# US 7,745,582 B2 · Isolation of immunoglobulins

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- Number: US 7,745,582 B2
- Document ID: US7745582B2
- Status: Granted
- Kind: B2
- Application number: 11/930,556
- Filed: 2007-10-31
- Published: 2008-06-05
- Granted: 2010-06-29
- Earliest claimed: 1996-08-30
- Assignees: UpFront Chromatography
- Inventors: Allan Otto Fog Lihme; Marie Bendix Hansen
- Web page: https://patentgrep.com/patent/US7745582B2

## CPC

- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- B01D 15/1807: {using counter-currents, e.g. fluidised beds}
- B01D 15/3804: {Affinity chromatography}
- B01J 20/3219: {involving a particular spacer or linking group, e.g. for attaching an active group}
- B01J 20/3242: {Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group}
- B01J 20/3253: {comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures}
- B01J 20/3255: {comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures}
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- 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)
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS

## Abstract

The present invention relates to a novel method for the isolation or purification of immunoglobulins (a special class of proteins) from a solution containing immunoglobulins, e.g. hybridoma cell culture supernatants, animal plasma or sera, or colostrum. The method includes the use of a minimum of salts, such as lyotropic salts, in the binding process and preferably also the use of small amounts of organic solvents in the elution process. The solid phase matrices, preferably epichlorohydrin activated agarose matrices, are functionalised with mono- or bicyclic aromatic or heteroaromatic ligands (molecular weight: at the most 500 Dalton) which, preferably, comprises an acidic substituent, e.g. a carboxylic acid. The matrices utilised show excellent properties in a “Standard Immunoglobulin Binding Test” and in a “Monoclonal Antibody Array Binding Test” with respect to binding efficiency and purity, and are stable in 1M NaOH.

## Claims

### Claim 1

A method for isolation of immunoglobulins from a solution comprising one or more immunoglobulins, said method comprising the steps of:
(i) contacting a solution containing one or more immunoglubulins which has a pH in the range of 2.0 to 10.0 and a total salt content corresponding to an ionic strength of at most 2.0 with a solid phase matrix, comprising a functionalised matrix backbone carrying acidic ligand groups wherein the acidic ligand groups comprise a mono- or bicyclic optionally substituted aromatic or heteroaromatic moiety and an acidic group having a pKa value of less than 6, and wherein criteria (a) and (b) are fulfilled:
(a) the solid phase matrix has a binding efficiency of at least 50% when tested at a pH in the range of 2.0 to 10.0 in the “Standard Immunoglobulin Binding Test” described herein; and
(b) the stability of the solid phase matrix in 1 M NaOH is such that incubation of the matrix in 1 M NaGH in the dark at room temperature for 7 days reduces the binding efficiency at a pH in the range of one pH unit lower than the binding maximum pH value to one pH unit higher than the binding maximum pH value, as determined in the “Standard Immunoglobulin Binding Test” described herein, with less than 25% compared to a corresponding non-incubated matrix;
whereby at least a part of the immunoglobulins becomes bound to the solid phase matrix;
(ii) separating the solid phase matrix having immunoglobulins bound thereto from the solution;
(iii) optionally washing the solid phase matrix; and
(iv) contacting the solid phase matrix with an eluent in order to liberate one or more of the immunoglobulins from the separated solid phase matrix.

### Claim 2 (depends on 1)

A method according to claim 1, wherein the acidic ligand group has a molecular weight below 200 Daltons.

### Claim 3 (depends on 1)

A method according to claim 1, wherein the acidic ligand groups are formed of a ligand-spacer conjugate.

### Claim 4 (depends on 3)

A method according to claim 3, wherein the ligand-spacer conjugate has a molecular weight below 500 Daltons.

### Claim 5 (depends on 3)

A method according to claim 3, wherein the acidic group is present on the spacer.

### Claim 6 (depends on 1)

A method according to claim 1, wherein the solid phase matrix has a binding efficiency of at least 50% when tested at a pH of 4.5 in the “Standard Immunoglobulin Binding Test”.

### Claim 7 (depends on 1)

A method according to claim 1, wherein the aromatic moiety is selected from benzene radicals and naphthalene radicals.

### Claim 8 (depends on 1)

A method according to claim 1, wherein the heteroaromatic moiety is a monocyclic heteroaromatic radical selected from the group consisting of thiophene, furan, pyran, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine and pyridazine radicals, or a bicyclic heteroaromatic radical selected from the group consisting of indole, purine, quinoline, benzofuran, benzimidazole and benzoxazole radicals.

### Claim 9 (depends on 1)

A method according to claim 1, wherein the acidic group is selected from the group consisting of a carboxylic acid group (COOH), sulfonic acid group (—SO2OH), sulfinic acid group (S(O)OH), phosphinic acid group (—PH(O)(OH)), phosphonic acid monoester group (—P(O)(OH)(OR)) and phosphonic acid group (—P(O)(OH)2).

### Claim 10 (depends on 1)

A method according to claim 1, wherein the aromatic moiety is a benzene ring.

### Claim 11 (depends on 1)

A method according to claim 1, wherein the total salt content of the solution containing the immunoglobulins corresponds to an ionic strength in the range of 0.05 to 2.0.

### Claim 12 (depends on 1)

A method according to claim 1, wherein the solution containing one or more immunoglobulins has an ionic strength in the range 0.05 to 1.4.

### Claim 13 (depends on 1)

A method according to claim 1, wherein the solution containing one or more immunoglobulins has an ionic strength in the range 0.05 to 1.0.

### Claim 14 (depends on 1)

A method according to claim 1, wherein the solution containing one or more immunoglobulins comprises lyotropic salts in a concentration of at the most 0.4 M.

### Claim 15 (depends on 1)

A method according to claim 1, wherein the eluent comprises less than 10% (v/v) of organic solvents.

### Claim 16 (depends on 1)

A method according to claim 1, wherein the washing of the solid phase comprises washing with an aqueous solution comprising a negatively charged detergent.

### Claim 17 (depends on 1)

A method according to claim 1, wherein the washing of the solid phase comprises washing with an inorganic or organic salt buffer comprising a negatively charged detergent.

### Claim 18 (depends on 1)

A method according to claim 1, wherein the solution containing one or more immunoglobulins comprises a negatively-charged detergent.

### Claim 19 (depends on 1)

A method according to claim 1, wherein the solution containing one or more immunoglobulins has a pH in the range of pH 3-7.