Methods for the production of purified recombinant human uteroglobin for the treatment of inflammatory and fibrotic conditions
First Claim
1. A bacterial expression system for the production of rhUG comprising a synthetic gene which codes for human UG, wherein the synthetic gene comprises Seq. ID. Nos. 1-4.
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Abstract
The present invention relates generally to the production of recombinant human uteroglobin (rhUG) for use as a therapeutic in the treatment of inflammation and fibrotic diseases. More particularly, the invention provides processes, including broadly the steps of bacterial expression and protein purification, for the scaled-up production of rhUG according to current Good Manufacturing Practices (cGMP). The invention further provides analytical assays for evaluating the relative strength of in vivo biological activity of rhUG produced via the scaled-up cGMP processes.
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Citations
106 Claims
- 1. A bacterial expression system for the production of rhUG comprising a synthetic gene which codes for human UG, wherein the synthetic gene comprises Seq. ID. Nos. 1-4.
- 3. A bacterial expression system for production of rhUG comprising a human cDNA sequence which codes for human UG wherein the gene further comprises Met-Ala-Ala at the N-terminus of the sequence.
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5. A method of producing a rhUG research seed bank comprising:
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a. inoculating onto a growth medium at least one colony of a bacterial strain comprising a rhUG expression system;
b. incubating the inoculated growth medium until a stationary phase is reached;
c. adding glycerol to the inoculated growth medium;
d. freezing the culture in aliquot portions; and
e. storing the frozen aliquot portions at a temperature below about −
50 C. - View Dependent Claims (6, 7, 8, 9)
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10. A method of producing a rhUG master cell bank comprising:
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a. inoculating a suitable incubating broth with an aliquot portion of a rhUG research seed bank to form a bacterial culture;
b. incubating the bacterial culture;
c. adding a cryopreservative to the bacterial culture to form a cryopreserved solution;
d. transferring a portion of the cryopreserved solution to a cryovial; and
e. storing the cryovial at a temperature below about −
60 C. - View Dependent Claims (11, 12, 13, 14)
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15. A method of producing a rhUG production cell bank comprising:
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a. inoculating a suitable incubating broth with an aliquot portion of a rhUG master cell bank to form a bacterial culture;
b. incubating the bacterial culture;
c. adding a cryopreservative to the bacterial culture to form a cryopreserved solution;
d. transferring a portion of the cryopreserved solution to a cryovial; and
e. storing the cryovial at a temperature below about −
60 C. - View Dependent Claims (16, 17, 18, 19)
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20. A method of expressing rhUG comprising the steps of:
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a. providing a production seed cell bank culture comprising an expression vector capable of expressing rhUG;
b. inoculating a broth medium with the production seed cell bank culture to form an inoculum;
c. incubating the bacterial culture formed in step b;
d. inoculating a large scale fermenter with the inoculum formed in step c to form a fermentation culture;
e. incubating the fermentation culture within the large scale fermenter;
f. adding an induction agent to the fermentation culture to induce the expression of rhUG; and
g. harvesting the fermentation culture after step f. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27)
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28. A method of expressing rhUG comprising the steps of:
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a. inoculating a large scale fermenter with an inoculum comprising an expression vector capable of expressing rhUG to form a fermentation culture;
b. incubating the fermentation culture within the large scale fermenter;
c. adding an induction agent to the fermentation culture to induce the expression of rhUG; and
d. harvesting the fermentation culture. - View Dependent Claims (29, 30, 31, 32, 33, 34)
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35. A method of purifying rhUG comprising the steps of:
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a. providing a bacterial cell paste comprising bacterial cells capable of overexpressing rhUG;
b. lysing the bacterial cell paste to form a supernatant;
c. filtering the supernatant formed in step b through a first nominal molecular weight cut off (NMWCO) membrane to form a first permeate;
d. concentrating the first permeate formed in step c by the use of a second NMWCO membrane;
e. loading the concentrated permeate formed in step d onto an anion exchange column to form a first eluate;
f. concentrating the first eluate formed in step e by the use of a third NMWCO membrane to form a second concentrate;
g. loading the second concentrate formed in step f onto a Hydroxyapatite (HA) column to form a second eluate;
h. separating host-derived proteins from the rhUG in the second eluate formed in step g to provide purified rhUG; and
i. recovering the purified rhUG formed in step h. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 80, 81, 82, 83, 84, 85)
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55. A method of purifying rhUG comprising the steps of:
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a. providing bacterial cells capable of overexpressing rhUG;
b. lysing the bacterial cells to form a supernatant liquid;
c. filtering the liquid through a molecular weight cut off (NMWCO) membrane;
d. loading the liquid onto an exchange column;
e. separating host-derived proteins from the rhUG to provide purified rhUG; and
f. recovering the purified rhUG. - View Dependent Claims (56, 57, 58, 59, 60)
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61. A method of producing a pharmaceutical grade rhUG drug substance comprising the steps of:
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a. providing a bacterial expression system capable of expressing rhUG;
b. inoculating a fermenter with an inoculum comprising the bacterial expression system to form a fermentation culture;
c. adding an induction agent to the fermentation culture to induce the expression of rhUG by the bacterial expression system;
d. harvesting the rhUG expressed in step c; and
e. purifying the rhUG harvested in step d, wherein the purifying step comprises the use of at least one filtration step and at least one exhange column.
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62. An assay method for determining the potency of recombinant human uteroglobin in a sample which comprises:
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(a) contacting a sample containing recombinant human uterogloblin with phospholipase A2, (b) introducing a labeled substrate to said sample, (c) separating product from sample, and (d) determining level of cleavage. - View Dependent Claims (63, 64, 65, 66, 67, 68, 69, 70)
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71. A method for measuring in vitro the anti-inflammatory effect arising from inhibition or blocking of secretory phopsholipase A2 enzymes by recombinant human uteroglobin, comprising:
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(a) contacting a sample containing recombinant human uterogloblin with phospholipase A2, (b) introducing labeled substrate to said sample, (c) separating product from sample, and (d) determining level of cleavage by scintillation counting.
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72. An assay method for assaying for the inhibition of secretory phopsholipase A2 activity by recombinant human uteroglobin, comprising:
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(a) contacting a sample containing recombinant human uterogloblin with phospholipase A2, (b) introducing labeled substrate to said sample, (c) separating product from sample, and (d) determining level of cleavage by scintillation counting.
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73. An assay method for determining the potency of recombinant human uteroglobin in a sample which comprises:
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(a) contacting a sample containing recombinant human uterogloblin with phospholipase A2, (b) introducing flourescently labeled substrate to said sample, (c) separating non-cleaved substrate from sample, and (d) determining amount of cleaved substrate by flourescence. - View Dependent Claims (74, 75, 76, 77, 78)
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79. A method for measuring in vitro the binding of recombinant human uteroglobin to fibronectin, comprising:
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(a) contacting a recombinant fragment of human fibronectin with a recombinant human CC10-HRP conjugate, (b) visualizing the assay to determine binding of recombinant human uteroglobin to the fibronectin fragment.
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- 86. A pharmaceutical composition comprising a purified recombinant human uteroglobin and a pharmaceutically acceptable carrier or diluent.
Specification