U.S. patent application number 10/771371 was filed with the patent office on 2004-10-21 for process for preparing purified nucleic acid and the use thereof.
This patent application is currently assigned to Roche Diagnostics GmbH. Invention is credited to Kuhne, Wolfgang.
Application Number | 20040209835 10/771371 |
Document ID | / |
Family ID | 8222465 |
Filed Date | 2004-10-21 |
United States Patent
Application |
20040209835 |
Kind Code |
A1 |
Kuhne, Wolfgang |
October 21, 2004 |
Process for preparing purified nucleic acid and the use thereof
Abstract
The invention relates to a nucleic acid preparation with a
content of below 1% protein, preferably below 0.1% protein, free of
ethidium bromide, phenol, cesium chloride and detergents based on
octyl phenol poly(ethylene glycol ether)n and with a content of
below 1 EU/mg DNA of endotoxins. Said preparation is suitable as a
drug particularly in gene therapy.
Inventors: |
Kuhne, Wolfgang; (Penzberg,
DE) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Roche Diagnostics GmbH
Mannheim
DE
|
Family ID: |
8222465 |
Appl. No.: |
10/771371 |
Filed: |
February 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10771371 |
Feb 5, 2004 |
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09117537 |
Aug 4, 1998 |
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6750333 |
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09117537 |
Aug 4, 1998 |
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PCT/EP97/00321 |
Jan 24, 1997 |
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Current U.S.
Class: |
514/44R ;
536/23.1 |
Current CPC
Class: |
A61K 48/005 20130101;
A61P 11/16 20180101; A61P 11/00 20180101; C12N 15/101 20130101;
A61K 48/0091 20130101; C12N 15/85 20130101 |
Class at
Publication: |
514/044 ;
536/023.1 |
International
Class: |
C07H 021/04; A61K
048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 6, 1996 |
EP |
96101628.4 |
Claims
1-10. (Canceled)
11. DNA preparation comprising a DNA, proteins in an amount of less
than about 0.1% DNA and endotoxins in an amount of less than about
1 EU/mg DNA.
12. The DNA preparation of claim 11, comprising endotoxins of less
than about 0.06 EU/mg DNA.
13. The DNA preparation of claim 11, comprising endotoxins of about
0.01 to 0.1 EU/mg DNA.
14. The DNA preparation of claim 11, wherein the DNA preparation is
free of ethidium bromide, phenol, cesium chloride,
octylphenolpoly(ethylene glycol ether)n detergents and MOPS
buffer.
15. The DNA preparation of claim 11, wherein the DNA can be
replicated in gram-negative bacteria.
16. The DNA preparation of claim 15, wherein the gram-negative
bacteria is Escherichia coli.
17. The DNA preparation of claim 11, wherein the DNA is plasmid
DNA.
18. The DNA preparation of claim 17, wherein the plasmid DNA is
capable of replication.
19. A pharmaceutical composition suitable for gene therapy
comprising a therapeutically effective amount of the DNA
preparation of claim 15 and a pharmaceutically acceptable
carrier.
20. The composition of claim 19, wherein the DNA preparation
contains endotoxins in an amount of less than about 0.06 EU/mg
DNA.
21. The composition of claim 19, wherein the DNA preparation
contains endotoxins in an amount of about 0.01 to 0.1 EU/mg
DNA.
22. The composition of claim 19, wherein the DNA is a plasmid
DNA.
23. The composition of claim 22, wherein the plasmid DNA is
encapsulated in liposomes.
24. A method for gene therapy of cystic fibrosis caused by the
absence of a normal first gene or the presence of a defective
second gene, comprising administering an effective amount of the
DNA preparation of claim 18 to a patient in need thereof, wherein
the plasmid DNA contains said normal first gene or a normal second
gene corresponding to the defective second gene.
25. A process for making the DNA preparation of claim 11,
comprising the following steps (a) providing gram-negative bacteria
containing said DNA; (b) lysing said bacteria to obtain a lysate,
wherein the lysate is a DNA-containing fraction and thereafter (c)
chromatographing said DNA-containing fraction on hydroxylapatite in
order to obtain said DNA preparation.
