Process for purifying a biological sample containing nucleic acids

Abstract

The present invention relates to a process for purifying nucleic acids, particularly plasmid DNA, from a liquid sample, which contains endotoxins in addition to nucleic acids. The invention involves a process for purifying nucleic acids from a biological sample containing nucleic acids, preferably plasmid DNA, comprising the following steps: a) providing a liquid sample comprising nucleic acids and endotoxins; b) precipitating the nucleic acids and the endotoxins or binding the nucleic acids and the endotoxins on a solid carrier and thereby separating them from the remaining components of the liquid sample; c) washing the precipitate or solid carrier from step b) at least once with a washing buffer comprising an alcohol to further remove remaining components; d) treating the nucleic acids and endotoxins separated in step b), and washed in step c), using a purifying buffer for at least partial removal of the endotoxin; e) optionally washing the precipitate or the solid carrier in step d) at least once with at least one washing composition, and f) optionally collecting the nucleic acids, optionally by eluting from the solid carrier or dissolving the precipitated nucleic acid; wherein the purifying buffer in step d) comprises an amino compound selected from the group consisting of triethylamine, triethanolamine, 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol, 1,3-bis[tris(hydroxymethyl)methvlamino]propane, 2-amino-2-(hydroxymethyl)-propane-1,3-diol, and individual salts thereof, an alcohol capable of dissolving the amino compound in a total amount of 30 to 70 % by weight based on the total weight of the purifying buffer, and water, wherein the purifying buffer has a pH in the range from 3.0 to 8.0.

Description

[0001] PROCESS FOR PURIFYING A BIOLOGICAL SAMPLE CONTAINING NUCLEIC ACIDS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a process for purifying nucleic acids, particularly plasmid DNA, from a liquid sample, which contains endotoxins in addition to nucleic acids.

[0004] BACKGROUND OF THE INVENTION

[0005] Purification of nucleic acid from biological samples, such as bacterial lysates, is well-known. As an example, the QIAprep Spin Miniprep (QIAGEN, Germany) is available for preparing a lysate of a bacterial sample using a silica membrane and then purifying the lysate by various centrifugation and washing steps.

[0006] Endotoxins are lipopolysaccharides (LPS) and are a constituent of the cell wall of gram-negative bacteria such as Escherichia coli, for example. The latter bacterium is very commonly used in biotechnological processes, and so endotoxins are frequently present as contaminants in many biotechnology products. In gene therapy and in the manufacture of pharmaceuticals, endotoxins can provoke excessive immune reactions in the living organism (in vivo) with potentially fatal results. Endotoxins have also been shown to have a negative effect upon transfection of nucleic acids in cell cultures (in vitro).

[0007] The removal of endotoxins from preparations of biotechnologically generated biologic macromolecules is a recognized problem in the literature. In many processes for purifying DNA, endotoxins are also separated, and they are a frequent contaminant of plasmid separation. Methods employed for purifying DNA are generally chromatographic procedures such as ion exchange and affinity chromatography or an extraction with detergents, of which the most prominent use Triton® X-114 (i.e. 1 ,1 ,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and salts of deoxycholic acid.

[0008] The QIAprep Spin Miniprep (Qiagen, Germany) and other systems based on chaotropic silica chemistry, as currently commercially available does not provide good separation of nucleic acids and endotoxins, although it does remove other components from e.g. a bacterial lysate.

[0009] EP3228710 discloses a process based on chaotropic chemistry for purifying nucleic acids from a biological sample also containing endotoxins using certain amino compounds in a concentration range between 200 and 1000 mM for creating different binding conditions for nucleic acids and endotoxins. These amino compounds include Tris (2-amino-2-(hydroxymethyl)-propane-1 ,3-diol) and Bis-Tris (2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1 ,3-diol). The skilled person will recognize that further improvements in the efficacy of the above-mentioned processes for purifying nucleic acids from a biological sample are desirable.

[0010] SUMMARY OF THE INVENTION

[0011] Accordingly, one aspect of the invention concerns a process for purifying nucleic acids from a biological sample containing nucleic acids, preferably plasmid DNA, comprising the following steps: a) providing a liquid sample comprising nucleic acids and endotoxins; b) precipitating the nucleic acids and the endotoxins or binding the nucleic acids and the endotoxins on a solid carrier and thereby separating them from the remaining components of the liquid sample; c) washing the precipitate or solid carrier from step b) at least once with a washing buffer comprising an alcohol to further remove remaining components; d) treating the nucleic acids and endotoxins separated in step b), and washed in step c), using a purifying buffer for at least partial removal of the endotoxin; e) optionally washing the precipitate or the solid carrier in step d) at least once with at least one washing composition, and f) optionally collecting the nucleic acids, optionally by eluting from the solid carrier or dissolving the precipitated nucleic acid; wherein the purifying buffer in step d) comprises an amino compound selected from the group consisting of triethylamine, triethanolamine, 2-[Bis(2-hydroxyethyl)amino]-2- (hydroxymethyl)propane-l ,3-diol, 1 ,3-bis[tris(hydroxymethyl)methvlamino]propane, 2-amino-2- (hydroxymethyl)-propane-l ,3-diol, and individual salts thereof, an alcohol capable of dissolving the amino compound in a total amount of 30 to 70 % by weight based on the total weight of the purifying buffer, and water, wherein the purifying buffer has a pH in the range from 3.0 to 8.0.

[0012] A further aspect of the invention concerns the use of a purifying buffer as defined herein for purifying nucleic acids from a biological sample containing nucleic acids, such as plasmid DNA, and endotoxins by at least partially removing the endotoxin.

[0013] Another aspect of the invention concerns a kit of parts for purifying nucleic acids from a biological sample containing nucleic acids, such as plasmid DNA, and endotoxins, wherein the kit comprises a purifying buffer as defined herein and a membrane and / or beads as defined herein. It has been found that increasing the concentration of the amino compound compared to the amine compound used in EP3228710 decreases the endotoxin content of the biological sample further.

[0014] DETAILED DESCRIPTION OF THE INVENTION

[0015] Definitions

[0016] In the context of the present invention, a “nucleic acid” is a biopolymer comprising nucleotides. Thus, “nucleic acid” comprises both RNA and DNA polymers.

[0017] In the context of the present invention, the term “endotoxin” is used interchangeably with the term “ li po po lys acch a ri de ” .

[0018] Chaotropic substances denature proteins, increase the solubility of non-polar substances in water and destroy hydrophobic interactions. In addition, they are also believed to mediate the binding of nucleic acids to selected solid phases. The Hofmeister series (also called lyotropic series) ranks the relative influence of ions on the physical behavior of water and aqueous processes, such as colloidal assembly or protein folding:

[0019] • CO32- > SO42- > S2O32- > H2PO4- > F- > Cl- > Br - > NO3- > |- > CIO4- > SCN' [for anions] and

[0020] • NH4+> K+> Na+> Li+> Mg2+> Ca2+> guanidinium [for cations].

