PURIFICATION OF NUCLEIC ACID FROM A SAMPLE CONTAINING NUCLEIC ACID AND ENDOTOXIN

Abstract

A process for purifying nucleic acids, especially plasmid DNA, from a nucleic acid-containing, biologic sample, consisting of the following steps: a) Preparation of a fluid sample containing nucleic acid and endotoxin each in dissolved form; b) Precipitation at least of nucleic acid and endotoxin from the fluid sample; c) Washing of the components of the fluid sample precipitated out in step b) in order to at least partially remove the endotoxin with at least one washing solution, which contains (i) at least one amine compound with at least two carbon atoms and with a molar mass of ≦500 g/mol and; (ii) at least one organic solvent different from the aforementioned amine compound and has a pH-value (20° C.) in the range from pH 3.0 to pH 8.5, and; d) Dissolution of the remaining nucleic acid from the washed, precipitated constituents from step c) using a dissolving buffer and collection of the dissolved nucleic acids in a separate receptacle.

The process is suitable for the effective and economical removal of endotoxins.

Claims

1.-15. (canceled)

16. A process for purifying nucleic acids from a biologic sample containing nucleic acid, comprising the following steps: a) preparation of a fluid sample containing nucleic acid and endotoxin each in dissolved form; b) precipitation of at least the nucleic acid and endotoxin from the fluid sample; c) washing of the precipitated constituents of the liquid sample from step b) to at least partially remove the endotoxin with at least one washing solution which contains (i) at least one amine compound with at least two carbon atoms and with a molar mass of ≦500 g/mol and; (ii) at least one organic solvent different from the at least one amine compound, and having a pH-value (20° C.) in the range from pH 3.0 to pH 8.5; and d) dissolution of the remaining nucleic acid derived from the washed, precipitated constituents from step c) by means of a dissolving buffer and capture of the dissolved nucleic acids in a separate receptacle, wherein the washing solution of step c) comprises the at least one amine compound in a concentration of 200 mmol/L to 1,000 mmol/L.

17. The process according to claim 16, wherein in step b) at least nucleic acid and endotoxin are precipitated by binding onto the surface of a solid carrier material, in particular by adsorption or precipitation, wherein the sample in step b) which is to be precipitated onto the carrier material is preferably displaced using a binding buffer.

18. The process according to claim 16, wherein in step b) at least nucleic acid and endotoxin are separated as solids by precipitation, whereby the precipitated nucleic acid and endotoxin as solids in particular i) are collected by filtration on the surface of a solid carrier material, or ii) compacted by centrifugation, and each is separated from the parent solution.

19. The process according to claim 17, wherein the carrier material is selected from the group consisting of mineral carrier materials, especially quartz fibers, silica, glass, aluminum oxide, zeolite, titanium dioxide, zirconium dioxide, and combinations of these.

20. The process according to claim 16, wherein the at least one amine compound of the washing solution in step c) is chosen from at least one compound of general formula (I), ##STR00003## wherein R.sup.1, R.sup.2 and R.sup.3 independently of each other stand for a hydrogen atom, a (C.sub.1 to C.sub.6)-alkyl group, a (C.sub.1 to C.sub.6)-monohydroxyalkyl group, a (C.sub.2 to C.sub.6)-dihydroxyalkyl group, a (C.sub.3 to C.sub.6)-trihydroxyalkyl group, with the requirement that the amine of formula (I) have a molar mass of ≦500 g/mol, and at least one of the residues R.sup.1, R.sup.2 and R.sup.3 differs from a hydrogen atom.

21. The process according to claim 16, wherein the at least one amine compound of the washing solution is selected from the group consisting of triethylamine, triethanolamine, 2-amino-2-(hydroxymethyl)-propane-1,3-diol, 2,2-bis(hydroxy-methyl)-2,2′,2″-nitrilotriethanol, 1,3-bis[tris(hydroxymethyl)methylamino]propane, and mixtures thereof.

22. The process according to claim 16, wherein the washing solution of step c) contains the at least one amine compound in a concentration of 200 mmol/L to 800 mmol/L.

23. The process according to claim 16, wherein the nitrogen atom of the amino group in the at least one amine compound is at least 50% protonated.

24. The process according to claim 16, wherein the washing solution of step c) contains as a solvent at least one C.sub.1-C.sub.6-monoalcohol, an isopropanol, or combinations thereof.

