# US 5,525,519 A · Method for isolating biomolecules from a biological sample with linear polymers

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- Number: US 5,525,519 A
- Document ID: US5525519A
- Status: Granted
- Kind: A
- Application number: 07/817,610
- Filed: 1992-01-07
- Granted: 1996-06-11
- Earliest claimed: 1992-01-07
- Assignees: Middlesex Sciences, Inc.
- Inventors: James E. Woiszwillo
- Web page: https://patentgrep.com/patent/US5525519A

## CPC

- C07K 16/065: {Purification, fragmentation}
- C07K 1/30: by precipitation
- Y10T 436/107497: Preparation composition [e.g., lysing or precipitation, etc.]
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

A method and composition for isolating biomolecules from a biological sample wherein the sample is mixed with a soluble, linear polymer, such as polyvinylpyrrolidone, to form a precipitate. The biomolecule of interest is found in the precipitate or is isolated from the supernatant.

## Claims

### Claim 1

A method of isolating at least one biomolecule from a biological sample comprising the steps of:
a) adding a sufficient amount of a solution of at least two soluble, linear polymers to a biological sample solution to form a precipitate, wherein a first polymer is polyvinylpyrrolidone and a second polymer is polyethylene glycol, and adjusting the solution to a predetermined pH; and
b) separating the precipitate from the supernatant; thereby isolating a biomolecule from the biological sample in either the supernatant or precipitate.

### Claim 2 (depends on 1)

The method of claim 1 wherein the pH of the solution is adjusted to a predetermined pH by adjusting the pH of the polymers with an amino acid before addition to the solution.

### Claim 3 (depends on 1)

The method of claim 1 wherein the pH of the solution is adjusted with an amino acid before or after addition of the polymers.

### Claim 4 (depends on 1)

The method of claim 1 wherein the polymers are added as an insoluble network of non-covalently linked polymer molecules.

### Claim 5 (depends on 1)

The method of claim 1 wherein the ratio of polymers to biological sample solution is approximately 8:1.

### Claim 6 (depends on 1)

The method of claim 1 wherein the polyvinylpyrrolidone has a molecular weight of approximately 40,000.

### Claim 7 (depends on 1)

The method of claim 1 wherein the first polymer is a 20% aqueous solution of polyvinylpyrrolidone.

### Claim 8 (depends on 1)

The method of claim 1 wherein the polyvinylpyrrolidone is first mixed with the polyethylene glycol and the polymer mixture is added to the sample.

### Claim 9 (depends on 8)

The method of claim 8 wherein the ratio of polyvinylpyrrolidone to polyethylene glycol in the polymer mixture is 1:1.

### Claim 10 (depends on 1)

The method of claim 1 wherein the polyvinylpyrrolidone has a molecular weight of approximately 40,000 and the polyethylene glycol has a molecular weight of approximately 3500.

### Claim 11 (depends on 1)

The method of claim 1 wherein the adjusted pH of the solution is between 4.0 and 9.2.

### Claim 12 (depends on 1)

The method of claim 1 wherein the biomolecule is selected from the group consisting of proteins, carbohydrates, non-protein hormones, and nucleic acids.

### Claim 13 (depends on 12)

The method of claim 12 wherein the protein is an immunoglobulin.

### Claim 14 (depends on 1)

The method of claim 1 wherein the biological sample solution is selected from the group consisting of blood serum, blood plasma, urine, and spinal fluid, fermentation fluid, lymph fluid, tissue culture fluid, and ascites fluid.

### Claim 15 (depends on 1)

The method of claim 1 wherein the polyethylene glycol has a molecular weight of approximately 3500.

### Claim 16

An assay for determining the albumin to globulin ratio in plasma comprising the steps of:
a) adding a sufficient amount of a polymer solution of polyvinylpyrrolidone and polyethylene glycol to the plasma to precipitate the globulin, leaving the albumin in the supernatant, wherein the pH of the plasma is adjusted to an approximately neutral pH;
b) separating the precipitate from the supernatant;
c) measuring the concentrations of globulin and albumin; and
d) calculating the ratio of albumin to globulin.

### Claim 17 (depends on 16)

The assay of claim 16 wherein the pH of the plasma is adjusted by adjusting the pH of the polymer solution to a predetermined value before addition to the plasma.

### Claim 18

A method for determining the disposition of a drug in serum proteins comprising the steps of:
a) adding a sufficient amount of an aqueous solution of polyvinylpyrrolidone and polyethylene glycol to serum to precipitate the globulin, wherein the pH of the serum is adjusted to a predetermined value;
b) separating the precipitate from the supernatant, thereby isolating globulin-bound drug and albumin-bound drug in either the precipitate or supernatant; and
c) analyzing the precipitate and supernatant to detect the drug.

### Claim 19

A method of isolating at least one protein from a biological sample solution comprising the steps of:
a) combining polyvinylpyrrolidone with polyethylene glycol to form a polymer mixture;
b) adjusting the pH of the polyvinylpyrrolidone and polyethylene glycol mixture to a predetermined value with an acid or base;
c) adding a sufficient amount of the pH-adjusted polyvinylpyrrolidone and polyethylene glycol mixture to the sample solution to form a precipitate; and
d) separating the precipitate from the supernatant, thereby isolating a protein from the biological sample in either the supernatant or precipitate.

### Claim 20 (depends on 19)

The method of claim 19 wherein the polyvinylpyrrolidone has a molecular weight of approximately 40,000.

### Claim 21 (depends on 19)

The method of claim 19 wherein the biological sample solution is selected from the group consisting of blood serum, blood plasma, urine, and spinal fluid, fermentation fluid, lymph fluid, tissue culture fluid, and ascites fluid.

### Claim 22 (depends on 19)

The method of claim 19 wherein the molecular weight of polyvinylpyrrolidone is approximately 40,000 and the molecular weight of polyethylene glycol is approximately 3500.

### Claim 23 (depends on 19)

The method of claim 19 wherein the ratio of polyvinylpyrrolidone to polyethylene glycol is 1:1.

### Claim 24 (depends on 19)

The method of claim 19 wherein the mixture is an insoluble network of non-covalently linked polymer molecules.

### Claim 25 (depends on 19)

The method of claim 19 wherein the concentration of polyvinylpyrrolidone and polyethylene glycol is approximately 20%.