Isolation of lactoferrin from milk

• US 5,861,491 A
• Filed: 10/16/1996
• Issued: 01/19/1999
• Est. Priority Date: 02/16/1994
• Status: Expired due to Term

• Current Assignee: Pharming Intellectual Property B.V. (Pharming Group N.V.)
• Original Assignee: Pharming BV (Pharming Group N.V.)
• Inventors: Van Veen, Harry H, Nuijens, Jan H
• Primary Examiner(s): LEITH, NANCY J
• Application Number:
  US08/693,274
• Time in Patent Office: 825 Days
• Field of Search: 530/395, 530/400, 530/413, 530/416, 530/832, 530/417, 800/2, 800/DIG. 1, 536/127, 710/690, 710/691, 710/692, 710/908
• US Class Current: 530/417
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A23C 9/1465 Chromatographic separation of protein or lactose fraction; Adsorption of protein or lactose fraction followed by elution
• CPC Class Codes: A23J 1/20 from milk, e.g. casein curds or cheese A23C; from whey
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A23V 2250/54248 Lactoferrin
• CPC Class Codes: A23V 2300/30 Ion-exchange
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/79 Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: Y10S 530/832 Milk; colostrum

Abstract

The invention provides methods for purification of human lactoferrin from milk, especially milk of nonhuman species, and for separation of human lactoferrin from undesired macromolecular species present in the milk, including separation from nonhuman lactoferrin species.

Claims

1. A method for the separation of human and bovine lactoferrin which comprises subjecting a mixture including the said lactoferrins to hydrophobic interaction chromatography, and eluting human lactoferrin separately from bovine lactoferrin.

2. The method of claim 1, wherein the mixture is milk or a milk fraction from a transgenic bovine expressing human lactoferrin in its milk.

3. The method of claim 1, wherein said hydrophobic interaction chromatography is carried out using a resin containing a ligand that comprises a phenyl group or a butyl group.

4. The method of claim 3, wherein the ligand comprises a phenyl group and said ligand is linked to an agarose support.

5. The method of claim 2, wherein the human lactoferrin is eluted in 40 mM NaPO₄ buffer.

6. A method for substantially purifying human lactoferrin from a mixture containing both human lactoferrin and bovine lactoferrin, said method comprising the steps of:

contacting the mixture containing human lactoferrin and bovine lactoferrin with a hydrophobic chromatography resin under elevated ionic strength conditions, whereupon the human lactoferrin associates with said resin to form human lactoferrin-resin complexes and the bovine lactoferrin associates with said resin to form bovine lactoferrin-resin complexes; and

,

eluting the human lactoferrin from the human lactoferrin-resin complexes under conditions in which the bovine lactoferrin is not eluted from the bovine lactoferrin-resin complexes, whereupon human lactoferrin substantially free from bovine lactoferrin is obtained.

7. The method of claim 6, wherein the mixture is milk or a milk fraction from a transgenic bovine expressing human lactoferrin in its milk.

8. The method of claim 6, wherein the hydrophobic chromatography resin contains a ligand that comprises a phenyl group or a butyl group.

9. The method of claim 8, wherein the ligand comprises a phenyl group and said ligand is linked to an agarose support.

10. The method of claim 8, wherein the human lactoferrin is eluted in 40 mM NaPO₄ buffer.

11. The method of claim 10, wherein the mixture comprises human lactoferrin and bovine lactoferrin and is adjusted to an ionic strength greater than that of 1M NH₄ SO₄.