# US 7,744,890 B2 · Methods and compositions with reduced opalescence

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- Number: US 7,744,890 B2
- Document ID: US7744890B2
- Status: Granted
- Kind: B2
- Application number: 11/974,312
- Filed: 2007-10-12
- Published: 2008-06-26
- Granted: 2010-06-29
- Earliest claimed: 2006-10-12
- Assignees: Wyeth
- Inventors: Pilarin Elizabeth Nichols; Donna L. Luisi
- Web page: https://patentgrep.com/patent/US7744890B2

## CPC

- A61K 39/39591: {Stabilisation, fragmentation}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Methods for reducing the opalescent appearance of a protein preparation by modifying the ionic strength of the preparation, as well as compositions, e.g., pharmaceutical compositions, of concentrated protein with decreased opalescence are disclosed. Purification methods which monitor and/or reduce the salt concentrations at selected steps are also disclosed.

## Claims

### Claim 1

A method for decreasing the opalescence of an antibody preparation, the method comprising:
(a) determining a second virial coefficient of an antibody in the antibody preparation; and
(b) if the second virial coefficient of the antibody is negative, reducing the ionic strength of the antibody preparation such that the opalescence of the antibody preparation is reduced.

### Claim 2 (depends on 1)

The method of claim 1, wherein the antibody comprises a human, humanized, chimeric, CDR-grafted, or an in vitro generated antibody, or an antigen-binding fragment thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein the antibody comprises an antibody having IgG1 or an IgG4 heavy chain constant region.

### Claim 4 (depends on 1)

The method of claim 1, wherein the second virial coefficient of the antibody is determined at a salt concentration ranging from about 50 to 200 mM.

### Claim 5 (depends on 4)

The method of claim 4, wherein the second virial coefficient of the antibody is determined to be about −1 to −10×10−3, or about −1 to −10×10−4 mol-mg/g2 at 100 mM sodium chloride.

### Claim 6 (depends on 1)

The method of claim 1, wherein the antibody is present at a concentration of about 5 to 300 mg/ml.

### Claim 7 (depends on 1)

The method of claim 1, wherein the ionic strength of the antibody preparation is reduced by decreasing the concentration of a salt present in the preparation.

### Claim 8 (depends on 7)

The method of claim 7, wherein the salt present in the preparation is selected from the group consisting of sodium chloride, calcium chloride, magnesium chloride and sodium phosphate.

### Claim 9 (depends on 7)

The method of claim 7, wherein the concentration of the salt is reduced to at least about two-, three-, five-, ten- or one hundred-fold lower than the initial concentration in the antibody preparation.

### Claim 10 (depends on 7)

The method of claim 7, wherein the ionic strength of the antibody preparation is reduced by replacing a first salt with a second salt, wherein the first salt is NaCl, and the second salt is selected from the group consisting of Na2HPO4, MgCl2, and CaCl2.

### Claim 11 (depends on 7)

The method of claim 7, wherein the ionic strength of the antibody preparation is reduced by replacing a first salt with a second salt, wherein the first salt is Na2HPO4, and the second salt is selected from the group consisting of MgCl2 and CaCl2.

### Claim 12 (depends on 1)

The method of claim 1, wherein the ionic strength of the antibody preparation is reduced by ultra-filtration or dialysis.

### Claim 13 (depends on 1)

The method of claim 1, wherein the ionic strength of the antibody preparation is reduced in a process of purifying the antibody, wherein the process of purifying the antibody comprises one or more of centrifugation, filtration, chromatography, lyophilization or reconstitution.

### Claim 14 (depends on 1)

The method of claim 1, further comprising evaluating the opalescence of the antibody preparation.

### Claim 15 (depends on 14)

The method of claim 14, wherein the opalescence of the antibody preparation is evaluated by determining the turbidity of the solution, a change in the second virial coefficient of the antibody, or a change in the formation of high molecular weight species.

### Claim 16 (depends on 15)

The method of claim 15, wherein the opalescence is reduced to about 2 or 1 European Pharmacopoeia standard or less.

### Claim 17 (depends on 15)

The method of claim 15, wherein the second virial coefficient changes from about −1 to −10×10−3 to about −1 to −10×10−4.

### Claim 18 (depends on 15)

The method of claim 15, wherein the high molecular weight species is reduced by an amount of about two-, three-, four-, five-, six-, seven-, eight-, nine-, ten-, fifty- or one hundred-fold.

### Claim 19 (depends on 1)

The method of claim 1, wherein the second virial coefficient of the antibody is determined by a static light scattering and Zimm analysis.

