# US 2014/0154693 A1 · Reagent usable for the isolation and/or purification of nucleic acids

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- Number: US 2014/0154693 A1
- Document ID: US20140154693A1
- Status: Published application
- Kind: A1
- Application number: 14/130,436
- Filed: 2012-07-04
- Published: 2014-06-05
- Earliest claimed: 2011-07-04
- Assignees: Qiagen GmbH
- Inventors: Peter Porschewski
- Web page: https://patentgrep.com/patent/US20140154693A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides a special reagent for the pretreatment of sample materials. The pretreatment with the reagent according to the invention enables the isolation of nucleic acids by a uniform method from very diverse sample materials, in particular different bioprocess samples, even if the nucleic acids are only present in small amounts in the sample materials. The reagent used for the pretreatment comprises, as key component, at least one compound comprising an amino group. The invention further relates to methods of purification of nucleic acids, in which the sample material is pretreated correspondingly, and suitable kits.

## Claims

### Claim at position 1

-31. (canceled)

### Claim 32

A method of isolation and/or purification of nucleic acids from a sample material, wherein the sample material is a bioprocess sample, the bioprocess sample is a purification fraction obtained in the purification process for a biotechnological product, and the nucleic acids to be isolated represent a contamination of the bioprocess sample, comprising:
(a) adding a reagent to the sample material, wherein the reagent comprises one or more compounds comprising an amino group selected from one or more of the following groups:
(i) polar-neutral proteinogenic α-aminocarboxylic acids,
(ii) nonpolar-hydrophobic proteinogenic α-aminocarboxylic acids,
(iii) basic α-aminocarboxylic acids,
(iv) non-proteinogenic aminocarboxylic acids selected from the group consisting of β-alanine, D-alanine, L-homoserine and D-valine,
(v) aminocarboxylic acid polymers comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vi) peptides from 2 to 4 aminocarboxylic acids, comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vii) a compound of the general formula R3R4N—CR1R2—COOH (I), wherein R1 and R2 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, aminoalkyl and thioalkyl, wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, di(hydroxyalkyl) and tri(hydroxyalkyl), wherein the alkyl component has a chain length from C1 to C5, is branched or unbranched, is saturated or unsaturated, and wherein at least one of R1, R2, R3 and R4 is not hydrogen,
(viii) a compound of the general formula R3R4N—CR5R6R7 (II), wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen and hydroxyalkyl, and wherein R5, R6 and R7 are selected independently of one another from the group consisting of hydrogen, alkyl and hydroxyalkyl,
(b) optionally mixing this reagent with the sample material,
(c) isolating and/or purifying nucleic acid(s) from the sample material that has been contacted with and/or mixed with the reagent, and
(d) optionally quantitatively and/or qualitatively detecting at least a portion of the isolated nucleic acid(s).

### Claim 33 (depends on 32)

The method of claim 32, wherein the polar-neutral porteinogenic α-aminocarboxylic acids of (a)(i) are asparagine, cysteine, glutamine, glycine, threonine and serine.

### Claim 34 (depends on 32)

The method of claim 32, wherein the nonpolar-hydrophobic proteinogenic α-aminocarboxylic acids of (a)(ii) are alanine, leucine, isoleucine, methionine, proline and valine.

### Claim 35 (depends on 32)

The method of claim 32, wherein the basic α-aminocarboxylic acids of (a)(iii) are arginine, lysine and ornithine.

### Claim 36 (depends on 32)

The method of claim 32, wherein the aminocarboxylic acid polymers of (a)(v) consist of the aminocarboxylic acids of (a)(i) to (a)(iv).

### Claim 37 (depends on 32)

The method of claim 32, wherein the aminocarboxylic acid polymer of (a)(v) are formed from an identical aminocarboxylic acid monomer.

### Claim 38 (depends on 32)

The method of claim 32, wherein the aminocarboxylic acid polymer of (a)(v) are formed from two or more different aminocarboxylic acid monomers.

### Claim 39 (depends on 32)

The method of claim 32, wherein the aminocarboxylic acid polymer of (a)(v) are in conjunction with halides.