25. The process according to claim 24, wherein the washing solution contains the C.sub.1-C.sub.6-monoalcohol in a total concentration of 20 to 80% by weight relative to the total weight of the washing solution.

26. The process according to claim 16, wherein the washing solution of step c) has a pH-value (20° C.) in the range from pH 3.0 to 7.5.

27. The process according to claim 16 wherein in the context of at least partial removal, in each case relative to the total quantity of endotoxin contained in the fluid sample, at least 50% by weight of the endotoxin is removed.

28. A method comprising utilizing a washing solution comprising (i) at least one amine compound with at least two carbon atoms and with a molar mass of ≦500 g/mol and (ii) at least one organic solvent different from the aforementioned amine compound having a pH-value (20° C.) in the range from pH 3.0 to pH 8.5, to at least partially remove endotoxin from a nucleic acid-containing sample wherein the washing solution comprises the at least on amine compound in a concentration of 200 mmol/L to 1,000 mmol/L.

29. The method according to claim 28 wherein the nucleic acid and the endotoxin are bound to the surface of a solid carrier material.

30. A kit for purifying nucleic acids comprising a washing solution, which contains (i) at least one amine compound with at least two carbon atoms and with a molar mass of ≦500 g/mol and (ii) at least one organic solvent different from the at least one amine compound, and having a pH-value (20° C.) in the range from pH 3.0 to pH 8.5, and at least one additional constituent selected from the groups consisting of an instruction manual to perform the process according to claim 16, a binding buffer, and a dissolving buffer to dissolve the nucleic acid at least partially cleansed of endotoxin, wherein the washing solution contains the at least one amine compound in a concentration of 200 mmol/L to 1,000 mmol/L.

Description

EXAMPLES

Example 1

[0147] An overnight culture of E. coli DH5α™, transformed with the plasmid pcDNA™3.1(+) was prepared in an ampicillin-containing nutrient medium. Varying 1 ml, 2 ml and 3 ml aliquots of the culture were centrifuged at 10,000×g for 5 min and the supernatant was discarded. The pellets were resuspended in a resuspension buffer containing EDTA, Tris and RNase A, lysed with a lysis buffer containing NaOH and SDS and neutralized with a neutralization buffer containing potassium acetate. The lysates were cleared by centrifugation at 10,000×g for 10 min and the clear supernatant was mixed with a guanidinium hydrochloride solution. The mixture was filled onto spin columns containing silica layers and flushed through the silica carrier material by centrifugation at 10,000×g for 1 min into a receptacle. Following this, each pelleted bacterial culture volume was filled onto spin columns with a washing solution with the following variations: [0148] no washing solution [0149] 30% isopropanol, 7 mM TRIS, 4.55 M guanidinium hydrochloride, pH 7.5 [0150] 50% isopropanol, 200 mM BIS-TRIS, pH 5.5

[0151] The columns were centrifuged at 10,000×g for 1 min, with the washing solution being flushed through the silica carrier material into a receptacle. This process was repeated once. Thereafter a washing step was performed using a known washing solution containing 80% ethanol. Again, in order to flush through the silica carrier material, it was centrifuged at 10,000×g for 1 min. To dry them the columns were centrifuged empty at 10,000×g for 1 min. The DNA was eluted after addition of 100 μl dissolving buffer (water with 5 mM Tris-Cl, pH 8.5) again by centrifugation at 10,000×g for 1 min. The yield was measured with a photometer by measuring extinction at 260 nm, and the endotoxin content measured using the pyrochrome LAL-Test (Cape Cod).

[0152] The following table shows the measured results

TABLE-US-00001 Endo- μg DNA/ relative EU/ toxin- Washing solution eluate yield/% μg removal/% 1 ml bacterial culture no special wash step 12.9 100%  125 0 30% isopropanol, 7 mM TRIS, 13.5 105%  15 88 4.55M Guanidinium hydro- chloride, pH 7.5 50% isopropanol + 200 mM 12.1 94% 14 89 BIS-TRIS, pH 5.5 2 ml bacterial culture without a special wash step 16.9 100%  632 0 30% isopropanol, 7 mM TRIS, 15.4 91% 102 84 4.55M Guanidinium hydro- chloride, pH 7.5 50% isopropanol + 200 mM 15.0 89% 14 98 BIS-TRIS, pH 5.5 3 ml bacterial culture without a special wash step 18.8 100%  914 0 30% isopropanol, 7 mM TRIS, 17.1 91% 128 86 4.55M Guanidinium hydro- chloride, pH 7.5 50% isopropanol + 200 mM 16.7 89% 12 99 BIS-TRIS, pH 5.5

[0153] Even the presence of small quantities of TRIS was sufficient to markedly reduce the endotoxin concentration of the eluate. No significant effect on the DNA yield was seen.