26. The method of claim 25, further comprising replicating said DNA
in gram-negative bacteria after step (a) and before step (b).
27. The method of claim 26, further comprising transfecting the
gram-negative bacteria with a cloning vector containing said DNA
before step (a).
28. The method of claim 26, further comprising eluting said
hydroxylapatite in step (c) with a solution of phosphate, citrate,
sulfate or divalent metal ions to obtain said DNA preparation.
29. The method of claim 25, further comprising, after step (b) and
before step (c), filtering the lysate to obtain a filtrate and
fractionating said filtrate by gel filtration to obtain said
DNA-containing fraction.
30. The method of claim 26, further comprising, after step (b) and
before step (c), filtering the lysate to obtain a filtrate and
fractionating said filtrate by gel filtration to obtain said
DNA-containing fraction.
31. The method of claim 27, further comprising, after step (b) and
before step (c), filtering the lysate to obtain a filtrate and
fractionating said filtrate by gel filtration to obtain said
DNA-containing fraction.
32. The method of claim 28, further comprising, after step (b) and
before step (c), filtering the lysate to obtain a filtrate and
fractionating said filtrate by gel filtration to obtain said
DNA-containing fraction.
Description
[0001] The invention concerns the preparation of purified nucleic
acid and its use especially in gene therapy.
[0002] Replicatable nucleic acid is usually produced by amplifying
replicatable plasmid DNA in gram-negative bacteria such as e.g. E.
coli. After lysis of the biomass (usually alkaline lysis with
lysozyme or ultrasound), it is centrifuged and the supernatant is
shaken out with phenol subsequently an ultracentrifugation on a
caesium chloride gradient is carried out (Birnboim & Doly,
Nucleic Acid Res. 7 (1979) 1513-1523, Garger et al., Biochem.
Biophys. Res. Comm. 117 (1983) 835-842). However, such preparations
contain endotoxins, phenol, caesium chloride and/or ethidium
bromide as a dye.
[0003] A further process is described in the QIAGEN.RTM. Plasmid
Handbook (Qiagen Inc., Chatsworth, USA) and EP-B 0 268 946.
According to this process the cell lysate obtained after a
conventional lysis is chromatographed on QIAGEN.RTM. -TIP, which
contains QIAGEN.RTM. resin (a support material based on silica).
The disadvantage of this process is that DNA binding proteins are
not completely detached from the DNA and therefore the purified
plasmid fraction contains proteins and in particular endotoxins
(from the membrane of the gram-negative host cells) in considerable
amounts.
[0004] In another process after alkaline lysis of the E. coli
biomass 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 Perseptive 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). Also in this case the
purified plasmid fraction contains proteins and in particular
endotoxins in considerable amounts.
[0005] In another process after alkaline lysis and subsequent
phenol/chloroform extraction it is possible to chromatograph by gel
filtration (McClung & Gonzales, Analyt. Biochem. 177 (1989)
378-382; Raymond et al., Analyt. Biochem. 173 (1988) 125-133). Even
after this purification the plasmid preparation contains impurities
and in particular phenol.
[0006] A process for the isolation and purification of nucleic
acids for use in gene therapy is described in WO 95/21177 in which
the purification is essentially carried out by centrifugation,
filtration, affinity chromatography or chromatography on an
inorganic chromatographic material with subsequent chromatography
on an ion exchanger. An additional removal of endotoxins can then
be achieved according to WO 95/21177 when the nucleic acid is
treated with an endotoxin removal buffer which contains 10%
Triton.RTM.X100 and 40 mmol/l MOPS buffer
(3-morpholino-1-propanesulf- onate buffer). A disadvantage of this
process is that the nucleic acid purified in this manner contains
impurities of Triton.RTM. and MOPS buffer. Although endotoxins can
be removed by this process to a content of ca. 100 EU/mg DNA
(Qiagen News 1/96, 3-5), it is not possible to remove endotoxins to
a greater extent by this process.
