Method of purifying recombinant human antithrombin to enhance the viral and prion safety profile
First Claim
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1. A method for purifying a molecular species of interest from a feedstream, comprising:
- (a) Filtering said feedstream by a tangential-flow filtration process (TFF) to produce a TFF permeate;
(b) Cycling said TFF permeate through a closed loop system until at least 50% of the said molecular species of interest is captured wherein said closed loop system further comprises a Heparin-affinity column;
(c) Nanofiltering said TFF/heparin eluate viral removal such that potential viral adventitious agents are removed;
(d) Removing unwanted molecular contaminants through the use of an anion exchange column;
(e) Utilizing a hydrophobic interaction column to eliminate unwanted or variant forms of said molecular species of interest; and
, (f) formulating said molecular species;
(g) lyophilizing said molecular species; and
, (h) Heating the purified lyophilized molecular species of interest to inactive viruses.
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Abstract
Methods of purifying antithrombin from a variety of source materials including from the milk of transgenic mammals to enhance its safety profile vis-à-vis the removal and/or the inactivation of contaminants. Contaminants would include particulate matter, viruses, and/or prions.
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Citations
83 Claims
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1. A method for purifying a molecular species of interest from a feedstream, comprising:
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(a) Filtering said feedstream by a tangential-flow filtration process (TFF) to produce a TFF permeate;
(b) Cycling said TFF permeate through a closed loop system until at least 50% of the said molecular species of interest is captured wherein said closed loop system further comprises a Heparin-affinity column;
(c) Nanofiltering said TFF/heparin eluate viral removal such that potential viral adventitious agents are removed;
(d) Removing unwanted molecular contaminants through the use of an anion exchange column;
(e) Utilizing a hydrophobic interaction column to eliminate unwanted or variant forms of said molecular species of interest; and
,(f) formulating said molecular species;
(g) lyophilizing said molecular species; and
,(h) Heating the purified lyophilized molecular species of interest to inactive viruses. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 77, 78, 79, 80, 81, 82, 83)
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73. A method of purifying a recombinant antithrombin III (rhAT) or a fragment thereof from a feedstream, comprising:
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Solubilizing said rhAT from a feedstream utilizing a tangential flow filtration process;
Washing said filtrate on a membrane with a PBS solution wherein said rhAT or fragment thereof remains in the retentate on the purification column;
Adding an aqueous solution to said rhAT remaining in the retentate to solubilize it;
Eluting said rhAT from said purification column; and
,Purifying out said rhAT from elution.
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74. A method for purifying a molecular species of interest from a feedstream, comprising:
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a) Filtering said feedstream by a tangential-flow filtration process (TFF) to produce a TFF permeate;
b) Cycling said TFF permeate to through a closed loop system until at least 50% of the said molecular species of interest is captured wherein said closed loop system further comprises a Heparin-affinity column;
c) Collecting a first eluate from said Heparin-affinity column and processing said first eluate through a first concentration step and a first diafiltration step;
d) Transferring said first eluate into a downstream processing and formulation area thereafter again transferring said first eluate after said first concentration step and said first diafiltration step to a ion exchange chromatography column to generate a second eluate;
e) Removing unwanted molecular contaminants through the use of an anion exchange column;
f) Processing said second eluate through an anionic exchange chromatography column;
g) Nanofiltering said TFF permeate ion step for viral removal such that potential adventitious agents are removed;
h) Processing said second eluate through a second concentration step and a second diafiltration step to generate a third eluate;
i) Transferring said product eluate through a nanofilter capable of removing viruses from said third eluate;
j) Utilizing a hydrophobic interaction column to eliminate unwanted or variant forms of said molecular species of interest;
k) Processing said product eluate through a third concentration step and a third diafiltration step to generate a product eluate;
l) Loading said product eluate on a anion column and thereafter eluting with a buffer; and
,m) Heating the purified molecular species of interest to inactive viruses. - View Dependent Claims (75, 76)
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Specification