Example 2

[0154] An overnight culture of E. coli DH5α™, transformed with the plasmid pc DNA™3.1(+) was prepared in an ampicillin-containing nutrient medium. 25 ml aliquots of the culture were centrifuged at 4,500×g for 10 min, and the supernatant was discarded. The pellets were resuspended in a resuspension buffer containing EDTA, Tris and RNase A, lysed with a lysis buffer containing NaOH and SDS and neutralized with a neutralization buffer containing potassium acetate. The lysates were cleared by centrifugation over a filter column containing polyethylene chips and silica at 3,000×g for 2 min and the clear effluent was mixed with a precipitation solution containing 85% tripropylene glycol and 2.5% ethanol. The mixture was filled onto columns containing silica layers and pulled through the silica carrier material into a receptacle by the application of a negative pressure of −0.3 bar. Following this, it was washed with washing solutions containing 200 mM or 400 mM TRIS or BIS-TRIS at pH 7 or pH 8 for TRIS and pH 5.5 or pH 6.5 for BIS-TRIS, as well as a control with no washing step, and washed with 50% isopropanol with no further additives and then rinsed with a known washing solution containing 80% ethanol. Again, a negative pressure of −0.3 bar was used to wash through the silica. To dry them, the columns were centrifuged empty at 10,000×g for 1 min. The DNA was eluted after addition of 200 μl dissolving buffer (water with 5 mM Tris-Cl, pH 8.5) again by centrifugation at 10,000×g for 1 min. The yield was measured with a photometer by measuring extinction at 260 nm, and the endotoxin content measured using the pyrochrome LAL-Test (Cape Cod).

[0155] The following table shows the measured results

TABLE-US-00002 μg DNA/ Washing solution eluate EU/μg Endotoxin removal % TRIS pH 7.0 200 mM 79 25 80 400 mM 73 13 89 TRIS pH 8.0 200 mM 73 18 86 400 mM 78 17 86 BIS-TRIS pH 5.5 200 mM 73 12 90 400 mM 74 18 85 BIS-TRIS pH 6.5 200 mM 70 5 96 400 mM 66 6 94 without wash step 89 123 0 50% Isopropanol 13

[0156] The washing solution not prescribed in this invention consisting of a solution of 50% isopropanol was not on its own able to hold the DNA on the column.

Example 3

[0157] An overnight culture of E. coli DH5α™, transformed with the plasmid pc DNA™3.1(+) was prepared in an ampicillin-containing culture medium. 50 ml aliquots of the culture were centrifuged at 4,500×g for 10 min, and the supernatant was discarded. The pellets were resuspended in a resuspension buffer containing EDTA, Tris and RNase A, lysed with a lysis buffer containing NaOH and SDS and neutralized with a neutralization buffer containing potassium acetate. The lysate was cleared by centrifugation over a filter column containing polyethylene chips and silica at 3,000×g for 2 min and the cleared effluent was mixed with a precipitation solution containing 85% tripropyleneglycol and 2.5% ethanol. The mixture was filled onto columns containing silica sheets and drawn into a receptacle by applying a negative pressure of −0.3 bar. Following this, washing solutions of the following constitution were filled onto the columns and drawn through the silica into a receptacle by applying a suction pressure of −0.3 bar: [0158] no washing step [0159] 50% isopropanol+200 mM BIS-TRIS, pH 6.0 (as in this invention) [0160] 50% isopropanol+100 mM NaCl [0161] 50% isopropanol+500 mM NaCl [0162] 50% isopropanol+1 M NaCl [0163] 50% isopropanol+1 M guanidine HCl [0164] 50% isopropanol+2 M guanidine HCl [0165] 50% isopropanol+3 M guanidine HCl [0166] 50% isopropanol+100 mM sodium acetate, pH 5.0 [0167] 50% isopropanol+250 mM sodium acetate, pH 5.0 [0168] 50% isopropanol+500 mM sodium acetate, pH 5.0

[0169] Finally, the columns were rinsed with a known washing solution containing 80% ethanol. Again, a suction pressure of −0.3 bar was used to wash through the silica carrier material. For drying, the columns were centrifuged empty at 10,000×g for 1 min. The DNA was eluted by the addition of 200 μl precipitation buffer (water with 5 mM Tris-Cl, pH 8.5), again whilst centrifuging at 10,000×g for 1 min. The yield was determined with a photometer by measuring the extinction at 260 nm, the endotoxin content by measurement using the pyrochrome LAL-Test (Cape Cod).