[0007] However, for a therapeutic application such as for example
for gene therapy a nucleic acid preparation is required which is as
free as possible of all impurities (in particular substantially
free of endotoxins). Above all the endotoxin content of plasmid
preparations has been hitherto an unsolved problem as described for
example by Cotten et al., Gene Therapy 1 (1994) 239-246. A reduced
endotoxin content (ca. 100 EU/mg DNA) can only be achieved by the
state of the art such as for example according to WO 95/21177 if
the nucleic acids are treated with non-ionic detergents such as
e.g. Triton (endotoxin removal buffer from WO 95/21177). However,
Triton.RTM. has a biological action such as e.g. lung changes or
reduction of blood pressure (Fiedler, "Lexikon der Hilfstoffe fur
Pharmazie und Kosmetik und angrenzende Gebiete (Band 9, 3rd
edition, 1989, Editio Cantor, DE)). The MOPS buffer which is
additionally required also contains a substance that is problematic
with regard to a therapeutic application.
[0008] The invention provides a nucleic acid preparation,
preferably a plasmid DNA, of high purity in which endotoxins are
substantially removed and preferably without ethidium bromide,
phenol, caesium chloride, polymyxin or non-ionic detergents and
also provides a simple and effective process for purifying such
nucleic acids in particular for removing endotoxins.
[0009] The invention concerns a nucleic acid that can be replicated
in gram-negative bacteria, preferably a plasmid DNA with a content
of less than 1% protein, preferably less than 0.1% protein and a
content of less than 1 EU/mg DNA, preferably 0.01-0.1 EU/mg DNA of
endotoxins. This plasmid DNA is preferably free of ethidium
bromide, phenol and caesium chloride, free of detergents based on
octylphenolpoly(ethylene glycol ether)n such as Triton.RTM.
detergents and also free of MOPS buffer substance and RNAse.
[0010] Amplification is understood as an increase in the copy
number of a nucleic acid (in particular DNA and plasmid DNA) based
on the replication of a vector. In this process numerous copies are
produced from a template. A vector is replicated which represents
the nucleic acid or which contains the cloned nucleic acid.
[0011] A plasmid DNA is understood as an extrachromosomal DNA
duplex molecule. The size of a DNA plasmid is usually 1 to more
than 200 kb and one to several hundred copies are present in host
cells. Plasmid DNA is usually amplified in gram-negative bacteria
such as e.g. E. coli and subsequently isolated. Plasmids are often
used to construct cloning vectors, for the transfection of
prokaryotic and eukaryotic cells. A therapeutic use is of especial
importance in connection with in vivo and ex vivo gene therapy.
Plasmid DNA that is used therapeutically preferably has a length of
5 to 20 kb, particularly preferably 5-10 kb and is double-stranded.
The plasmid DNA can be linearized or circularly closed. Preferably
DNA is used that is essentially circularly closed.
[0012] Consequently the invention additionally concerns a
pharmaceutical composition containing a nucleic acid according to
the invention, preferably plasmid DNA, in a therapeutically
effective amount and optionally additional pharmaceutical auxiliary
substances, fillers or additives.
[0013] Endotoxins are lipopolysaccharides from gram-negative
bacteria. Endotoxins can have a pyrogenic effect in mammals and
induce an endotoxin shock. The main toxic component of endotoxins
is lipid A, the polysaccharide moiety mediating the water
solubility and the lipid moiety having the toxic effect. The
biological effect of endotoxins in mammals are in particular a
hypersensitization as well as other reactions which are accompanied
by fever.
[0014] Plasmid DNA is amplified by standard methods in E. coli i.e.
a gram-negative bacterium. After fermentation the biomass obtained
in this process is lysed and the cells are lysed. In this process
the endotoxins are released from the cell membrane. This means that
after amplification of nucleic acids, in particular of plasmid DNA,
in gram-negative bacteria and in particular in E. coli it is
necessary to remove endotoxins if it is intended to use this
plasmid DNA therapeutically.
[0015] Depending on the application doses of 50 .mu.g to 10 mg and
more are used or planned for a therapeutic application of
replicatable nucleic acids, in particular of plasmid DNA. The dose
amount depends on the disease and type of administration. In an
aerosol, e.g. for the treatment of cystic fibrosis, doses of 400
.mu.g and more are used. This applies likewise to plasmid DNA
encapsulated in a lipid complex (e.g. in liposomes). In order to
provide such amounts of replicatable nucleic acid that can be used
therapeutically, it is necessary to produce the replicatable
nucleic acid on a large scale. For this fermentation preparations
are expedient with 1-5 kg biomass from which 1-5 g nucleic acid can
be isolated.