[0021] "Early" members of the series, i.e. members on the left, increase the surface tension of the solvent and decrease the solubility of non-polar molecules, such as proteins (so-called "salting ouf' / aggregation), while the "later" members (on the right) decrease the solvent's surface tension and increase the solubility of non-polar molecules (so-called "salting in'Vsolubilizing). The specific order of two ions in the series may vary depending upon the scientific reference, in particular for ions positioned in the middle of the series. It is, however, generally accepted that iodide, perchlorate, (iso)thiocyanate, and / or guanidinium ions are chaotropic ions, while sulphate and ammonium ions, for example, are non-chaotropic ions (also known as kosmotropic ions). Thus, in the context of the present invention, a chaotrope is in particular a water-soluble salt comprising iodide, perchlorate, (iso)thiocyanate, and / or guanidinium ions, such as for example sodium perchlorate, sodium iodide, potassium iodide, guanidinium (iso)thiocyanate, and guanidine hydrochloride.

[0022] Liquid sample containing nucleic acids and endotoxins

[0023] Biological samples, such as samples extracted from bacteria, e.g. gram-negative bacteria, contain nucleic acids. When a biological sample is extracted from gram-negative bacteria, this is typically done by lysis. Gram-negative bacteria contain endotoxins in their cell wall. Lysates from gramnegative bacteria therefore typically comprise endotoxins in addition to nucleic acids.

[0024] A biological sample is used to make the liquid nucleic acid- and endotoxin-containing sample. Any biological material, which contains both nucleic acids and endotoxins is suitable, including bacterial cultures, animal or human tissues, tissue components, bodily fluids such as saliva, sputum, cerebrospinal fluid, whole blood, serum or plasma. Bacteria, yeasts and other fungi or viruses are also considered to be suitable sources, as are PCR-amplification reactions which contain primer and DNA fragments, or cell culture remnants. Artificial sample material, e.g. with synthetically or in- vitro generated nucleic acids, also falls into the area of applicability of the present invention.

[0025] The nucleic acids contained in a biological sample may have many different origins. If originating from bacteria, the nucleic acids will typically be DNA, such as plasmid DNA. Thus, in one embodiment, the nucleic acids are DNA. In a further embodiment, the nucleic acids are plasmid DNA.

[0026] As mentioned above, a lysate from gram-negative bacteria contains both nucleic acids and endotoxins. Thus, in one embodiment, the liquid sample comprising nucleic acids and endotoxins is a bacterial lysate. In a further embodiment, the liquid sample comprising nucleic acids and endotoxins is a bacterial lysate comprising DNA. In still a further embodiment, the liquid sample comprising nucleic acids and endotoxins is a bacterial lysate comprising plasmid DNA.

[0027] Typically, each of the nucleic acids and endotoxins is dissolved in the liquid sample, which is typically an aqueous sample. Thus, in one embodiment, each of the nucleic acids and endotoxins is dissolved in the liquid sample. In a further embodiment, the liquid sample is an aqueous sample. In still a further embodiment, the liquid sample is an aqueous sample wherein each of the nucleic acids and endotoxins is dissolved.

[0028] In the process of the present invention, a liquid sample containing nucleic acids and endotoxins is provided as a first step. Said provided sample may be an unpurified bacterial lysate, or it may be a purified bacterial lysate, where components other than nucleic acids and endotoxins have already been removed.

[0029] One or more of the following statements may apply to the bacterial lysate: i) the lysate is obtained by conducting an alkaline lysis, ii) the lysate is neutralized using an acidic solution, preferably an acetate or citrate buffer, iii) the lysate is cleared from cell debris, and / or iv) the lysate is cleared from any precipitate resulting from the lysis or any subsequent step, such as a neutralisation with an acidic solution. A typical way of preparing a bacterial lysate includes (re-)suspending pelleted bacteria in Buffer P1 (Qiagen), adding Buffer P2 (Qiagen), and neutralizing with neutralization Buffer S2 (Qiagen).

[0030] Lysis conditions may vary depending on the type of nucleic acid to be isolated, in particular if one is interested in only isolating one type of nucleic acid, especially if it is a specific one, such as plasmid DNA. As such, lysis conditions for subsequent RNA isolation may be different from lysis conditions for subsequent DNA isolation. In case two types of nucleic acids, such as RNA and DNA, are isolated from the same sample, there may be a combined lysis procedure for both types of nucleic acids. The same applies to the binding conditions. DNA and RNA may be bound together and separated later, e.g. by selective elution, or, alternatively, RNA and DNA may be separated by using selective binding conditions. However, the binding conditions used for endotoxin removal may be the same or similar for RNA and DNA, as demonstrated in the examples.

[0031] Solid carrier

[0032] In the process of the present invention, the liquid sample containing nucleic acids and endotoxins is typically provided without purification. Step b) is a preparatory step serving to remove components other than nucleic acids and endotoxins. Such removal may inter alia be achieved using the QIAprep Spin Miniprep (Qiagen, Germany). The QIAprep membrane is an example of a suitable solid carrier for use in step b).

[0033] Different types of solid carriers are suitable for use in the present invention. Without being bound by a particular theory, the nucleobases of the nucleic acids bind better to a (partial) negatively charged surface. The solid carriers used in the method of the invention therefore preferably have a negatively charged surface. Different types of negatively charged surfaces of solid carriers are known in the art. These include e.g. solid carriers with a silica surface, solid carriers with a carboxylate-modified surface. Accordingly, in one embodiment of the invention, the negatively charged surface of the solid carrier is a silica or carboxylate-modified surface. In a further embodiment, the negatively charged surface of the solid carrier is a carboxylate-modified surface. In still a further embodiment, the negatively charged surface of the solid carrier is a silica surface. It is also possible to use solid carriers with a surface having a mixture of both, a silica and carboxylate-modified surface.

[0034] Depending on how the negatively charged surface is produced, it may not be necessary that the complete surface is covered with negative charges, but there may also be areas which are not negatively charged. The surface only needs to comprise enough negative charges to sufficiently bind the nucleic acids. Consequently, in one embodiment, 80% or more of the carrier surface should be covered with negative charges. In a further embodiment, 85% or more of the carrier surfaces are covered with negative charges. In still a further embodiment, 90% or more of the carrier surfaces are covered with negative charges. In yet a further embodiment, 95% or more of the carrier surfaces are covered with negative charges. In another embodiment, 100% of the carrier surfaces are covered with negative charges.

[0035] Without being bound by a particular theory, it has been found that the surface properties of the solid carrier are more important than the particular geometry of the solid carrier in order to bind the nucleic acids and endotoxins. The solid carrier according to the invention may be either a membrane or particles, such as beads, e.g. magnetic beads. Thus, in one embodiment, the solid carrier is a membrane or particles. In another embodiment, the solid carrier is a membrane. In still another embodiment, the solid carrier is a silica membrane. Silica membranes are also sometimes referred to as “glass fiber filters”. Examples of a suitable membrane is contained in the QIAprep Spin Miniprep Kit (silica membrane)(Qiagen, Germany), the NucleoSpin Plasmid Mini Kit (silica membrane) (Macherey-Nagel, Germany), or the Zyppy Plasmid Miniprep (silica membrane)(Zymo Research, USA) for isolation of plasmid DNA. Other examples include the QIAamp and RNeasy Kits (all QIAGEN, Germany) for gDNA and RNA isolation, respectively, or other NucleoSpin Kits, e.g. the NucleoSpin Tissue, Clean up, and RNA Kits for gDNA, DNA clean-up, and RNA, respectively (all Macherey-Nagel, Germany). Most of these systems are available in a 96-well plate format, as well, using the same binding principle and silica membrane as in the spin column format.

