CLARIFICATION OF TRANSGENIC MILK USING DEPTH FILTRATION

• US 20100056757A1
• Filed: 04/03/2009
• Published: 03/04/2010
• Est. Priority Date: 02/16/2006
• Status: Abandoned Application

• Current Assignee: GTC Biotherapeutics, Inc.
• Original Assignee: GTC Biotherapeutics, Inc.
• Inventors: Perreault, Mark A.
• Application Number:
  US12/417,832
• Publication Number:
  US 20100056757A1
• Time in Patent Office: Days
• US Class Current: 530/387.100
• CPC Class Codes: C07K 1/34 by filtration, ultrafiltration or reverse osmosis
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA

Abstract

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.

Claims

1. A method for separating a protein of interest from a feedstream, comprising:

(a) filtering said feedstream by a depth filtration process that separates said protein of interest from said feedstream on the basis of size;

(b) performing a viral inactivation step that inactivates at least 90% of viral particles or fragments;

(c) performing an aseptic filtration step for the filtered feedstream; and

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(d) clarifying the remaining material through additional downstream processing

wherein material that is retained by a filter during the depth filtration process is trapped both upon a surface of the filter and within a matrix of the filter.

5. The method of claim 1, wherein the feedstream containing the protein of interest is composed of the milk from a transgenic mammal.

6. The method of claim 5, wherein casein proteins are disassociated from the rest of the feedstream through the use of EDTA.

7. The method of claim 6, further comprising wherein casein micelles are aggregated through the use of an ammonium sulfate solution.

12. The method of claim 1, wherein said protein of interest has a molecular weight of between 1 and 1000 kDa.

21. The method of claim 1, wherein said protein of interest is selected from the group consisting of proteins, immunoglobulins, polypeptides, antibodies, peptides and glycoproteins.

28. The method of claim 1, wherein said additional downstream processing comprises cation chromatography.

29. The method of claim 1, wherein said additional downstream processing comprises size exclusion chromatography.

30. The method of claim 1, wherein said additional downstream processing comprises reverse phase chromatography.

33. The method of claim 1, wherein the depth filtration process is carried out at the protein of interest'"'"'s isoelectric pH.

34. 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 IX, Factor X, fibrinogen, glucocerebrosidase, glutamate decarboxylase, human serum albumin, recombinant human albumin, insulin, myelin basic protein, lactoferrin, lactoglobulin, lysozyme, lactalbumin, proinsulin, soluble CD4, component and complex of soluble CD4, and tissue plasminogen activator.

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39. A method for separating a protein of interest from a transgenic milk feedstream, comprising:

(a) filtering said milk feedstream by a depth filtration process that separates said protein of interest from said feedstream on the basis of size;

(b) performing a viral inactivation step that inactivates at least 90% of viral particles or fragments;

(c) performing an aseptic filtration step for the filtered feedstream; and

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(d) clarifying the remaining material through downstream processing;

wherein material that is retained by a filter during the depth filtration process is trapped both upon a surface of the filter and within a matrix of the filter.

43. The method of claim 39, wherein said milk feedstream is diluted with an ammonium sulfate solution.

44. The method of claim 39, wherein casein proteins are disassociated from the rest of the feedstream through the use of EDTA.

45. The method of claim 44, further comprising wherein casein micelles are aggregated through the use of an ammonium sulfate solution.

50. The method of claim 39, wherein said protein of interest has a molecular weight of between 1 and 1000kDa.

59. The method of claim 39, wherein said protein of interest is a selected from the group consisting of proteins, immunoglobulins, polypeptides, antibodies, peptides and glycoproteins.

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74. A method for isolating a protein of interest from a feedstream comprising:

collecting a feedstream sample;

diluting the feedstream sample with ammonium sulfate resulting in the precipitation of contaminants, wherein the pH of the collected feedstream is above pH 5.0

subjecting the collected feedstream to microfiltration in a filtration unit with a mean pore size ranging from 1.5 micron to 15 micron;

subjecting the filtrate to tangential flow microfiltration with a limit of separation of 5,000 to 50,000 daltons.

75. The method of claim 74, wherein the contaminants removed include micelles and caseins.

76. The method of claim 74, wherein the feedstream is milk.

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