[0016] The invention also concerns a process for the production of
a plasmid DNA with a content of less than 1 EU/mg DNA, preferably
0.01-0.1 EU/mg DNA of endotoxins which is characterized in that
plasmid DNA is replicated in gram-negative bacteria, the biomass is
lysed and the soluble components are chromatographed on
hydroxylapatite and subsequently the said plasmid DNA is isolated.
Before chromatography on hydroxylapatite it is preferably to carry
out an ion exchange chromatography which essentially removes RNA
and foreign proteins. This can optionally remove further impurities
and achieve a content of nucleic acid of less than 1% protein,
preferably less than 0.1% protein, free of ethidium bromide, phenol
and caesium chloride. Such a preparation is also preferably free of
detergents based on octylphenol poly(ethylene glycol ether)n and
MOPS buffer substance.
[0017] The process according to the invention enables numerous
impurities to be avoided or removed which plasmid DNA contains if
it is produced by a process familiar to a person skilled in the
art. Above all it is surprisingly possible to drastically reduce
the endotoxin content in a simple manner.
[0018] In the process according to the invention an outstanding
removal of endotoxins is achieved by the chromatography with
hydroxylapatite. This is all the more surprising since
chromatography on hydroxylapatite is only used in the literature to
separate DNA and RNA (Johnson & Ilan, Analyt. Biochem. 132
(1983) 20-25).
[0019] The chromatographic effect of hydroxylapatite is essentially
based on the interaction between calcium.sup.2+ groups and the
negative charge of the nucleic acid to be purified and to a lesser
extent on the interaction of the nucleic acid to be purified with
PO.sub.4.sup.3- groups on the surface of crystalline
hydroxylapatite (cf e.g. Protein Purification Methods, Ed. by Elv.
Harries and S. Angal, Oxford University Press 1989, 238-244).
Chromatography on hydroxylapatite can be essentially referred to as
an ion exchanger step for nucleic acids in which the bound DNA
cannot be eluted from the hydroxylapatite matrix by a
simple-increase of ionic strength (e.g. NaCl) but rather by
increasing the concentration, preferably of phosphate or citrate,
divalent metal ions and/or EDTA.
[0020] In the process according to the invention endotoxins and the
nucleic acid to be purified are firstly bound to hydroxylapatite
primarily via dipole-dipole interactions in the chromatography on
hydroxylapatite (e.g. HA-ceramic, BioGel HPHT, Bio-Gel HT/HTP from
Biorad DE, HA-Ultrogel from IBF or HA spheroidal from BDH,
Macrosorb C from Sterling Organics). The equilibration is usually
carried out at neutral pH in phosphate buffer. A denaturing agent
is preferably added, as in the subsequent washing of the column.
Surprisingly it is possible to displace the nucleic acid from its
binding to hydroxylapatite with phosphate, citrate or calcium ions
whereas the endotoxins remain bound. Instead of calcium, the
displacement of the nucleic acid from its binding to
hydroxylapatite can be achieved with other divalent metal ions
which can replace calcium in the apatite such as e.g. Mg, Fe, Mn.
For the elution the ion concentration is preferably 100 mmol/l or
more. Ion concentrations between 100 and 500 mmol/l or 200 and 500
mmol/l are particularly preferred. A solution containing phosphate
ions (e.g. phosphate buffer) is particularly preferably used.
Before the elution (without denaturing agent) it is expedient to
wash (with denaturing agent). It has turned out to be advantageous
to for example use a phosphate of sulfate solution (100-200 mmol/l)
to which a denaturing agent (e.g. urea or guanidine hydrochloride)
has been added at a DNA-denaturing concentration (e.g. 6 mol/l
urea).
[0021] In a preferred embodiment an ultrafiltration is additionally
carried out after the chromatography on hydroxylapatite.