[0036] In yet another embodiment, the solid carrier is beads. In still another embodiment, the solid carrier is magnetic or non-magnetic beads, with modified or non-modified surfaces. Non-modified surfaces comprise so-called silica beads with surface characteristics similar to the membranes mentioned above. Examples are the MagAttract Suspension G (QIAGEN, Germany), NucleoMag B-Beads (Macherey-Nagel, Germany), MagBinding Beads (Zymo Research, USA), and Mag G beads (Perfinity Biosciences). A particular kind of magnetic beads with modified surfaces is known as solid-phase reverse immobilization (SPRI) beads. Thus, in a further embodiment, the beads are SPRI beads. SPRI beads are commercially available and include Sera-Mag™ and Sera-Mag™ SpeedBeads (Sigma Aldrich), AMPure XP (Beckmann Coulter), PCRCIean™ (Aline Biosciences), MagSi (AMSBIO), Axygen™ AxyPrep (Fischer Scientific), QIAseq (QIAGEN) and DNA IQ™ (Promega).

[0037] The carriers with bound nucleic acids and endotoxins used in the invention are supposed to be separated from the remaining composition containing the remaining components. This separation may be accomplished in various ways, including methods utilizing centrifugal forces or gravity, optionally supported by vacuum or pressure, and methods utilizing magnetic forces (in the embodiment comprising magnetic beads), or any combination of those means. The beads, whether magnetic or non-magnetic, may also be contained in a column when the liquid sample is added or the beads with already bound nucleic acids and endotoxins may be added to a column for the purpose of separating the beads from the remaining composition. In case a liquid permeable closure of the column is used at its lower end, like a membrane, frit or similar, this allows binding, washing and / or eluting the nucleic acids and endotoxins within the column in a flow-through process.

[0038] Binding buffer - chaotropic or non-chaotropic chemistry

[0039] The precipitation of nucleic acids or binding to a solid carrier in step b) may advantageously be carried out in the presence of a binding / neutralization buffer. This has historically been done using chaotropic chemistry, i.e. by using chaotropic salts. It has also been done successfully using non- chaotropic chemistry, i.e. by using non-chaotropic salts. One example disclosing the binding of nucleic acids to silica surfaces is US 8,679,744 B2, which discloses the use of cationic detergents.

[0040] An example of a neutralization buffer providing chaotropic binding chemistry is the Buffer N3 (containing guanidium hydrochloride) from Qiagen (Hilden, Germany). An example of a non- chaotropic neutralization buffer is the Buffer S3 (containing potassium acetate) from Qiagen (Hilden, Germany), which together with the Buffer BB (containing cetyltrimethylammonium bromide - also known as CTAB) binding buffer provides a non-chaotropic binding environment.

[0041] Washing buffer

[0042] The endotoxin removal should, as much as possible, only remove endotoxins without significantly removing the nucleic acids. It has been found that the use of an alcoholic washing buffer, such as a washing buffer comprising ethanol, prior to the removal of endotoxins increases the nucleic acid yield in that it reduces the amount of nucleic acids removed together with the endotoxins.

[0043] Thus, the washing buffer comprises an alcohol. Given that the liquid sample is typically an aqueous sample, the alcohol comprised in the washing buffer should preferably be miscible with water. Hence, in one embodiment, the alcohol comprised in the washing buffer is miscible with water. In a further embodiment, the alcohol comprised in the washing buffer is a Ci to C3 alcohol. In yet a further embodiment, the alcohol comprised in the washing buffer is a Ci to C3 monoalcohol. In still a further embodiment, the alcohol comprised in the washing buffer is selected from methanol, ethanol, n-propanol, 2-propanol, and any mixture thereof. In another embodiment, the alcohol comprised in the washing buffer is selected from ethanol, n-propanol, 2-propanol, and any mixture thereof. In yet another embodiment, the alcohol comprised in the washing buffer is ethanol.

[0044] The amount of alcohol comprised in the washing buffer may vary. In one embodiment, the total amount of alcohol comprised in the washing buffer is 30 to 90% by weight based on the total weight of the washing buffer, such as in the range 50 to 90%, e.g. in the range 60 to 85%, 70 to 85%, or 75 to 83%.

[0045] Suitable washing compositions include commercially available washing compositions, for example Buffer PE, Buffer AW1 , Buffer AW2, Buffer MW1, Buffer MW2, or Buffer PB (all Qiagen, Germany) or mixtures thereof. One example of a suitable washing buffer comprising an alcohol is the Buffer PE comprised in the QIAprep Miniprep kit.

[0046] The pH of the washing buffer is preferably non-acidic. As such, the pH of the washing buffer is higher than 7. In one embodiment, the pH of the washing buffer is in the range 7 to 10, such as in the range 7.2 to 9, e.g. in the range 7.5 to 8.5.

[0047] Purifying buffer

[0048] Once the components of the liquid sample have been reduced to nucleic acids and endotoxins and have been washed with the washing buffer, the endotoxins are to be removed at least partially from the sample in step d). The at least partial removal of the endotoxins is achieved in step d) by treating the nucleic acids and endotoxins using a purifying buffer. Without being bound by a particular theory, the purifying buffer creates conditions favourable to the binding of nucleic acids to the solid carrier while at the same time creating conditions unfavourable to the binding of the endotoxins to the solid carrier. The main components of the purifying buffer are an amino compound, a pH of 3.0 to 8.0, and an alcohol.

[0049] The amino compound is selected from the group consisting of triethylamine, triethanolamine, 2- [Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1 ,3-diol (“Bis-Tris”), 1 ,3- bis[tris(hydroxymethyl)methvlamino]propane (“Bis-Tris-propane”), 2-amino-2-(hydroxymethyl)- propane-1 ,3-diol (“Tris”), and individual salts thereof. The amino compound may also be a mixture of one or more of these compounds, such as a mixture of Tris and a salt thereof. In one embodiment, the amino compound is selected from the group consisting of 2-[Bis(2- hydroxyethyl)amino]-2-(hydroxymethyl)propane-1 ,3-diol, 1 ,3- bis[tris(hydroxymethyl)methylamino]propane, 2-amino-2-(hydroxymethyl)-propane-1 ,3-diol, and individual salts thereof. In a further embodiment, the amino compound comprises 2-amino-2- (hydroxymethyl)-propane-l ,3-diol, a salt of 2-amino-2-(hydroxymethyl)-propane-1 ,3-diol, or a mixture thereof.

