Clarification of transgenic milk using depth filtration
View Patent ↗Processes and apparati are provided for separating molecules of interest from a mixture by depth filtration (DF). The DF of the invention is useful in the clarification and processing of various feedstreams for the removal of a molecule of interest. According to a preferred embodiment, a transgenic milk feedstream is stabilized and particulate matter such as fat, casein miscelles and bacteria are removed. An aseptic filtration step was also developed to remove any bacteria remaining in a clarified transgenic milk feedstream.
1. A method for separating a protein of interest from a feedstream, comprising:
filtering said feedstream by a depth filtration process that separates a protein of interest from said feedstream on the basis of particulate size,
wherein said feedstream containing the protein of interest is composed of milk from a transgenic mammal,
wherein milk casein micelles are aggregated by dilution with an ammonium sulfate solution, and
wherein the feedstream and the ammonium sulfate solution are blended prior to filtration by an in line static mixer in fluid communication with a filtration element of said depth filtration process.
2. The method of claim 1 , wherein the depth filtration process comprises a filter matrix capable of trapping particles in the range of about 1-5 microns.
3. The method of claim 1 , wherein the depth filtration process comprises a filter matrix capable of trapping particles ranging from 5 microns to 50 microns.
4. The method of claim 1 wherein the depth filtration process comprises a filter matrix capable of trapping particles ranging from 10 microns to 25 microns.
5. The method of claim 1 , further comprising dissociating the milk casein micelles from the rest of the feedstream through the use of EDTA.
6. The method of claim 1 , further comprising wherein at least 3 liters/ft2 of feedstream containing a protein of interest can be processed in 2 hours at 60 LMH.
7. The method of claim 1 , further comprising wherein 3 liters/ft2 of feedstream containing a protein of interest can be processed in 2-4 hours at 30-60 LMH.
8. The method of claim 1 , wherein the depth filtration process comprises a filter medium composed of one or more of the following materials: metallic, polymeric, inorganic, or organic materials.
9. The method of claim 1 , wherein said protein of interest has a molecular weight of between 1 and 1000 kDa.
10. The method of claim 1 , wherein said feedstream is diluted before depth filtration.
11. The method of claim 1 , wherein said protein of interest is bioactive.
12. The method of claim 1 , wherein the condition of said milk is selected from the group consisting of:
a) raw;
b) diluted;
c) treated with a buffer solution;
d) chemically treated; and
e) concentrated.
13. The method of claim 1 , wherein the depth filtration process is carried out at a permeation flux of less than 30 lmh.
14. The method of claim 1 , wherein the depth filtration process is carried out at a pH above that at which said protein of interest does not precipitate.
15. The method of claim 1 , further comprising a fractionation step utilizing a cellulose filtration membrane.
16. The method of claim 1 wherein said protein of interest is a biological entity selected from the group consisting of proteins, immunoglobulins, polypeptides, peptides and glycoproteins.
17. The method of claim 1 , wherein the depth filtration is performed at a temperature of 4° C. to 40° C.
18. The method of claim 17 , wherein the depth filtration is performed at a temperature of 15° C. to 30° C.
19. The method of claim 18 , wherein the depth filtration is performed at a temperature of 20° C. to 25° C.
20. The method of claim 1 , further comprising after the filtering step an anion chromatography step.
21. The method of claim 1 , further comprising after the filtering step a cation chromatography step.
22. The method of claim 1 , further comprising after the filtering step a size exclusion chromatography step.
23. The method of claim 1 , further comprising after the filtering step a reverse phase chromatography step.
24. The method of claim 1 , wherein the feedstream has a concentration ranging from 0.25×to 4×natural milk.
25. The method of claim 1 , wherein the feedstream has a concentration ranging from 0.5×to 3×natural milk.
26. The method of claim 1 , wherein the depth filtration process is carried out at the protein of interest's isoelectric pH.
27. The method of claim 1 , wherein the protein of interest is selected from the group consisting of alpha-1-antitrypsin, alkaline phosphatase, angiogenin, antithrombin, chitinase, extracellular superoxide dismutase, Factor VIII, Factor TX, Factor X, fibrinogen, glucocerebrosidase, glutamate decarboxylase, human serum albumin, insulin, myelin basic protein, lactoferrin, lactoglobulin, lysozyme, lactalbumin, proinsulin, soluble CD4, component and complex of soluble CD4, and tissue plaminogen activator.
28. The method of claim 1 , wherein said feedstream is treated with a solution selected from the group consisting of:
a) water;
b) a buffered aqueous salt solution;
c) chelating agent;
d) acid solution; and
e) alkali solution.