[0022] In order to produce the nucleic acid according to the
invention the plasmids which represent or contain the nucleic acid
are usually amplified in gram-negative bacterial cultures. Such
methods are known to a person skilled in the art and are described
for example by Sambrook et al. (1989), Molecular Cloning: A
Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press
and by F.M. Ausubel et al., eds. (1991), Current Protocols in
Molecular Biology, Wiley Interscience, New York. For this the
bacterial cultures which contain the plasmids are firstly
subcultured and subsequently cultured in a suitable medium
optionally with addition of a selection agent.
[0023] The biomass is also lysed by methods familiar to a person
skilled in the art (mechanical or enzymatic lysis, see e.g.
Birnboim and Doly, Nucleic Acids Research 7 (1979) 1513-1423)
without addition of RNAse. It is possible to omit shaking out with
phenol if the proteins are separated by chromatography on an anion
exchanger. After lysis and separation of the insoluble components,
preferably by centrifugation and filtration over a filter candle (5
.mu.m pores), the cell supernatant is preferably firstly
chromatographed on an anion exchanger to remove proteins. Suitable
anion exchangers are anion exchangers based on agarose such as for
example Q-Sepharose. Other suitable anion exchangers are based on
polymethacrylate (Macroprep/Bio-Rad, Germany),
polystyrene/divinylbenzene (Poros/Perseptive, HyperD/Biosepra,
Source/Pharmacia) or silica gel on the surface of which
diethylaminoethyl (DEAE) or dimethylaminoethyl (DMAE) groups are
for example bound.
[0024] In order to optimize the purification effect, the nucleic
acid is eluted by means of a high salt gradient e.g. NaCl gradient
(preferably 0.65 mol/l-0.85 mol/l) in TE buffer. This surprisingly
enables numerous impurities (RNA, protein) to be separated in one
step.
[0025] It is also preferable to carry out an additional
isopropanol/ethanol precipitation, preferably after the
hydroxylapatite chromatography, to minimize the bioburden and for
desalting. Subsequently the nucleic acid according to the invention
can be bottled under sterile. conditions.
[0026] The following examples describe the invention in more
detail.
EXAMPLE 1
Isolation of Nucleic Acid from E. coli Biomass
[0027] E. coli biomass containing plasmid DNA is lysed by an
alkaline lysis and the released plasmid DNA is chromatbgraphed over
Q-Sepharose and HA-Ceramic. The eluate is desalted by an
isopropanol/ethanol precipitation and concentrated and the plasmid
DNA precipitate is resuspended in TE buffer.
[0028] Resuspension buffer: 50 mmol/l Tris/HCl, 10
mmol/EDTA-Na.sub.2, pH 8.0.+-.0.2
[0029] Potassium acetate buffer: 3 mol/l potassium acetate buffer
pH 5.5
[0030] 60 g biomass (wet, E. coli) from the fermenter is filled
into depyrogenized centrifuge beakers. 750 ml resuspension buffer
is added and it is stirred slowly (ca. 35 rpm) for at least 24
hours at 5.+-.4.degree. C. until the biomass is completely
suspended. During this process the temperature of the suspension is
slowly increased to 25.degree. C.
[0031] 750 ml 0.2 mol/l NaOH/1% SDS is added to the suspension
while stirring at ca 80 rpm and incubated for 5 minutes at
25.degree. C. 750 ml potassium acetate buffer (see above) is added
while stirring and the temperature of the biomass is lowered as
rapidly as possible to 4.degree. C.
[0032] The biomass is centrifuged for 30 minutes at 26000.times.g
and 4.degree. C. The supernatant which contains the plasmid DNA is
decanted and filtered clear over a 5 .mu.m filter candle.
[0033] Chromatography on Q-Sepharose ff:
[0034] TE buffer: 10 mmol/l Tris-HCl, 1 mmol/l EDTA pH
8.0.+-.0.2
[0035] Equilibration/wash buffer=gradient buffer A: 10 mmol/l
Tris-HCl, 1 mmol/l EDTA, 0.65 mol/l NaCl pH 8.0.+-.2.
[0036] Gradient buffer B: 10 mmol/l Tris-HCl, 1 mmol/l EDTA, 0.85
mol/l NaCl pH 8.0.+-.0.2.