### Claim 40 (depends on 32)

The method of claim 32, wherein the aminocarboxylic acid polymer of (a)(v) is selected from the group consisting of poly-L-lysine, poly-L-lysine hydrobromide, poly-L-lysine hydrochloride, poly(L-glutamine, L-alanine), poly(L-glutamine, L-lysine) hydrobromide, and poly-L-ornithine.

### Claim 41 (depends on 32)

The method of claim 32, wherein the peptides of (a)(vi) consist of the aminocarboxylic acids of (a)(i) to (a)(iv).

### Claim 42 (depends on 32)

The method of claim 32, wherein the peptides of (a)(vi) consist of identical aminocarboxylic acids of one of (a)(i) to (a)(iv).

### Claim 43 (depends on 32)

The method of claim 32, wherein the peptides of (a)(vi) consist of two or more different aminocarboxylic acids of (a)(i) to (a)(iv).

### Claim 44 (depends on 32)

The method of claim 32, wherein the peptides of (a)(v) are glycine dimers, glycine trimmers, and glycine tetramers.

### Claim 45 (depends on 32)

The method of claim 32, wherein the compound of (a)(vii) is N-(tri(hydroxymethyl)methyl)glycine (tricine) or N,N-bis(2-hydroxyethyl)glycine (bicine).

### Claim 46 (depends on 32)

The method of claim 32, wherein at least one of R5, R6 and R7 of the compound of (a)(viii) is a hydroxyalkyl, and wherein the alkyl component has a chain length from C1 to C5, is branched or unbranched, and is saturated or unsaturated.

### Claim 47 (depends on 32)

The method of claim 32, wherein the compound of (a)(viii) is Tris(hydroxymethyl)-aminomethane (Tris).

### Claim 48 (depends on 32)

The method according to claim 32, wherein the biotechnological product is a biopharmaceutical, the contaminating nucleic acid to be isolated is a nucleic acid of the host cell that was used for the production of the biopharmaceutical and/or is a viral nucleic acid, and step (d) is performed to analyse whether such contaminating nucleic acids are contained in the bioprocess sample.

### Claim 49 (depends on 32)

The method of claim 32, wherein the method comprises at least one of the following features:
I. the reagent used in step (a) is added to the sample material does not contain a chaotropic agent, or
II. if at least one chaotropic agent containing reagent is added in step (c), the addition of the at least one chaotropic agent occurs for the first time in the absence of a branched or unbranched alkanol with 1 to 5 carbon atoms, and
wherein the sample material with the nucleic acids to be isolated and/or purified is an aqueous composition that is a purification fraction from a purification process for a biotechnologically produced product comprising biomolecules and/or organisms, wherein the biomolecules and/or organisms have at least one of the following features:
i. the biomolecules and/or organisms are selected from the group consisting of proteins, viruses and/or vaccines,
ii. the biomolecules and/or organisms are pharmaceutically active substances, and
iii. the biomolecules are biotechnologically produced molecules that are produced by genetically modified or unmodified organisms.

### Claim 50 (depends on 32)

The method of claim 32, wherein step (c) comprises at least the following sub-steps:
(i) optionally degrading proteins and/or contaminating sample constituents by adding one or more enzymes, optionally in combination with a chaotropic agent containing reagent, in the absence of a branched or unbranched alkanol, to the composition of the sample material and the reagent resulting from step (a) or from steps (a) and (b),
(ii) adding a formulation comprising at least one chaotropic compound and/or at least one non-ionic detergent,
(iii) optionally mixing and/or incubating the mixture of sub-step (ii),
(iv) for adjusting the binding conditions for binding nucleic acid(s) to a carrier material, adding a formulation comprising
at least one branched or unbranched alkanol with one to five carbon atoms,
optionally additionally a chaotropic compound, and/or
optionally a non-ionic detergent,
(v) binding the nucleic acid(s) to the carrier material, wherein the carrier material is used in particle form, as non-woven fabric, as fibres, as gel matrix, as membrane, or as column packing material,
(vi) optionally washing the bound nucleic acid(s) with a washing reagent, and
(vii) optionally eluting the bound nucleic acid(s) with an elution reagent,
wherein the one or more enzymes of sub-step (i) and the formulation of sub-step (ii) or the mixture of the one or more enzymes of sub-step (i) and the formulation of sub-step (ii) can be added in variable order to the composition of the sample material and the reagent resulting from step (a) or from steps (a) and (b).