[0050] Suitable salts for the amino compounds disclosed herein are known in the art. Such suitable salts include hydrochloride, acetate, citrate, and phosphate salts. Thus, in one embodiment, the amino compound comprises a hydrochloride salt, an acetate salt, a citrate salt, and / or a phosphate salt. In still a further embodiment, the amino compound comprises a salt of 2-amino-2-(hydroxymethyl)- propane-1 ,3-diol, said salt being a hydrochloride, acetate, citrate, or phosphate salt. The concentration of the amino compound may vary. It has, however, been found that the total concentration range of the amino compound in the range 1.1 to 5.0 mol / L provides improved purification of the nucleic acids compared to the prior art. Accordingly, in one embodiment, the purifying buffer comprises the amino compound having a total concentration in the range of 1 .1 to 5.0 mol / L. In a further embodiment, the purifying buffer comprises the amino compound having a total concentration in the range of 1 .2 to 4.0 mol / L. In another embodiment, the purifying buffer comprises the amino compound having a total concentration in the range of 1 .3 to 3.0 mol / L. In still another embodiment, the purifying buffer comprises the amino compound having a total concentration in the range of 1 .4 to 2.5 mol / L. In yet another embodiment, the purifying buffer comprises the amino compound having a total concentration in the range of 1 .5 to 2.0 mol / L.

[0051] The alcohol comprised in the purifying buffer should be capable of dissolving the amino compound, said alcohol being present in a total amount of 30 to 70 % by weight based on the total weight of the purifying buffer. Given that the liquid sample is typically an aqueous sample, the alcohol comprised in the purifying buffer should preferably be miscible with water. Thus, in one embodiment, the alcohol comprised in the purifying buffer is miscible with water. In a further embodiment, the alcohol comprised in the purifying buffer is a Ci to C3 alcohol. In yet a further embodiment, the alcohol comprised in the purifying buffer is a Ci to C3 mono-alcohol. In still a further embodiment, the alcohol comprised in the purifying buffer is selected from methanol, ethanol, n-propanol, 2-propanol, and any mixture thereof. In another embodiment, the alcohol comprised in the purifying buffer is selected from ethanol, n-propanol, 2-propanol, and any mixture thereof. In still another embodiment, the alcohol comprised in the purifying buffer is selected from n-propanol, 2-propanol, and any mixture thereof. In yet another embodiment, the alcohol comprised in the purifying buffer is 2-propanol.

[0052] In one embodiment, the total amount of alcohol comprised in the purifying buffer is 40 to 60% by weight based on the total weight of the purifying buffer. In a further embodiment, the total amount of alcohol comprised in the purifying buffer is 45 to 55% by weight based on the total weight of the purifying buffer. In still a further embodiment, the total amount of alcohol comprised in the purifying buffer is about 50% by weight based on the total weight of the purifying buffer.

[0053] The pH of the purifying buffer may affect the amount of removed endotoxin. It has been found that the pH range of 3.0 to 8.0 is a suitable range for the process of the present invention. In one embodiment, the pH of the purifying buffer is in the range from 4.0 to 8.0. In another embodiment, the pH of the purifying buffer is in the range from 5.0 to 7.5. In still another embodiment, the pH of the purifying buffer is in the range from 5.5 to 7.0. In yet another embodiment, the pH of the purifying buffer is about 6.5.

[0054] In another embodiment the purifying buffer comprises 2-[Bis(2-hydroxyethyl)amino]-2- (hydroxymethyl)propane-l ,3-diol, 1 ,3-bis[tris(hydroxymethyl)methvlamino]propane, 2-amino-2- (hydroxymethyl)-propane-l ,3-diol, individual salts thereof, or a mixture thereof having a total concentration in the range of 1 .2 to 4.0 mol / L, n-propanol or 2-propanol in a total amount of 40 to 60% by weight based on the total weight of the purifying buffer, and has a pH in the range from 5.0 to 7.5. In a further embodiment the purifying buffer comprises 2-amino-2-(hydroxymethyl)-1 ,3- propanediol, a salt of 2-amino-2-(hydroxymethyl)-propane-1 ,3-diol, or a mixture thereof having a total concentration in the range of 1 .2 to 3.0 mol / L, 2-propanol in a total amount of 40 to 60% by weight based on the total weight of the purifying buffer, and has a pH in the range from 5.5 to 7.0.

[0055] Collecting the purified nucleic acid

[0056] After washing with the purifying buffer, the remaining components from step d), i.e. the precipitate or the solid carrier, may be washed in step e) one or more times with a washing composition, which may be the same or different from the washing composition used in step c). In one embodiment, the optional washing step e) is included. In a further embodiment, the optional washing step e) is not included. Suitable washing compositions for steps c) and / or e) include commercially available washing compositions, for example Buffer PE, Buffer AW1 , Buffer AW2, Buffer MW1 , Buffer MW2, or Buffer PB (all Qiagen, Germany) or mixtures thereof.

[0057] The optionally washed composition resulting from step e) or from step d) may contain nucleic acids bound to the solid carrier. This composition may be used as such in a downstream application, or the precipitated or bound nucleic acids may be collected first in step f). In order to collect the nucleic acids bound to the solid carrier in step f), the solid carrier will typically be eluted with a suitable aqueous composition capable of solubilizing nucleic acids. Such dissolving compositions are known in the art and are commonly referred to as “TE buffers”, including e.g. Buffer TE (Qiagen, Germany). As an example, a TE buffer contains 5 to 10 mM Tris in combination with up to 1 mM ethylenediaminetetraacetic acid (EDTA). Pure water or any aqueous buffer with low contents of salts will also elute the nucleic acids.

[0058] Use of the washing and purifying buffers

[0059] The washing and purifying buffer according to the invention have not been disclosed in the art for use in a process as defined herein. Thus, a further aspect of the invention concerns the use of a washing buffer and a purifying buffer according to the invention for purifying nucleic acids from a biological sample containing nucleic acids, such as plasmid DNA, and endotoxins by at least partially removing the endotoxin.

[0060] The process according to the invention is effective in removing endotoxins. In one embodiment, at least 60% by weight of the endotoxins separated in step b), and washed in step c), are removed in step d). This percentage is determined by comparing the amount of endotoxins remaining after carrying the process of the invention with an identical process leaving out step d). In a further embodiment, at least 70% by weight of endotoxins are removed in step d). In still a further embodiment, at least 80% by weight of endotoxins are removed in step d). In yet a further embodiment, at least 90% by weight of endotoxins are removed in step d). In another embodiment, at least 93% by weight of endotoxins are removed in step d). In still another embodiment, at least 95% by weight of endotoxins are removed in step d). In yet another embodiment, at least 96% by weight of endotoxins are removed in step d). In a further embodiment, at least 97% by weight of endotoxins are removed in step d). In still a further embodiment, at least 98% by weight of endotoxins are removed in step d). In yet a further embodiment, at least 99% by weight of endotoxins, such as 100%, are removed in step d).

[0061] The process according to the invention does not remove endotoxins at the expense of the nucleic acids. Thus, in one embodiment, at least 60% by weight of the nucleic acids, separated in step b), and washed in step c), are retained in step d), such as at least 70%, 80% or 90%, e.g. at least 93%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0062] Kit of parts

[0063] The solid carrier as defined herein may be provided together with the remaining components, e.g. various buffers as defined herein, for use in the process according to the invention. The washing buffer and the purifying buffer as defined herein have not previously been provided together with the remaining components used in the process according to the invention. Accordingly, another aspect of the invention concerns a kit of parts for purifying nucleic acids from a biological sample containing nucleic acids, such as plasmid DNA, and endotoxins, wherein the kit comprises a washing buffer and a purifying buffer as defined herein and a solid carrier as defined herein.