[0170] The following measurements were obtained:

TABLE-US-00003 μg DNA/ relative endotoxin- Washing solution eluate yield/% EU/μg removal/% No wash step 635 100%  473 0 50% isopropanol + 200 mM 597 94% 34 93 BIS-TRIS, pH 6.0 50% isopropanol + 100 mM 625 98% 448 5 sodium chloride 50% isopropanol + 500 mM 603 95% 511 0 sodium chloride 50% isopropanol + 1000 Mm 617 97% 305 36 sodium chloride 50% isopropanol + 1M 617 97% 317 33 guanidine HCl 50% isopropanol + 2M 612 96% 464 2 guanidine HCl 50% isopropanol + 3M 586 92% 154 67 guanidine HCl 50% isopropanol + 100 mM 641 101%  369 22 sodium acetate, pH 5.0 50% isopropanol + 250 mM 644 101%  388 18 sodium acetate, pH 5.0 50% isopropanol + 500 mM 606 95% 275 42 sodium acetate, pH 5.0

[0171] It can be clearly seen that in all cases the salt concentration was sufficient to keep the DNA on the column during the washing step. A significant removal of endotoxin was neither achieved by salt nor by isopropanol alone, nor by means of a pH-related effect. Acetate can also only incompletely separate the endotoxin.

Comparative Tests Against WO 2005/111059 A2

[0172] The following comparative tests show a comparison of the invention to the state of the art in the form of WO 2005/111059 A2. Initially two washing solutions were made as per WO 2005/111059 A2:

Washing Solution 1:

[0173]

TABLE-US-00004 100 mM Tris 1.211 g/100 mL.sup.  4.5M guanidine hydrochloride .sup. 43.0 g/100 mL Dissolve in H.sub.2O Adjustment to pH 6.9 with acetic acid 25% isopropanol 25 mL/100 mL to 100 mL with H.sub.2O

Washing Solution 2:

[0174]

TABLE-US-00005 10 mM Tris 0.121 g/100 mL 10 mM NaCl 0.058 g/100 mL Dissolve in H.sub.2O Adjustment to pH 8.0 with HCl 80% ethanol .sup. 80 mL/100 mL to 100 mL with H.sub.2O

[0175] For the following tests, the following chemicals were also used:

Resuspension buffer: 50 mM Tris-Cl, 10 mM EDTA, pH 8.0, 400 μg/mL RNase A
Lysis buffer: 1% SDS, 200 mM NaOH
Neutralization buffer: 2.8 M potassium acetate, pH 5.1
Binding buffer: 8 M guanidine hydrochloride
Precipitation solution: 85% tripropyleneglycol, 2.5% ethanol
Endotoxin removal buffer: 8% triethanolamine, 50% isopropanol, pH 6.0
Alcoholic wash buffer: 2.5 mM Tris, 20 mM NaCl, 80% ethanol, pH 7.5

Comparative Test 1

[0176] 300 mL LB-medium and 100 μg/mL Ampicillin were inoculated with E. coli Top 10, containing the plasmid pc DNA™3.1 (+) and incubated overnight at 37° C. on a shaker. After 16 hours of incubation an OD600 of 4.6 was measured. The culture was divided into six 50 mL aliquots which were separated by centrifugation at 6,000×g for 5 min into a bacterial pellet and supernatant.

[0177] The supernatants were discarded and the bacterial pellets were each taken up into 5 mL of resuspension buffer. Lysis was achieved by the addition of 5 mL lysis buffer to each and incubation at room temperature for 2 minutes. A precipitate was produced by the addition of 5 mL neutralization buffer to each, which was removed by means of centrifugation over a filter column containing PE-filter chips and silica paper at 3,000×g for 2 minutes. The clear supernatants containing the released plasmids were pooled, mixed by vortexing and re-aliquoted into 6×12 mL samples.

[0178] 3 mL of binding buffer were added to each 12-mL master lysate and mixed by vortexing. The concentration of chaotropic salt at effectively 1.6 M guanidine hydrochloride corresponded to the concentration recommended in D1.