### Claim 51 (depends on 50)

The method of claim 50, wherein the one or more enzymes of sub-step (i) are proteinase K.

### Claim 52 (depends on 50)

The method of claim 50, wherein the non-ionic detergent is selected from the group consisting of polyoxyethylene-fatty alcohol ethers that comprise a fatty alcohol moiety having 6 to 22 carbon atoms and a polyoxyethylene moiety containing 2 to 150 (CH2CH2O) units.

### Claim 53 (depends on 50)

The method of claim 50, wherein sub-step (ii) is for adjusting or predefining the binding conditions for binding nucleic acids to the carrier material.

### Claim 54 (depends on 50)

The method of claim 50, wherein the non-ionic detergents of sub-step (iv) are selected from the group consisting of polyoxyethylene-fatty alcohol ethers that comprise a fatty alcohol moiety having 6 to 22 carbon atoms and a polyoxyethylene moiety containing 2 to 150 (CH2CH2O) units.

### Claim 55 (depends on 50)

The method of claim 50, wherein the carrier material of sub-step (v) contains silica, glass fibre or polyalkylene.

### Claim 56 (depends on 55)

The method of claim 55, wherein the polyalkylene is PP, PE/PP, or PE.

### Claim 57 (depends on 50)

The method of claim 50, wherein the carrier material is used in the form of beads.

### Claim 58 (depends on 57)

The method of claim 57, wherein the carrier material is used in the form of magnetic particles or magnetic beads.

### Claim 59 (depends on 50)

The method of claim 50, wherein the washing reagent of sub-step (vi) comprises at least one branched or unbranched alcohol with one to five carbon atoms, optionally additionally a chaotropic compound, and/or optionally a non-ionic surfactant.

### Claim 60 (depends on 50)

The method of claim 50, wherein the elution reagent of sub-step (vii) comprises at least one complexing agent in a buffered milieu.

### Claim 61 (depends on 32)

The method of claim 32, further comprising:
(1) carrying out lysis of cells that are contained in the sample before adding the reagent to the sample material according to step (a),
(2) purifying the sample material to remove cell fragments and/or molecular and/or ionic constituents of the sample material in one or more successive steps before adding the reagent to the sample material according to step (a), and/or
(3) incubating the composition of the sample material and the reagent resulting from step (a) or the composition of the sample material and the reagent after mixing resulting from process steps (a) and (b).

### Claim 62 (depends on 32)

The method of claim 32, wherein the biotechnologically produced biomolecules are produced by genetically modified or unmodified organisms.

### Claim 63 (depends on 62)

The method of claim 62, wherein the genetically modified or unmodified organisms are selected from the group consisting of cell lines of mammals, cell lines of birds, cell lines of insects, cell lines of fishes, cell lines of plants, and cloned microorganisms.

### Claim 64 (depends on 63)

The method of claim 63, wherein the cell lines of mammals are selected from the group consisting of human cell lines, cell lines from horse cells, cell lines from bovine cells, cell lines from pig cells, cell lines from kangaroo cells, cell lines from sheep cells, cell lines from monkey cells, cell lines from dog cells, cell lines from cat cells, cell lines from marten cells, cell lines from rabbit cells, cell lines from hamster cells, cell lines from rat cells, and cell lines from mouse cells.

### Claim 65 (depends on 63)

The method of claim 63, wherein the cell lines of mammals are the cell lines Hek293 (human embryonic kidney), HeLa (human cervical cancer), Vero (kidney of a normal adult African green monkey), MDCK (canine Cocker Spaniel kidney), CHO (Chinese hamster ovary cells), SP2 (mouse myeloma), or NSO (mouse myeloma).