[0064] The kit of parts may also conveniently comprise any other buffer and / or eluting composition used in the process according to the invention. Thus, in one embodiment, the kit of parts according to the invention further comprises a TE buffer. In still a further embodiment, the kit of parts according to the invention further comprises one or more different washing compositions and a TE buffer.

[0065] BRIEF DESCRIPTION OF THE FIGURES

[0066] Figure 1

[0067] Yields of QIAprep and Plasmid Plus Mini preparations with two variants of the purifying buffer according to the invention (1 .5M and 1 .75M Tris, pH 6.8 in 50% isopropanol), with and without upstream washing with Buffer PE. M&N refers to the commercially available “Detoxification Buffer” from Macherey-Nagel.

[0068] Figure 2

[0069] 1% agarose-gel of pBlueScript plasmid DNA (10 pl eluate each) isolated with QIAprep with different washing conditions. Figure 3

[0070] Endotoxin contamination of pBS plasmid DNA isolated with QIAprep with different first washing conditions. Data were normalized on pg of isolated plasmid DNA. Upper panel: overview of the effect of endotoxin removal with and without Tris-containing purifying buffers. Lower panel: higher resolution to demonstrate increasing endotoxin removal at higher Tris concentrations.

[0071] Figure 4

[0072] Endotoxin content of different plasmids isolated from different E. coli strains with magnetic beads and QIAprep with and without endotoxin removal washing step. Data were normalized on pg of isolated plasmid DNA.

[0073] Figure 5

[0074] Endotoxin content of high- and low-copy plasmids isolated with the non-chaotropic Plasmid Plus chemistry with the endotoxin removal buffer ETR supplied with the Plasmid Plus kit compared to the purifying buffers according to the invention, (a): scale up to 45 EU / pg, (b): scale up to 1 EU / pg to visualize the very low endotoxin content after washing with the purifying buffers according to the invention.

[0075] SPECIFIC EMBODIMENTS

[0076] 1. A process for purifying nucleic acids, preferably for removing endotoxins, from a biological sample containing nucleic acids, preferably plasmid DNA, comprising the following steps: a) providing a liquid sample comprising nucleic acids and endotoxins; b) precipitating the nucleic acids and the endotoxins or binding the nucleic acids and the endotoxins on a solid carrier and thereby separating them from the remaining components of the liquid sample; c) washing the precipitate or solid carrier from step b) at least once with a washing buffer comprising an alcohol to further remove remaining components; d) treating the nucleic acids and endotoxins separated in step b), and washed in step c), using a purifying buffer for at least partial removal of the endotoxin; e) optionally washing the precipitate or the solid carrier remaining in step d) at least once with at least one washing composition, and f) optionally collecting the nucleic acids, optionally by eluting from the solid carrier or dissolving the precipitated nucleic acid; wherein the purifying buffer in step d) comprises an amino compound selected from the group consisting of triethylamine, triethanolamine, 2-[Bis(2-hydroxyethyl)amino]-2- (hydroxymethyl)propane-l ,3-diol, 1 ,3-bis[tris(hydroxymethyl)methvlamino]propane, 2- amino-2-(hydroxymethyl)-propane-1 ,3-diol, and individual salts thereof, an alcohol capable of dissolving the amino compound in a total amount of 30 to 70 % by weight based on the total weight of the purifying buffer, and water, wherein the purifying buffer has a pH in the range from 3.0 to 8.0.

[0077] 2. The process according to embodiment 1 , wherein the alcohol in the washing buffer is a Ci to C3 alcohol, preferably a Ci to C3 mono-alcohol, such as methanol, ethanol, n-propanol, 2-propanol, or a mixture thereof.

[0078] 3. The process according to embodiment 2, wherein the alcohol in the washing buffer is ethanol.

[0079] 4. The process according to any one of embodiments 1 to 3, wherein the alcohol in the washing buffer is present in the amount of 30 to 90% (w / w), such as in the range 50 to 90%, e.g. in the range 60 to 85%, 70 to 85%, or 75 to 83%.

[0080] 5. The process according to any one of the preceding embodiments, wherein the nucleic acids and endotoxins are bound on a solid carrier material in step b).

[0081] 6. The process according to any one of the preceding embodiments, wherein the solid carrier is a membrane or particles, such as beads, preferably magnetic beads.

[0082] 7. The process according to embodiment 6, wherein the surface of the membrane or of the particles is a negatively charged surface, such as a silica surface or a carboxylate-modified surface.

[0083] 8. The process according to any one of the preceding embodiments, wherein the purifying buffer comprises the amino compound having a total concentration in the range of 1 .1 to 5.0 mol / L, such as in the range of 1 .2 to 4.0 mol / L, e.g. in the range of 1 .3 to 3.0 mol / L, such as in the range of 1 .4 to 2.5 mol / L, e.g. in the range of 1 .5 to 2.0 mol / L.

[0084] 9. The process according to any one of the preceding embodiments, wherein the alcohol in the purifying buffer is a Ci to C3 alcohol, preferably a Ci to C3 mono-alcohol, such as methanol, ethanol, n-propanol, 2-propanol, or a mixture thereof.

[0085] 10. The process according to any one of the preceding embodiments, wherein the total amount of alcohol in the purifying buffer is 40 to 60% by weight based on the total weight of the purifying buffer, such as 45 to 55%, e.g. 50%.

[0086] 11. The process according to any one of the preceding embodiments, wherein the pH of the purifying buffer is in the range from 4.0 to 8.0, such as from 5.0 to 7.5, e.g. from 5.5 to 7.0, such as about 6.5.

[0087] 12. The process according to any one of the preceding embodiments, wherein the purifying buffer comprises an amino compound selected from the group consisting of 2-[Bis(2- hydroxyethyl)amino]-2-(hydroxymethyl)propane-1 ,3-diol, 1 ,3- bis[tris(hydroxymethyl)methvlamino]propane, 2-amino-2-(hydroxymethyl)-propane-1 ,3-diol, and individual salts thereof, as well as any mixture thereof.

[0088] 13. The process according to any one of the preceding embodiments, wherein the amino compound is selected from the group consisting of 2-amino-2-(hydroxymethyl)-propane-

[0089] 1 ,3-diol, a salt of 2-amino-2-(hydroxymethyl)-propane-1 ,3-diol, individual salts thereof, and any mixtures thereof.

[0090] 14. The process according to any one of the preceding embodiments, wherein the amino compound comprises a salt of 2-amino-2-(hydroxymethyl)-propane-1 ,3-diol, said salt being a hydrochloride, acetate, or phosphate salt.

[0091] 15. The process according to any one of the preceding embodimens, wherein the purifying buffer comprises 2-amino-2-(hydroxymethyl)-1 ,3-propanediol, a salt of 2-amino-2- (hydroxymethyl)-propane-l ,3-diol, or a mixture thereof having a total concentration in the range of 1.2 to 2.0 mol / L, 2-propanol in a total amount of 40 to 60% by weight based on the total weight of the purifying buffer, and has a pH in the range from 5.5 to 7.0.

[0092] 16. The process according to any one of the preceding embodiments, wherein the nucleic acids and endotoxins are each dissolved in the liquid sample provided in step a).