### Claim 20

A method for reducing the formation of high molecular weight species in an antibody preparation, the method comprising:
(a) providing an antibody preparation comprising an antibody determined to have a negative second virial coefficient;
(b) reducing the ionic strength of the antibody preparation such that the high molecular weight species in the antibody preparation are reduced.

### Claim 21 (depends on 20)

The method of claim 20, wherein the antibody comprises a human, humanized, chimeric, CDR-grafted, or an in vitro generated antibody, or an antigen-binding fragment thereof.

### Claim 22 (depends on 20)

The method of claim 20, wherein the antibody comprises an IgG1 or an IgG4 heavy chain constant region.

### Claim 23 (depends on 20)

The method of claim 20, wherein the antibody of the antibody preparation has a negative second virial coefficient in a salt concentration ranging from about 50 to 200 mM.

### Claim 24 (depends on 23)

The method of claim 23, wherein the second virial coefficient is about −1 to −10×10−3, or about −1 to −10×10−4 mol-mg/g2 in an antibody preparation containing 100 mM sodium chloride.

### Claim 25 (depends on 20)

The method of claim 20, wherein the antibody is present at a concentration of about 5 to 300 mg/ml.

### Claim 26 (depends on 20)

The method of claim 20, wherein the ionic strength of the antibody preparation is reduced by decreasing the concentration of a salt present in the preparation.

### Claim 27 (depends on 26)

The method of claim 26, wherein the salt present in the preparation is selected from the group consisting of sodium chloride, calcium chloride, magnesium chloride and sodium phosphate.

### Claim 28 (depends on 26)

The method of claim 26, wherein the concentration of the salt is reduced to at least about two-, three-, five-, ten- or one hundred-fold lower than the initial concentration in the antibody preparation.

### Claim 29 (depends on 26)

The method of claim 26, wherein the ionic strength of the antibody preparation is reduced by replacing a first salt with a second salt, wherein the first salt is NaCl, and the second salt is selected from the group consisting of Na2HPO4, MgCl2, and CaCl2.

### Claim 30 (depends on 26)

The method of claim 26, wherein the ionic strength of the antibody preparation is reduced by replacing a first salt with a second salt, wherein the first salt is Na2HPO4, and the second salt is selected from the group consisting of MgCl2 and CaCl2.

### Claim 31 (depends on 20)

The method of claim 20, wherein the ionic strength of the antibody preparation is reduced by ultra-filtration or dialysis.

### Claim 32 (depends on 20)

The method of claim 20, wherein the ionic strength of the antibody preparation is reduced in a process of purifying the antibody, wherein the process of purifying the antibody comprises one or more of centrifugation, filtration, chromatography, lyophilization or reconstitution.

### Claim 33 (depends on 20)

The method of claim 20, further comprising evaluating the opalescence of the antibody preparation.

### Claim 34 (depends on 33)

The method of claim 33, wherein the opalescence of the antibody preparation is evaluated by determining the turbidity of the solution, a change in the second virial coefficient of the antibody, or a change in the formation of high molecular weight species.

### Claim 35 (depends on 34)

The method of claim 34, wherein the opalescence is reduced to about 2 or 1 European Pharmacopoeia standard or less.

### Claim 36 (depends on 34)

The method of claim 34, wherein the second virial coefficient changes from about −1 to −10×10−3 to about −1 to −10×10−4.

### Claim 37 (depends on 20)

The method of claim 20, wherein the high molecular weight species is reduced by an amount of about two-, three-, four-, five-, six-, seven-, eight-, nine-, ten-, fifty- or one hundred-fold.

### Claim 38

A method of decreasing the formation of high molecular weight species in an antibody preparation, the method comprising:
(a) providing an antibody preparation comprising an antibody determined to have a negative second virial coefficient in 100 mM sodium chloride solution;
(b) reducing the ionic strength in the antibody preparation such that the formation of high molecular weight species in the antibody preparation is reduced.

### Claim 39

A method of improving the efficiency of a purification process of an antibody in an antibody preparation, the method comprising:
evaluating whether the antibody in the antibody preparation has a negative second virial coefficient at one or more salt concentrations;
reducing the ionic strength in the antibody preparation, such that the high molecular weight species in the antibody preparation is reduced, thereby improving the efficiency of a purification process of an antibody preparation.

### Claim 40 (depends on 39)

The method of claim 39, wherein the ionic strength of the antibody preparation is reduced by one or more steps selected from filtration, replacing a salt used in the antibody preparation with a lower opalescent inducer, or reducing the concentration of a salt present in the purification process.