### Claim 66 (depends on 63)

The method of claim 63, wherein the genetically modified or unmodified organisms are Saccharomyces cerevisiae.

### Claim 67

A method for preparing a bioprocess sample for subsequent nucleic acid isolation and/or purification, wherein the bioprocess sample is a purification fraction obtained in a purification process for a biotechnologically produced product and wherein the target nucleic acid represents a contamination of the bioprocess sample, comprising:
(a) pretreating the bioprocess sample with a reagent, wherein the reagent comprises at least one or more compounds with an amino group selected from one or more of the following groups:
a. (i) polar-neutral proteinogenic α-aminocarboxylic acids,
(ii) nonpolar-hydrophobic proteinogenic α-aminocarboxylic acids,
(iii) basic α-aminocarboxylic acids,
(iv) non-proteinogenic aminocarboxylic acids selected from the group comprising β-alanine, D-alanine, L-homoserine and D-valine,
(v) aminocarboxylic acid polymers comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vi) peptides from 2 to 4 aminocarboxylic acids, comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vii) a compound of the general formula R3R4N-CR1R2-COOH (I), wherein R1 and R2 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, aminoalkyl and thioalkyl, and wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, di(hydroxyalkyl) and tri(hydroxyalkyl), wherein the alkyl component has a chain length from C1 to C5, is branched or unbranched, is saturated or unsaturated, and wherein at least one of R1, R2, R3 and R4 is not hydrogen, and
(viii) a compound of the general formula R3R4N-CR5R6R7 (II), wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen and hydroxyalkyl, and wherein R5, R6 and R7 are selected independently of one another from the group consisting of hydrogen, alkyl and hydroxyalkyl,
and optionally one or more of the following components:
b. at least one detergent,
c. at least one complexing agent,
d. at least one protein.

### Claim 68

(canceled)

### Claim 69 (depends on 32)

The method of claim 32, wherein step (c) comprises one or both of the following steps:
(c)(1) adding at least one lysis reagent to the bioprocess sample pretreated in step (a), wherein the lysis reagent comprises at least one proteolytic enzyme and/or a chaotropic agent, and
(c)(2) isolating the target nucleic acid by binding the nucleic acid(s) to a carrier material.

### Claim 70 (depends on 69)

The method of claim 69, wherein a binding reagent that comprises an alcohol and/or a chaotropic compound is added in step (c)(2).

### Claim 71 (depends on 32)

The method of claim 32, having one or more of the following features:
a. the biotechnologically produced product is a biopharmaceutical,
b. the bioprocess sample comprises a biopharmaceutical, wherein the biopharmaceutical is a pharmaceutically active substance comprising one or more biomolecules,
c. the biotechnologically produced product is a biopharmaceutical, wherein said biopharmaceutical is not a nucleic acid,
d. the bioprocess sample comprises the purified and/or formulated biotechnologically produced product.

### Claim 72 (depends on 71)

The method of claim 71, wherein the biopharmaceutical of c. is a peptide or protein.

### Claim 73 (depends on 71)

The method of claim 71, wherein the purified and/or formulated biotechnologically produced product of d. is a biopharmaceutical.

### Claim 74 (depends on 32)

The method of claim 32, wherein the purification fraction has one or more of the following characteristics:
a. the purification fraction is essentially cell-free,
b. the purification fraction is a chromatographic eluate, and/or
c. the purification fraction comprises a biopharmaceutical as the biotechnologically produced product, one or more buffer substances and/or salts, and the target nucleic acids to be isolated.

### Claim 75 (depends on 32)

The method of claim 32, having at least one of the following characteristics:
a. the nucleic acid(s) comprises DNA from a host cell and/or host organism used for the production of the biotechnologically produced product, and
b. the nucleic acid(s) is a nucleic acid of the host cell used for producing the biotechnologically produced product and/or is a viral nucleic acid.

### Claim 76 (depends on 75)

The method of claim 75, wherein the host cell of a. is an immortalized host cell.