[0179] The batches were loaded onto columns filled with three layers of silica paper (diameter of the filter: 7.25 mm) and were to have been drawn through the silica membrane by the application of a vacuum (900 mbar reduction of pressure compared to normal pressure). However, after a few milliliters the columns blocked completely.

Comparative Test 2

[0180] 300 mL LB-medium plus 100 μg/mL Ampicillin were inoculated with E. coli Top 10 containing the plasmid pcDNA™3.1 (+) and incubated on a shaker at 37° C. over night. After 16 hours of incubation an OD600 of 4.6 was measured. The culture was divided into six 50 mL aliquots which were separated into bacterial pellets and supernatant by centrifugation at 6,000×g for 5 min.

[0181] The supernatants were discarded and the bacterial pellets were each taken up into 5 mL of resuspension buffer. Lysis was achieved by the addition to each of 5 mL lysis buffer and incubation at room temperature for 2 minutes. A precipitate was produced by the addition to each of 5 mL neutralization buffer, which was removed by means of centrifugation over a filter column containing PE-filter chips and silica paper at 3,000×g for 2 minutes. The clear supernatants containing the released plasmids were pooled, mixed by vortexing and re-aliquoted into 6×12 mL samples. To each 12 ml of master lysate was added 6 mL of precipitation solution and these were mixed by vortexing.

[0182] The batches were loaded onto columns filled with three layers of silica paper (diameter of the filter: 7.25 mm) and drawn through the silica membrane by the application of a vacuum (300 mbar reduction of pressure compared to normal pressure). When the solution had completely run through, as the first part of a threefold analysis each solution was washed with 2 mL of washing solution as per WO 2005/111059 A2, then with 4 mL washing solution as per WO 2005/111059 A2. As alternatives in the threefold analysis each solution was washed with 2 mL of the endotoxin removal buffer and then with 4 mL of alcoholic washing buffer. The washing steps were all carried out under a vacuum pressure of 0.3 bar in comparison to normal pressure.

[0183] All six batches were freed of ethanol residue by centrifugation at 11,000×g for 1 min. Elution was achieved by the addition of 500 μL H2O to each and centrifugation at 11,000×g for 1 min.

[0184] The eluted DNA was measured photometrically (spectrum from 200 nm to 300 nm) and the endotoxin content measured by means of the chromogenic LAL-Test (Pyrochrome, 260260PA-25, Pyroquant Diagnostik Ltd.). A control standard endotoxin (EC010-5, Pyroquant Diagnostik Ltd.) was used as quantification standard. For the measurement, the eluate was diluted 1:1,000 with water.

Result of Comparative Test

[0185] This example shows that the washing solutions known from WO 2005/111059 A2 require special conditions, for example a larger filter area or the presence of detergent in the binding buffer. These requirements lead to increased complexity, which can be considered a disadvantage in sequential processing.

Result of Comparative Test 2

[0186] The photometric measurement as per FIG. 1 gave the following results:

TABLE-US-00006 Invent.sup.n. Compar.sup.n. Invent.sup.n. Compar.sup.n invent.sup.n. Compar.sup.n. μg DNA/eluate A260/A280 A260/A230 454 12 1.86 1.76 2.28 1.89 452 12 1.86 1.78 2.28 1.93 431 9 1.86 1.77 2.29 1.89 446 11 1.86 1.77 2.28 1.90

[0187] In FIG. 1, the results according to the invention are shown as a continuous line and those of the process according to WO 2005/111059 A2 as a dashed line. It is apparent that the use of wash buffer 1 and wash buffer 2 from WO 2005/111059 A2 removed a majority of the DNA. The purity conditions indicate clean DNA in all cases (Soil: A260/A280=1.8 to 1.9, A260/A230=1.9 to 2.3).

LAL-Test:

[0188]

TABLE-US-00007 EU/ μg DNA/ eluate eluate EU/μg Invent.sup.n 4796 454 11 3965 452 9 3178 431 7 3980 446 9 Comp.sup.n 9840 12 795 10207 12 823 10259 9 1207 10102 11 911

[0189] Although the DNA quantity in WO 2005/111059 A2 is markedly less (approx. 2.5% compared to the invention), the content of endotoxin is around 250% higher. It follows that a way has been found to separately precipitate DNA and endotoxins, and then remove the precipitated endotoxins using the solution described in this invention.