Isolation of lactoferrin from milk

• US 5,919,913 A
• Filed: 03/09/1995
• Issued: 07/06/1999
• 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: Nuyens, Jan H., Van Veen, Harry H.
• Primary Examiner(s): Degen, Nancy
• Application Number:
  US08/406,271
• Time in Patent Office: 1,580 Days
• Field of Search: 530/395, 530/400, 530/413, 530/416, 530/832, 800/2, 800/DIG. 1, 536/127, 210/690, 210/691, 210/692, 210/908
• US Class Current: 530/395
• 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 substantially purifying human lactoferrin from milk, comprising the steps of:

contacting milk or a milk fraction containing human lactoferrin with a strong cation exchange resin under elevated ionic strength conditions which are at least 10 mM higher in monovalent cation concentration than milk for a binding period forming human lactoferrin-resin complexes;

removing the milk or milk fraction which remains unbound and recovering the human lactoferrin-resin complexes; and

eluting the human lactoferrin from the recovered human lactoferrin-resin complexes with a salt solution having an ionic strength sufficient to elute the human lactoferrin from the human lactoferrin-resin complexes.

2. A method according to claim 1, wherein said step of contacting said milk or milk fraction with said strong cation exchange resin is conducted with a limiting amount of said strong cation exchange resin which has a binding capacity approximately equal to or less than the expected amount of human lactoferrin in the milk or milk fraction.

3. A method according to claim 1, wherein the milk or milk fraction is adjusted to approximately 0.35-0.4M salt by the addition of NaCl or KCl prior to contacting the milk or milk fraction with said strong cation exchange resin.

4. A method according to claim 1, wherein transgenic bovine milk whey is the milk fraction used for contacting with said strong cation exchange resin.

5. A method according to claim 1, comprising the further intermediate step of washing the recovered human lactoferrin-resin complexes with a wash buffer prior to eluting he human lactoferrin.

6. A method of claim 5, wherein the recovered human lactoferrin-resin complexes are poured into a column.

7. A method according to claim 1, wherein the strong cation exchange resin is selected from the group consisting of Mono S™

and S Sepharose™

.

8. A method according to claim 7, wherein human lactoferrin is eluted and separated from bovine lactoferrin by a salt gradient.

9. A method according to claim 1, wherein the salt solution for eluting the human lactoferrin is at least 0.4M NaCl or at least 0.4M KCl.

10. A method according to claim 1, wherein the human lactoferrin is eluted in a stepwise mode comprising an elution step having a salt solution which substantially elutes human lactoferrin without substantially eluting bovine lactoferrin.

11. A method according to claim 1, comprising the further step of recovering the eluted human lactoferrin, loading the recovered human lactoferrin on a strong cation exchange resin or concanavalin A resin, and eluting the human lactoferrin from the resin in substantially purified form.

12. A method according to claim 1, wherein the step of contacting the milk or milk fraction with the strong cation exchange resin is performed across a dialysis membrane which is substantially permeable to human lactoferrin.

13. A method according to claim 1, wherein the elevated ionic strength conditions which are at least 10 mM higher in monovalent cation concentration than milk are produced by the addition of exogenous NaCl or KCl and/or buffer to milk or a milk fraction to which a detergent has or has not been added.

14. A method according to claim 1, wherein human lactoferrin is purified from milk or a milk fraction containing bovine lactoferrin, whereby the human lactoferrin is isolated from bovine lactoferrin by differential elution with a salt solution.

15. The method of claim 1 further comprising the step of mixing the human lactoferrin with a carrier to form a pharmaceutical composition.

16. A method for purifying native human lactoferrin from hLF degradation products in a sample, comprising the step of contacting the sample with a strong cation exchange resin under binding conditions and eluting the native human lactoferrin as a resolvable peak by applying a salt gradient or in a stepwise mode and recovering the peak consisting essentially of native human lactoferrin having an intact amino terminal sequence.

17. A method according to claim 16, wherein the degradation products comprise human lactoferrin species which lack amino-terminal amino acids compared to a full-length human lactoferrin protein.

18. A method according to claim 16, wherein the degradation products elute at a lower salt concentration than that at which native human lactoferrin elutes.

19. A method for separating human lactoferrin from bovine lactoferrin in a sample, comprising contacting the sample with a strong cation exchange resin and eluting the human lactoferrin from the resin with a salt solution which preferentially elutes human lactoferrin but does not substantially elute bovine lactoferrin concomitantly with the human lactoferrin.

20. A method according to claim 19, wherein hLF and bLF are separated by stepwise increases of salt concentration.

21. A method according to claim 19, wherein hLF and bLF are separated by elution with a salt gradient.

22. A method according to claim 19, wherein the strong cation exchange resin is Mono S™

or Fast Sepharose S™

, the binding conditions comprise elevated ionic strength which is at least 10 mM higher in monovalent cation concentration than milk, and elution of human lactoferrin is accomplished with a salt solution which preferentially elutes human lactoferrin but does not substantially elute nonhuman lactoferrin concomitantly with the human lactoferrin.

23. A method for isolating substantially pure recombinant human lactoferrin from the milk of a transgenic animal harboring a transgene which encodes the expression and secretion of recombinant human lactoferrin in its milk, comprising the steps of:

obtaining milk or a milk fraction of said transgenic animal containing recombinant human lactoferrin;

contacting said milk or milk fraction with a strong cation exchange resin under conditions for binding of the human lactoferrin to the resin;

eluting the recombinant human lactoferrin from the strong cation exchange resin with a stepwise salt gradient or linear salt gradient.

24. A method according to claim 23, wherein the strong cation exchange resin is Mono S™

or Fast Sepharose S™

, the binding conditions comprise elevated ionic strength which is at least 10 mM higher in monovalent cation concentration than milk, and elution of human lactoferrin is accomplished by eluting with a salt solution of at least 0.5M NaCl.

25. A method according to claim 23, wherein the binding conditions are the milk or milk fraction adjusted to approximately 0.35-0.4M NaCl and elution is accomplished with a salt solution consisting essentially of at least 0.4M NaCl or KCl.

26. A method for removing LPS from a solution, comprising the step of contacting a solution containing LPS with a resin comprising immobilized hLF and recovering the portion of the solution that is bound to the resin comprising immobilized hLF.

27. A method of claim 26, wherein the resin comprising immobilized hLF is hLF covalently linked to Sepharose.