### Claim 77 (depends on 75)

The method of claim 75, wherein the target nucleic acid of b. is host cell DNA.

### Claim 78 (depends on 32)

The method of claim 32, having one or more of the following characteristics:
a. the method has an LOD (limit of detection) of at least 1 pg or at least 0.1 pg.
b. the isolation and/or detection of the isolated target nucleic acid is quantitative, and/or
c. the recovery rate of the isolated target nucleic acid is at least 50%.

### Claim 79 (depends on 77)

The method of claim 77, wherein the recovery rate of the isolated target nucleic acid of c. is at least 75% or at least 85%.

### Claim 80 (depends on 32)

The method of claim 32, having one or more of the following characteristics:
a. the nucleic acid(s) is isolated from different purification fractions obtained from a purification process for a biotechnologically produced product,
b. remaining nucleic acid(s) of the cells used for the production of the biopharmaceutical and/or other nucleic acid contaminants are detected in the purified end product and/or in one or more of the bioprocess samples obtained in the course of the purification process, and/or
c. the nucleic acid(s) is isolated from various or each purification fraction of the purification process, including the finally purified end product.

### Claim 81 (depends on 32)

The method according to claim 32, having one or more the following characteristics:
a. the method is performed for monitoring the course of purification,
b. the method is performed for analysing that the amount of contaminating nucleic acid(s) contained in the purified end product does not exceed the desired and/or permissible amount, and/or
c. at least a portion of the isolated nucleic acid(s) is detected and it is determined whether the amount of contaminating target nucleic acid in the analysed bioprocess sample is above or below a permissible and/or desired maximum limit.

### Claim 82 (depends on 32)

The method of claim 32, wherein the reagent of step (a) also comprises at least one or more of the following components:
at least one detergent or mixtures of detergents,
at least one complexing agent or mixtures of complexing agents, and/or
at least one protein or mixtures of proteins.

### Claim 83 (depends on 82)

The method of claim 82, wherein the detergent is selected from the group of non-ionic surfactants consisting of alkylglucosides and/or polyoxyethylene-alkylphenyl ethers.

### Claim 84 (depends on 82)

The method of claim 82, wherein the protein is a globular protein.

### Claim 85 (depends on 84)

The method of claim 84, wherein the globular protein is albumin or globulin.

### Claim 86 (depends on 83)

The method of claim 83, wherein the alkylglucoside is a polysorbate.

### Claim 87 (depends on 86)

The method of claim 86, wherein the polysorbate is polysorbate 20 (Tween 20), polysorbate 40, or polysorbate 80.

### Claim 88 (depends on 83)

The method of claim 83, wherein the polyoxyethylene-alkylphenyl ether is octoxynol 9 (Triton X-100) or Nonidet P-40.

### Claim 89 (depends on 82)

The method of claim 82, wherein the complexing agent(s) is/are selected from the group consisting of ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(aminoethylether)-N,N-tetraacetic acid (EGTA), and ethylenediamine disuccinic acid (EDDS).

### Claim 90 (depends on 82)

The method of claim 82, wherein the protein(s) is/are selected from one or more of the following groups:
a. albumin selected from animal, plant and/or human albumin, and
b. globulin selected from animal, plant, and/or human globulin.

### Claim 91 (depends on 90)

The method of claim 90, wherein the albumin is bovine serum albumin (BSA).

### Claim 92 (depends on 90)

The method of claim 90, wherein the globulin is γ-globulin.