[0037] The decanted centrifuge supernatant is adjusted to 49-50
mS/cm conductivity by addition of ca. 350 ml TE buffer/l
centrifugation supernatant and cooled to 5.degree..+-.4.degree. C.
The whole chromatography is carried out at this temperature. The
centrifugation supernatant is applied to the equilibrated column at
5-8 column volumes (CV)/h. Subsequently the column is washed at a
flow rate of 5-8 CV/h with ca. 8 CV 10 mmol/l Tris-HCl, 1 mmol/l
EDTA, 0.65 mol/l NaCl pH 8.0.+-.0.2.
[0038] Elution
[0039] A gradient is applied to the column (5 CV buffer A, 5 CV
buffer B) and the eluate is fractionated at a flow rate of 5-8
CV/h. The detection is carried out at 254 nm. The pre-peak
(impurities) is separated from the main peak (plasmid DNA) by
collecting the main peak from the increasing flank onwards in a
separate vessel. The endotoxin content of the eluate is between
1200 and 12000 EU/mg plasmid DNA.
[0040] Chromatography on Hydroxylapatite (HA Ceramic)
[0041] The chromatography is carried out at 5.+-.4.degree. C.
[0042] Equilibration buffer: 0.1 mol/l potassium phosphate, 6 mol/l
urea pH 7.0.+-.0.2
[0043] wash buffer 1: 0.15 mol/l potassium phosphate, 6 mol/l urea
pH 7.0.+-.0.2
[0044] wash buffer 2: 0.02 mol/l potassium phosphate buffer pH
7.0.+-.0.2
[0045] elution buffer: 0.5 mol/l potassium phosphate pH
7.0.+-.0.2
[0046] The detection is carried out at 254 nm with a UV
detector/plotter unit. A 1% product solution (plasmid DNA) measured
with a calibrated photometer is used as a calibration solution.
[0047] The Q-Sepharose.RTM. pool is adjusted to a final
concentration of 1.1 mmol/l calcium chloride and applied to the
equilibrated column at a flow rate of 5-8 CV/h.
[0048] Subsequently the column is consecutively washed at a flow
rate of 5-8 CV/h with:
[0049] 1. 0.1 mol/l potassium phosphate, 6 mol/l urea pH
7.0.+-.0.2, until absorbance is no longer detectable on the
detector.
[0050] 2. 2-4 CV, 0.15 mol/l potassium phosphate, 6 mol/l urea pH
7.0.+-.0.2
[0051] 3. 5 CV, 0.02 mol/l potassium phosphate pH 7.0.+-.0.2
[0052] Following the wash steps it is eluted with 0.5 mol/l
potassium phosphate buffer pH 7.0.+-.0.1 at a flow rate of 5-6
CV/h.
[0053] The peak is collected, heated to 25.degree. C. and 10% of
it's volume of 4 mol/l KCl solution is added. Subsequently 0.7
parts by volume (relative to the volume of the eluate) of
isopropanol is added, the solutions are mixed and incubated for
5-10 minutes at 25.degree. C. It is centrifuged for 30 minutes at
.gtoreq.20,000.times.g, the plasmid DNA being in the
precipitate.
[0054] 20 ml 70% ethanol is added to the precipitate and it is
again centrifuged for 10-15 minutes at .gtoreq.20,000.times.g at
4.degree. C.
[0055] The precipitate which contains the plasmid DNA is
resuspended in TE buffer (10 mmol/l Tris-HCl, 1 mmol/l EDTA pH
8.0+0.2) and adjusted to a plasmid concentration of 1 mg/ml. The
endotoxin content is typically less than 0.06 EU/mg DNA and between
0.01 and 0.06 EU/mg DNA.
[0056] The endotoxin content is determined by adding a limulus
amoebocyte lysate solution (LAL solution) to the solution to be
examined. Endotoxins result in a gel formation in this mixture.
[0057] No gel formation should occur in the negative control
preparations and in the positive control preparations as well as in
the sample solutions supplemented with two .lambda. control
standard endotoxin a gel formation must occur.