[0093] 17. The process according to any one of the preceding embodiments, wherein the liquid sample provided in step a) is a bacterial lysate.

[0094] 18. The process according to embodiment 17, wherein: i) the lysate is obtained by conducting an alkaline lysis, ii) the lysate is neutralized using an acidic solution, preferably an acetate or citrate buffer, iii) the lysate is cleared from cell debris, and / or iv) the lysate is cleared from any precipitate resulting from the lysis or any subsequent step, such as a neutralisation with an acidic solution.

[0095] 19. The process according to any one of the preceding embodiments, wherein the nucleic acids are DNA.

[0096] 20. The process according to embodiment 19, wherein the nucleic acids are plasmid DNA.

[0097] 21 . The process according to any one of the preceding embodiments, wherein at least 60% by weight of the endotoxins separated in step b), and washed in step c), are removed in step d), such as at least 70%, 80% or 90%, e.g. at least 93%, 95%, 96%, 97%, 98%, 99%, or 100%. 22. The process according to any one of the preceding embodiments, wherein at least 60% by weight of the nucleic acids, separated in step b), and washed in step c), are retained in step d), such as at least 70%, 80% or 90%, e.g. at least 93%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0098] 23. Use of a washing buffer and a purifying buffer as defined in any one of embodiments 1 to 22 for purifying nucleic acids from a biological sample containing nucleic acids, such as plasmid DNA, and endotoxins by at least partially removing the endotoxin.

[0099] 24. A kit of parts for purifying nucleic acids from a biological sample containing nucleic acids, such as plasmid DNA, and endotoxins, wherein the kit comprises a washing buffer and a purifying buffer as defined in any one of embodiments 1 to 22 and a solid carrier as defined in any one of embodiments 6 and 7.

[0100] EXAMPLES

[0101] Materials

[0102] Buffers P1 , P2, N3, S3, PB, PE, EB, MW1 , and TE were acquired from QIAGEN, Germany.

[0103] Example 1 - Improving yields with an upstream alcohol washing step

[0104] The use of the purifying buffer according to the invention may cause a loss of yield of nucleic acids compared to the yield without using the purifying buffer. It was found that a washing step with an alcoholic washing buffer prior to the removal of endotoxins increases the yield of nucleic acids.

[0105] Tris and alcohol concentrations indicated below are the final concentrations in the purifying buffer. The Tris-stock solution used to prepare the final purifying buffer was brought to pH 6.8 with hydrochloric acid.

[0106] Method:

[0107] (a) - chaotropic

[0108] A frozen pellet from 1 .5 ml over-night culture of DH5a / pCMVB (E coli strain / plasmid) was resuspended in 250 pl Resuspension Buffer P1 (QIAGEN) and carefully mixed with 250 pl Lysis Buffer P2 (QIAGEN) and incubated at ambient temperature for 5 minutes. 350 pl Neutralization Buffer N3 (QIAGEN) was added and mixed.

[0109] The crude lysate was centrifuged for 10 minutes at 13000 rpm in a Sigma benchtop centrifuge. 800 pl of the then cleared lysate (equivalent to 941 pl of the original culture) were transferred into a QIAprep spin column (part of the QIAprep Spin Miniprep Kit) and centrifuged at 13000 rpm for 1 minute in an Eppendorf tabletop centrifuge 5417C. The flow-through was discarded.

[0110] (b) - non-chaotropic

[0111] Pelleted bacteria were resuspended in 200 pl of Buffer P1 . 200 pl of Buffer P2 was added, followed by mixing by inversion and incubation at room temperature for 3 minutes. 200 pl of Buffer S3 (neutralization buffer) was added to the lysate, immediately followed by inverting 4-6 times. The lysate was not incubated on ice.

[0112] The crude lysate was centrifuged for 10 minutes at 13000 rpm in a Sigma benchtop centrifuge. 800.

[0113] 200 pl of Buffer BB (binding buffer) was added to the cleared lysate, followed by mixing by inverting 4-6 times. This mixture was then transferred to a Qiagen Plasmid Plus Midi spin column. By applying vacuum, the solution was drawn through the column, resulting in the binding of the plasmid to the column.

[0114] (a) and (b)

[0115] The columns were washed with 700 pl Buffer PE (comprising ethanol), and then 700 pl of purifying buffer variants - according to the invention - were added: a) 1.5 M Tris, pH 6.8 b) 1.75 M Tris, pH 6.8

[0116] All purifying buffers contained 50% (w / w) isopropanol.

[0117] The spin columns were centrifuged at 13000 rpm for 1 minute and the flow-through discarded. The membranes were washed by applying 750 pl Buffer PE (QIAGEN) to the column and again centrifuged at 13000 rpm for 1 minute. The flow-through was discarded and the spin columns were centrifuged for another minute to dry the membrane.

[0118] The spin columns were then transferred into a new 1 .5 ml microcentrifuge tube and 100 pl Buffer TE (QIAGEN) was added to elute the plasmid DNA. After incubation for 1 minute at ambient temperature, the spin columns were centrifuged for 1 minute at 13000 rpm.

[0119] Every experiment was done in duplicate. As a reference, preparations without the purifying buffer and a commercially available endotoxin removal buffer, using triethanolamine (“Detoxification Buffer”, Macherey-Nagel, Germany), was done.

[0120] Results:

[0121] The results (Figure 1) show the beneficial effect of an additional up-stream washing step with an alcohol-containing buffer and brings the yield on the same level as a commercially available endotoxin removal buffer, but at the same time improving the removal of endotoxins (as further demonstrated below in Example 2).

[0122] Example 2 - Effect of purifying buffer at varying Tris concentrations

[0123] Endotoxin content in plasmid preparation can vary significantly between different E. coli strains and the type of plasmid.

[0124] The Tris and alcohol concentrations indicated below are the final concentrations in the purifying buffer. The Tris-stock solution used to prepare the final purifying buffer was brought to pH 6.5 with hydrochloric acid, except for the 2M Tris solution. Due to concentration / volume limitations, pH could only be lowered to 7.5.

[0125] Method:

[0126] A frozen pellet from 50 ml over-night culture of DH5a / pBS (E. coli strain / plasmid) was resuspended in 12.5 ml Resuspension Buffer P1 (QIAGEN) and carefully mixed with 12.5 ml Lysis Buffer P2 (QIAGEN) and incubated at ambient temperature for 5 minutes. 17.5ml Neutralization Buffer N3 (QIAGEN) was added and mixed.

[0127] The crude lysate was centrifuged for 15 minutes at 5000 rpm in a Sigma benchtop centrifuge. 800 pl of the then cleared lysate (equivalent to 941 pl of the original culture) were transferred into a QIAprep spin column (part of the QIAprep Spin Miniprep Kit) and centrifuged at 13000 rpm for 1 minute in an Eppendorf tabletop centrifuge 5417C. The flow-through was discarded and 750 pl of purifying buffer variants - according to the invention - were added: a) 2.0 M Tris, pH 7.5 b) 1 .5 M Tris, pH 6.5 c) 1 M Tris, pH 6.5 d) 0.5 M Tris, pH 6.5; e) No endotoxin purifying step (sample “PE”). All purifying buffers contained 50% (w / w) isopropanol.