### Claim 93 (depends on 32)

The method of claim 32, wherein
a. the reagent used for step (a) has at least one of the following features:
i. the total concentration of the one or more compounds comprising an amino group in the reagent is 5 mM to 500 mM, 25 mM to 350 mM, or 50 mM to 250 mM,
ii. the reagent comprises one or more detergents, wherein
(a) the total concentration of the detergent(s) in the reagent is 0.1% (v/v) to 5% (v/v), 0.2% (v/v) to 4% (v/v), 0.5% (v/v) to 3% (v/v), or 1% (v/v) to 3% (v/v),
(b) the detergent is Triton X-100 at a total concentration in the reagent of 0.2% (v/v) to 0.5% (v/v),
(c) the detergent is Tween 20 at a total concentration in the reagent of 1% (v/v) to 5% (v/v), or
(d) the detergent is a non-ionic surfactant.
iii. the reagent comprises complexing agent(s), and the total concentration of the complexing agent(s) in the reagent is 10 mM to 500 mM, or 10 mM to 100 mM,
iv. the reagent comprises protein(s), wherein
(a) the total concentration of the protein(s) in the reagent is 1 mg/mL to 100 mg/mL, 2 mg/mL to 25 mg/mL, 2 mg/mL to 10 mg/mL, or 4 mg/mL to 10 mg/mL, or
(b) the protein(s) are IgG and/or BSA, and the total concentration of the protein(s) is 2 mg/mL to 25 mg/mL,
v. the pH value of the reagent is in the range from pH 3 to pH 9,
b. the components of the reagent in the reaction mixture of the sample material and the reagent have at least one of the following features:
i. the total concentration of the one or more compounds comprising an amino group in the reaction mixture is 2.5 mM to 400 mM, 12.5 mM to 280 mM, or 25 mM to 200 mM,
ii. the reagent comprises one or more detergents, wherein
(a) the total concentration of the detergent(s) in the reaction mixture is 0.05% (v/v) to 4% (v/v), 0.1% (v/v) to 3% (v/v), 0.25% (v/v) to 2.5% (v/v), or 5% (v/v) to 2.5% (v/v),
(b) the detergent is Triton X-100 at a total concentration in the reaction mixture of 0.1% (v/v) to 0.4% (v/v),
(c) the detergent is Tween 20 at a total concentration in the reaction mixture of 0.5% (v/v) to 4% (v/v), or
(d) the detergent is a non-ionic surfactant,
iii. the reagent comprises complexing agent(s), and the total concentration of the complexing agent(s) in the reaction mixture is 5 mM to 400 mM, or 5 mM to 80 mM,
iv. the reagent comprises protein(s), wherein
(a) the total concentration of the protein(s) in the reaction mixture is 0.5 mg/mL to 80 mg/mL, 1 mg/mL to 20 mg/mL, 1 mg/mL to 8 mg/mL, or 2 mg/mL to 8 mg/mL, or
(b) the protein(s) are IgG and/or BSA, and the total concentration of the protein(s) in the reaction mixture is 1 mg/mL to 20 mg/mL
c. the reagent is mixed with the sample material in a ratio of Vreagent to Vsampie material in the range of 1:4 to 4:1, 1:2 to 2:1, or 1:1, and/or
d. the reagent does not contain an aqueous formulation that contains phosphate and/or citrate in an overall concentration of ≧5 mM.

### Claim 94 (depends on 32)

The method of claim 32, wherein the reagent comprises at least one or more compounds comprising an amino group selected from one or more of the following groups i. to viii:
i. asparagine, cysteine, glutamine, glycine, threonine, and serine,
ii. alanine, leucine, isoleucine, methionine, proline, and valine,
iii. arginine, lysine, and ornithine,
iv. β-alanine, D-alanine, L-homoserine, and D-valine,
v. poly-L-lysine, poly-L-lysine hydrobromide, poly-L-lysine hydrochloride, poly(L-glutamine, L-alanine), poly(L-glutamine, L-lysine) hydrobromide, and poly-L-ornithine,
vi. glycine dimers, glycine trimers, glycine tetramers, and the dipeptide (Ala-Glu),
vii. N-(tri(hydroxymethyl)methyl)glycine (tricine) and N,N-bis(2-hydroxyethyl)glycine (bicine), and/or
viii. Tris(hydroxymethyl)-aminomethane (Tris).

### Claim 95 (depends on 32)

The method of claim 32, wherein the compound comprising an amino group is N-(tri(hydroxymethyl)methyl)glycine (tricine), N,N-bis(2-hydroxyethyl)glycine (bicine), or Tris(hydroxymethyl)-aminomethane (Tris).