[0058] The first dilution step of the solution of active substance
for which these criteria apply and in which no gel formation occurs
is used to calculate the endotoxin content of the solution of
active substance solution according to the following formula:
E=V.times..lambda.(EE/ml)
[0059] E: endotoxin content
[0060] V: dilution factor
[0061] .lambda.: lysate sensitivity (EE/ml)
EXAMPLE 2
[0062] Plasmid Preparation According to the State of the Art
[0063] The plasmid preparation is carried out analogously to
Birnboim et al., Nucl. Acids Res. 7 (1979) 1513-1523 and Meth.
Enzymol. 100 (1983) 243-255. Accordingly the bacterial cells are
lysed in NaOH/SDS in the presence of RNase. It is centrifuged and
the supernatant which contains the plasmid DNA is processed
further. The supernatant is loaded onto a pre-equilibrated
Qiagen.RTM. column.
[0064] The bacterial mass is resuspended in 4 ml buffer (100
.mu.g/ml RNase A, 50 mmol/l Tris-HCl, 10 mmol/l EDTA, pH 8.0). 4 ml
lysis buffer (200 mmol/l NaOH, 1% SDS) is added and it is incubated
for 5 minutes at room temperature. Subsequently 4 ml neutralisation
buffer (3 mol/l potassium acetate, pH 5.5) is added and it is
incubated for 15 minutes at 4.degree. C. It is centrifuged for 30
minutes at 30,000.times.g at the same temperature and the
supernatant is processed further. A Qiagen.RTM. column is
equilibrated with 4 ml equilibration buffer (750 mmol/l NaCl, 50
mmol/l MOPS, 15% ethanol, pH 7.0, 0.15% Triton.RTM.X 100) and the
supernatant is applied to the column. It is washed with 1 mol/l
NaCl, 50 mmol/l MOPS, 15% ethanol, pH 7.0 and eluted with 5 ml
elution buffer (1.25 mol/l NaCl, 50 mmol/l Tris-HCl, 15% ethanol,
pH 8.5).
[0065] The eluate is precipitated with isopropanol (0.7 vol) and
centrifuged for 30 minutes at 15,000.times.g at 4.degree. C. The
DNA pellet is washed in 70% ethanol and again centrifuged.
Subsequently the pellet is resolubilized in 10 mmol/l Tris-HCl, 1
mmol/l EDTA, pH 8.0.
[0066] The endotoxin content of such a plasmid preparation is
typically 300-3000 EU/mg. Using an endotoxin removal buffer
according to WO 95/21177 and Qiagen news 1/96, p. 3-5 the endotoxin
content can be further reduced to ca. 100 EU/mg.
[0067] List of References
[0068] Ausubel, F. M., et al. eds. (1991), Current Protocols in
Molecular Biology, Wiley Interscience, New York
[0069] Birnboim, H. C. and Doly, J., Nucleic Acids Research 7
(1979) 1513-1523
[0070] Birnboim, H. C., et al., Meth. Enzymol. 100 (1983)
243-255
[0071] Chandra et al., Analyt. Biochem. 203 (1992) 169-172
[0072] Cotten et al., Gene Therapy 1 (1994) 239-246
[0073] Dion et al., J. Chrom. 535 (1990) 127-147
[0074] European Patent EP-B 0 268 946
[0075] Fiedler, Lexikon der Hilfsstoffe fur Pharmazie und Kosmetik
und angrenzende Gebiete (Vol. 9, 3rd edition, 1989, Editio Cantor,
GER)
[0076] Garger et al., Biochem. Biophys. Res. Comm. 117 (1983)
835-842
[0077] Johnson & Ilan, Analyt. Biochem. 132 (1983) 20-25
[0078] McClung & Gonzales, Analyt. Biochem. 177 (1989)
378-382
[0079] Protein Purification Methods, Ed. by Elv. Harries and S.
Angal, Oxford University Press 1989, 238-244
[0080] QIAGEN NEWS 1/96, 3-5
[0081] QIAGEN Plasmid Handbook (Qiagen Inc., Chatsworth, USA)
[0082] Raymond et al., Analyt. Biochem. 172 (1988) 125-133
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