[0128] The spin columns were centrifuged at 13000 rpm for 1 minute and the flow-through discarded. The membranes were washed by applying 750 pl Buffer PE (QIAGEN) to the column and again centrifuged at 13000 rpm for 1 minute. The flow-through was discarded and the spin columns were centrifuged for another minute to dry the membrane.

[0129] The spin columns were then transferred into a new 1 .5 ml microcentrifuge tube and 100 pl Buffer TE (QIAGEN) was added to elute the plasmid DNA. After incubation for 1 minute at ambient temperature, the spin columns were centrifuged for 1 minute at 13000 rpm.

[0130] Every experiment was done in duplicate.

[0131] The isolated plasmid DNA was analyzed for yield and structural integrity by agarose-gel electrophoresis (see Fig. 2) as well as endotoxin content (see Fig. 3).

[0132] Endotoxin content was analyzed with the Kinetic-QCL™ Kinetic Chromogenic LAL Assay from Lonza according to the manufacturer’s instructions.

[0133] The commercially available endotoxin removal buffer “Detoxification Buffer ERB” (Macherey-Nagel, Germany) was used as a “positive control” for endotoxin removal.

[0134] Results:

[0135] With a strain yielding endotoxin contaminations of >600 EU / pg, a Tris / isopropanol-containing purifying buffer is able to reduce the amount of endotoxin contamination about 100-fold.

[0136] Furthermore, the results also clearly show that Tris concentrations >1 M further are advantageous and reduce the level of endotoxins compared to Tris concentrations of 1 .0 M and below but do not have any influence on quality and yield of the isolated plasmid DNA as shown by agarose gel electrophoresis.

[0137] The commercially available endotoxin removal buffer (the “positive control”) provides inferior endotoxin removal compared to the invention.

[0138] Example 3 - Using magnetic beads as the solid carrier

[0139] Different E. coli strains were tested with two different bead concentrations. 1 ml E. coli cultures were inoculated and grown over night. Plasmids were isolated following an automated protocol on a KingFisher Flex instrument (Thermo Fisher Scientific). The following strains and plasmids were tested:

[0140] 1) XL1 blue / pBlueScript (pBS)

[0141] 2) DH5a / pCMVb

[0142] 3) HB101 / pBR322

[0143] 4) HB101 / pBlueScript

[0144] The 96-well plates on the KingFisher Flex instrument were loaded with the following compositions:

[0145] 1) 300pl carboxylated magnetic beads (prepared by in-house synthesis, equivalent to Sera- Mag™) (for capturing E. coli cells)

[0146] 2) 500 pl Buffer P1 I Buffer P2 (1 :1) (for lysis)

[0147] 3) 15 pl or 30pl Mag G suspension in 300pl water (for plasmid binding)

[0148] 4) 800 pl Buffer MW1 (wash 1)

[0149] 5) 800 pl Buffer PE (wash 2) or 1 .5 M Tris 1 50 % Isopropanol, pH 6.5 (purifying endotoxin removal buffer)

[0150] 6) 800 pl Buffer PE (wash 3)

[0151] 7) 100 pl Buffer TE (Elution)

[0152] After step 2), the protocol was paused and 350 pl Buffer N3 (neutralization, contains chaotrop for binding) was added manually.

[0153] Then the protocol was continued.

[0154] All samples were subjected to compositions 1) to 7) in that order, with composition 5) either being the same PE washing buffer used in composition 6) or the purifying buffer according to the invention.

[0155] The protocol according to Example 2 was carried out in parallel as reference.

[0156] Endotoxin measurement:

[0157] Endotoxin content was analyzed with the Kinetic-QCL™ Kinetic Chromogenic LAL Assay (Lonza, Switzerland) according to the manufacturer’s instructions. The resulting endotoxin units (EU) were normalized to EU / pg yield. Results:

[0158] The results are shown in Figure 4. The results clearly show that the method according to the invention also results in efficient endotoxin removal with beads instead of silica membranes, at different bead concentrations.

[0159] Example 4 - Endotoxin removal with non-chaotropic chemistry

[0160] In addition to the use of chaotropic salts to bind nucleic acids to mineral surfaces such as silica, which was used in the examples above, the nucleic acids may also be bound with non-chaotropic salts, such as those containing cationic detergents.

[0161] Method:

[0162] Plasmid was isolated using the Qiagen Plasmid Plus kit.

[0163] Pelleted bacteria were resuspended in 2 ml of Buffer P1 . The following strains were used:

[0164] DH5a / pCMVb (high-copy plasmid): 25 ml culture

[0165] DH5a / pBR322 (low-copy plasmid): 50 ml culture

[0166] 2 ml of Buffer P2 was added, followed by mixing by inversion and incubation at room temperature for 3 minutes. 2 ml of Buffer S3 (neutralization buffer; containing potassium acetate) was added to the lysate, immediately followed by inverting 4-6 times. The lysate was not incubated on ice.

[0167] The lysate was transferred to a QI Afilter cartridge (part of the Qiagen Plasmid Plus kit) and incubated at room temperature for 10 minutes. The lysate was cleared by vacuum filtering through the cartridge filter.

[0168] 2 ml of Buffer BB (binding buffer; containing CTAB) was added to the cleared lysate, followed by mixing by inverting 4-6 times. This mixture was then transferred to a Qiagen Plasmid Plus Midi spin column. By applying vacuum, the solution was drawn through the column, resulting in the binding of the plasmid to the column. The column containing the bound plasmid was subsequently purified with 0.7 ml of a purifying buffer. The following purifying buffers were used

[0169] • Buffer ETR (the buffer that is currently used as the endotoxin removal buffer provided with the kit)

[0170] • 3 M Tris, pH 6.8 (purifying buffer according to the invention)

[0171] • 3.5 M Tris, pH 6.8 (purifying buffer according to the invention)

[0172] After purification, the column was washed with 0.7 ml Buffer PE, and the flow-through was discarded. The DNA was finally eluted using 200 pl of Buffer EB (10 mM Tris Ci, pH 8.5).

[0173] Endotoxin content was analyzed with the Kinetic-QCL™ Kinetic Chromogenic LAL Assay from Lonza according to the manufacturer’s instructions.

[0174] The resulting endotoxin units (EU) were normalized to EU / pg yield.

[0175] Results

[0176] The resulting endotoxin units are shown in Figure 5 on (a) a scale up to 45 EU / pg and (b) up to 1 EU / pg to visualize the very low endotoxin content when using the purifying buffer according to the invention. The data demonstrate that the new purifying buffer(s) according to the invention works for all types of silica (glass / mineral) surfaces independent of the original binding chemistry (chaotropic or non-chaotropic) and is able to improve the quality of isolated plasmid DNA by significantly reducing the amount of endotoxin contamination in the preparation.

[0177] Example 5 - Demonstrating the process for isolating RNA

[0178] The process of the invention is shown to be useful in the isolation of not only DNA but also RNA in this example.

[0179] Tris and alcohol concentrations indicated below are the final concentrations in the purifying buffer. The Tris-stock solution used to prepare the final purifying buffer was brought to pH 6.5 with hydrochloric acid.