### Claim 96 (depends on 32)

The method of claim 32, wherein the reagent used in step (a) comprises at least glycine, Gly-Gly, tricine and/or bicine as the one or more compounds comprising an amino group, at least one complexing agent, and at least one or at least two detergents, and optionally at least one protein.

### Claim 97 (depends on 96)

The method of claim 96, wherein the reagent used in step (a) comprises a polyoxyethylene-alkylphenyl ether and/or a polysorbate as detergent.

### Claim 98 (depends on 96)

The method of claim 96, wherein the reagent used in step (a) comprises at least one protein.

### Claim 99 (depends on 98)

The method of claim 98, wherein the protein is a globular protein.

### Claim 100 (depends on 98)

The method of claim 98, wherein the protein is IgG or BSA.

### Claim 101 (depends on 32)

The method of claim 32, wherein the reagent used in step (a) does not have any lysing properties.

### Claim 102

A reagent for isolation and/or purification of nucleic acids from a sample material, in particular for preparation of the sample material, wherein the sample material is a bioprocess sample and wherein the bioprocess sample is a purification fraction obtained in a purification process for a biotechnologically produced product and wherein the nucleic acids to be isolated represent a contamination of the bioprocess sample, which comprises the following components:
a. at least one or more compounds comprising an amino group, selected from one or more of the following groups:
i. polar-neutral proteinogenic α-aminocarboxylic acids, preferably asparagine, cysteine, glutamine, glycine, threonine and serine,
ii. nonpolar-hydrophobic proteinogenic α-aminocarboxylic acids, preferably alanine, leucine, isoleucine, methionine, proline and valine,
iii. basic α-aminocarboxylic acids, preferably arginine, lysine and ornithine,
iv. non-proteinogenic aminocarboxylic acids, selected from the group comprising β-alanine, D-alanine, L-homoserine and D-valine,
v. aminocarboxylic acid polymers comprising at least one of the aminocarboxylic acids mentioned in (i.) to (iv.), preferably consisting of the aminocarboxylic acids mentioned in (i.) to (iv.), preferably formed from identical, two or several different aminocarboxylic acid monomers, preferably in conjunction with halides, preferably selected from the group poly-L-lysine, poly-L-lysine hydrobromide, poly-L-lysine hydrochloride, poly(L-glutamine, L-alanine), poly(L-glutamine, L-lysine) hydrobromide, poly-L-ornithine,
vi. peptides from 2 to 4 aminocarboxylic acids, comprising at least one of the aminocarboxylic acids mentioned in (i.) to (iv.), preferably consisting of the aminocarboxylic acids mentioned in (i.) to (iv.), preferably selected from identical or different aminocarboxylic acids mentioned in (i.) to (iv.), preferably glycine dimers, glycine trimers and glycine tetramers,
vii. a compound of the general formula R3R4N—CR1R2—COOH (I), wherein R1 and R2 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, aminoalkyl and thioalkyl, and wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, di(hydroxyalkyl) and tri(hydroxyalkyl), wherein the alkyl component has a chain length from C1 to C5, is branched or unbranched, is saturated or unsaturated, and wherein at least one of R1, R2, R3 and R4 is not hydrogen, preferably selected from the group N-(tri(hydroxymethyl)methyl)glycine(tricine) and N,N-bis(2-hydroxyethyl)glycine (bicine),
viii. a compound of the general formula R3R4N—CR5R6R7 (II), wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen and hydroxyalkyl, and wherein R5, R6 and R7 are selected independently of one another from the group consisting of hydrogen, alkyl and hydroxyalkyl, wherein preferably at least one of R5, R6 and R7 is a hydroxyalkyl, and wherein the alkyl component has a chain length from C1 to C5, is branched or unbranched, is saturated or unsaturated, wherein the compound is preferably Tris(hydroxymethyl)-aminomethane (Tris), and
b. at least one detergent or mixtures of detergents, preferably selected from the group of non-ionic surfactants comprising alkylglucosides and/or polyoxyethylene-alkylphenyl ethers,
c. optionally at least one complexing agent or mixtures of complexing agents, and
d. at least one protein or mixtures of proteins, wherein the protein is selected from the group of albumins and/or globulins.

