution 100 6-12
Concentrate 1 100 0.06 - 0.60
Concentrate 2 100 0.06 - 0.60
Concentrate 3 100 0.06 - 0.60
Concentrate 4 100 0.06 - 0.60
'Concentrate 5 100 < 0.06
Diafiltration buffer - < 0.06