[0180] Method: RNA was isolated from 1 ml E. coli DH5a cells using the QIAGEN RNeasy Mini Kit (Cat. No.: 74104). The protocol is in accordance with the RNAprotect Bacteria Reagent Handbook (01 / 2020). Protocol 1 (enzymatic Lysis) was used unchanged for the lysis of the bacterial cells, and a modified Protocol 7 (RNA isolation) was used for the isolation of total RNA from the bacteria.

[0181] Lysis:

[0182] The bacterial cells (1.2*108cells / ml) were lysed using 100 pl of Buffer TE (containing lysozyme) and 350 pl of Buffer RLT.

[0183] Isolation:

[0184] 700 pl of lysate (including precipitate) was transferred to an RNeasy Mini spin column placed in a 2 ml collection tube. The lid was closed, and the tube centrifuged for 15 s at 10,000 rpm. The flow- through was discarded.

[0185] The same tube was used in the following step, where 700 pl of Buffer RW1 (containing ethanol and a guanidine salt) were added to the RNeasy Mini spin column. The lid was closed, and the tube centrifuged for 15 s at 10,000 rpm. The flow-through and tube were discarded.

[0186] 500 pl of purifying buffer were added. The purifying buffer had a Tris / HCI concentration of 1 .75 M at a pH of 6.5, as well as 50% isopropanol. The lid was closed, and the tube centrifuged for 15 s at 10,000 rpm to wash the spin column membrane. The flow-through was discarded.

[0187] 500 pl of Buffer RPE were added to the RNeasy spin column. The lid was closed, and the tube was centrifuged for 15 s >10,000 rpm to wash the spin column membrane. The flow-through was discarded. In the comparative experiments, the previous step with the purifying buffer was replaced with this step, leading to two washings with Buffer RPE.

[0188] The RNeasy Mini spin column was placed in a new 1 .5 ml collection tube and 30-50 pl of RNase- free water were added directly to the spin column membrane. The lid was closed, and the tube centrifuged for 15 s at 10,000 rpm to elute the RNA.

[0189] Endotoxin measurement: Endotoxin content was analyzed with the Kinetic-QCL™ Kinetic Chromogenic LAL Assay (Lonza, Switzerland) according to the manufacturer’s instructions. The resulting endotoxin units (EU) were normalized to EU / pg yield. Results:

[0190] The duplicate results for two washings with Buffer RPE (comparative) and purifying buffer followed by Buffer RPE (according to the invention) are shown in Fig. 6 and demonstrate that the invention is applicable to all types of nucleic acids, including RNA.

Claims

CLAIMS1. A process for purifying nucleic acids, preferably for removing endotoxins, from a biological sample containing nucleic acids, preferably plasmid DNA, comprising the following steps: a) providing a liquid sample comprising nucleic acids and endotoxins; b) precipitating the nucleic acids and the endotoxins or binding the nucleic acids and the endotoxins on a solid carrier and thereby separating them from the remaining components of the liquid sample; c) washing the precipitate or solid carrier from step b) at least once with a washing buffer comprising an alcohol to further remove remaining components; d) treating the nucleic acids and endotoxins separated in step b), and washed in step c), using a purifying buffer for at least partial removal of the endotoxin; e) optionally washing the precipitate or the solid carrier remaining in step d) at least once with at least one washing composition, and f) optionally collecting the nucleic acids, optionally by eluting from the solid carrier or dissolving the precipitated nucleic acid; wherein the purifying buffer in step d) comprises an amino compound selected from the group consisting of triethylamine, triethanolamine, 2-[Bis(2-hydroxyethyl)amino]-2- (hydroxymethyl)propane-l ,3-diol, 1 ,3-bis[tris(hydroxymethyl)methvlamino]propane, 2- amino-2-(hydroxymethyl)-propane-1 ,3-diol, and individual salts thereof, an alcohol capable of dissolving the amino compound in a total amount of 30 to 70 % by weight based on the total weight of the purifying buffer, and water, wherein the purifying buffer has a pH in the range from 3.0 to 8.0.

2. The process according to claim 1 , wherein the alcohol in the washing buffer is a Ci to C3 alcohol, preferably a Ci to C3 mono-alcohol, such as methanol, ethanol, n-propanol, 2- propanol, or a mixture thereof.

3. The process according to claim 2, wherein the alcohol in the washing buffer is ethanol.

4. The process according to any one of claims 1 to 3, wherein the alcohol in the washing buffer is present in the amount of 30 to 90% (w / w), such as in the range 50 to 90%, e.g. in the range 60 to 85%, 70 to 85%, or 75 to 83%.

5. The process according to any one of the preceding claims, wherein the nucleic acids and endotoxins are bound on a solid carrier material in step b).

6. The process according to any one of the preceding claims, wherein the solid carrier is a membrane or particles, such as beads, preferably magnetic beads.

7. The process according to any one of the preceding claims, wherein the purifying buffer comprises the amino compound having a total concentration in the range of 1.1 to 5.0 mol / L, such as in the range of 1 .2 to 4.0 mol / L, e.g. in the range of 1 .3 to 3.0 mol / L, such as in the range of 1 .4 to 2.5 mol / L, e.g. in the range of 1 .5 to 2.0 mol / L.

8. The process according to any one of the preceding claims, wherein the alcohol in the purifying buffer is a Ci to C3 alcohol, preferably a Ci to C3 mono-alcohol, such as methanol, ethanol, n-propanol, 2-propanol, or a mixture thereof.

9. The process according to any one of the preceding claims, wherein the purifying buffer comprises an amino compound selected from the group consisting of 2-[Bis(2- hydroxyethyl)amino]-2-(hydroxymethyl)propane-1 ,3-diol, 1 ,3- bis[tris(hydroxymethyl)methvlamino]propane, 2-amino-2-(hydroxymethyl)-propane-1 ,3-diol, and individual salts thereof, as well as any mixture thereof.

10. The process according to any one of the preceding claims, wherein the amino compound is selected from the group consisting of 2-amino-2-(hydroxymethyl)-propane-1 ,3-diol, a salt of 2-amino-2-(hydroxymethyl)-propane-1 ,3-diol, individual salts thereof, and any mixtures thereof.

11. The process according to any one of the preceding claims, wherein the liquid sample provided in step a) is a bacterial lysate.

12. The process according to claim 17, wherein: i) the lysate is obtained by conducting an alkaline lysis, ii) the lysate is neutralized using an acidic solution, preferably an acetate or citrate buffer, iii) the lysate is cleared from cell debris, and / or iv) the lysate is cleared from any precipitate resulting from the lysis or any subsequent step, such as a neutralisation with an acidic solution.

13. The process according to any one of the preceding claims, wherein the nucleic acids are DNA.

14. Use of a washing buffer and a purifying buffer as defined in any one of claims 1 to 13 for purifying nucleic acids from a biological sample containing nucleic acids, such as plasmid DNA, and endotoxins by at least partially removing the endotoxin.

15. A kit of parts for purifying nucleic acids from a biological sample containing nucleic acids, such as plasmid DNA, and endotoxins, wherein the kit comprises a washing buffer and a purifying buffer as defined in any one of claims 1 to 13 and a solid carrier as defined in claim 6.

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