### Claim 103 (depends on 102)

A kit for isolating and/or purifying nucleic acids from a bioprocess sample, comprising the reagent of claim 102, and optionally at least one or more of the following components:
a. a carrier material, preferably containing silica, glass fibre, polyalkylenes, e.g. PP, PE/PP, PE, for separating the target nucleic acids, wherein the carrier material is used in particle form, as non-woven fabric, as fibres, as gel matrix, as membrane, preferably as column packing material, wherein the particles are preferably in the form of beads, preferably as magnetic particles, more preferably as magnetic beads,
b. one or more formulations that serve as lysis reagent, preferably comprising at least one chaotropic compound and/or at least one detergent from the group of non-ionic detergents, preferably selected from the group of polyoxyethylene-fatty alcohol ethers, which comprises a fatty alcohol moiety that has 6 to 22 carbon atoms, and comprises a polyoxyethylene moiety that contains 2 to 150 (CH2CH2O) units,
c. one or more enzymes, preferably proteinase K, DNase and/or RNase,
d. one or more formulations that serve as binding reagent, preferably comprising at least one chaotropic compound and/or at least one detergent from the group of non-ionic detergents, preferably selected from the group of polyoxyethylene-fatty alcohol ethers, which comprises a fatty alcohol moiety that has 6 to 22 carbon atoms, and comprises a polyoxyethylene moiety that contains 2 to 150 (CH2CH2O) units, and optionally at least one branched or unbranched alcohol with one to five carbon atoms,
e. one or more formulations that serve as washing reagent, preferably comprising at least one branched or unbranched alcohol with one to five carbon atoms, and optionally a chaotropic compound, and/or optionally a detergent from the group of non-ionic surfactants, and
f. one or more formulations that serve as elution reagent, preferably comprising at least one complexing agent in a buffered milieu.

### Claim 104

A method for detecting at least one target nucleic acid(s) from different bioprocess samples obtained from a purification process for a biotechnological product, wherein the different bioprocess samples are purification fractions obtained in the purification process, and wherein the at least one target nucleic acid(s) to be detected represents a contamination of the different bioprocess samples, comprising:
(a) pretreating the different bioprocess samples with a reagent, wherein the reagent comprises at least one or more compounds with an amino group selected from one or more of the following groups:
(i) polar-neutral proteinogenic α-aminocarboxylic acids,
(ii) nonpolar-hydrophobic proteinogenic α-aminocarboxylic acids,
(iii) basic α-aminocarboxylic acids,
(iv) non-proteinogenic aminocarboxylic acids selected from the group comprising β-alanine, D-alanine, L-homoserine and D-valine,
(v) aminocarboxylic acid polymers comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vi) peptides from 2 to 4 aminocarboxylic acids, comprising at least one of the aminocarboxylic acids of (a)(i) to (a)(iv),
(vii) a compound of the general formula R3R4N-CR1R2-COOH (I), wherein R1 and R2 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, aminoalkyl and thioalkyl, and wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen, alkyl, hydroxyalkyl, di(hydroxyalkyl) and tri(hydroxyalkyl), wherein the alkyl component has a chain length from C1 to C5, is branched or unbranched, is saturated or unsaturated, and wherein at least one of R1, R2, R3 and R4 is not hydrogen, and
(viii) a compound of the general formula R3R4N-CR5R6R7 (II), wherein R3 and R4 are selected independently of one another from the group consisting of hydrogen and hydroxyalkyl, and wherein R5, R6 and R7 are selected independently of one another from the group consisting of hydrogen, alkyl and hydroxyalkyl;
(b) optionally mixing the reagent with the different bioprocess samples;
(c) isolating and/or purifying the at least one target nucleic acid(s) from the different bioprocess samples that are contacted with and/or mixed with the reagent; and
(d) quantitatively and/or qualitatively detecting at least a portion of the isolated or purified target nucleic acid(s).