CELL-BASED BIOIDENTITY TEST FOR INSULIN

1 . A method for determining the potency of an insulin or insulin analog in a sample comprising:

(a) providing a cell culture of recombinant cells capable of gluconeogenesis, wherein the recombinant cells comprise a nucleic acid molecule encoding a reporter molecule operably linked to the promoter for the glucose-6-phosphate catalytic-subunit-encoding gene C (G6PC);

(b) mixing equal aliquots of the cell culture with a series of dilutions of cell culture medium comprising the insulin or insulin analog to provide a series of assay cell cultures and incubating the series of assay cell cultures for about 15 to 27 hours;

(c) detecting the reporter molecule in the series of assay cell cultures to provide data for the assay cell culture and generating a dose response curve from the data; and

(d) comparing the dose response curve to a reference dose response curve obtained from one or more series of reference assay cell cultures determined by a method comprising

(i) mixing equal aliquots of the cell culture with a series of dilutions of a reference cell culture medium comprising a known amount of the insulin or insulin analog to provide a series of reference assay cell cultures,

(ii) incubating the series of reference assay cell cultures for about 15 to 27 hours, and

(iii) detecting the reporter molecule in the series of reference assay cell cultures to provide data for the reference assay cell culture and generating the reference dose response curve from the data, to provide the potency of the insulin or insulin analog in the sample.

2 . (canceled)

3 . The method of claim 1 , wherein the recombinant cells are H4IIE rat hepatoma cells or HepG2 human hepatoma cells, which comprise the nucleic acid molecule encoding the reporter molecule operably linked to the promoter for the glucose-6-phosphate catalytic-subunit-encoding gene C (G6PC).

4 . The method of claim 1 , wherein the promoter is a human G6PC promoter comprising the nucleotide sequence set forth SEQ ID NO: 1.

5 . The method of claim 1 , wherein the reporter molecule is an enzyme and a substrate for the enzyme is provided to the culture to detect the reporter molecule.

6 . The method of claim 5 , wherein the enzyme is luciferase and the substrate is luciferin.

7 - 9 . (canceled)

10 . A method for releasing a manufacturing batch or lot of drug product comprising insulin or insulin analog, comprising:

(a) providing a manufacturing batch or lot of a drug product;

(b) obtaining a sample from the batch or lot and subjecting the sample to a cell-based bioidentity test to determine whether the batch or lot can be released, wherein the bioidentity test comprises

(i) mixing equal aliquots of a cell culture of recombinant cells with a series of dilutions of cell culture medium comprising the sample to provide a series of assay cell cultures and mixing equal aliquots of the cell culture with a series of dilutions of a reference cell culture medium comprising a known amount of the insulin or insulin analog to provide a series of reference assay cell cultures;

(ii) incubating the series of assay cell cultures and the series of reference cell cultures for about 15 to 27 hours;

(iii) detecting the reporter molecule in the series of assay cell cultures to provide data for the assay cell culture and generating a sample dose response curve from the data and detecting the reporter molecule in the series of reference assay cell cultures to provide data for the reference assay cell culture and generating a reference dose response curve from the data; and

(c) comparing the sample dose response curve to the reference dose response curve and releasing the batch or lot when the difference between the sample dose response curve is within a predetermined range from the reference dose response curve;

wherein the recombinant cells capable of gluconeogenesis, wherein the recombinant cells comprise a nucleic acid molecule encoding a reporter molecule operably linked to the promoter for the glucose-6-phosphate catalytic-subunit-encoding gene C (G6PC).

11 . (canceled)

12 . The method of claim 10 , wherein the recombinant cells are H4IIE rat hepatoma cells or HepG2 human hepatoma cells, which comprise the nucleic acid molecule encoding the reporter molecule operably linked to the promoter for the glucose-6-phosphate catalytic-subunit-encoding gene C (G6PC).

13 . The method of claim 10 , wherein the promoter is a human G6PC promoter comprising the nucleotide sequence set forth SEQ ID NO: 1.

14 . The method of claim 10 , wherein the reporter molecule is an enzyme and a substrate for the enzyme is provided to the culture to detect the reporter molecule.

15 . The method of claim 14 , wherein the enzyme is luciferase and the substrate is luciferin.

16 - 18 . (canceled)

19 . A provides a method for determining the bioidentity of an insulin or insulin analog in a sample comprising:

(a) providing a cell culture of recombinant cells capable of gluconeogenesis, wherein the recombinant cells comprise a nucleic acid molecule encoding a reporter molecule operably linked to the promoter for the glucose-6-phosphate catalytic-subunit-encoding gene C (G6PC);

(b) mixing equal aliquots of the cell culture with a series of dilutions of cell culture medium comprising the insulin or insulin analog to provide a series of assay cell cultures and incubating the series of assay cell cultures for about 15 to 27 hours;

(c) detecting the reporter molecule in the series of assay cell cultures to provide data for the assay cell culture and generating a dose response curve from the data; and

(d) comparing the dose response curve to a reference dose response curve obtained from one or more series of reference assay cell cultures determined by a method comprising

(i) mixing equal aliquots of the cell culture with a series of dilutions of a reference cell culture medium comprising a known amount of the insulin or insulin analog to provide a series of reference assay cell cultures,

(ii) incubating the series of reference assay cell cultures for about 15 to 27 hours, and

(iii) detecting the reporter molecule in the series of reference assay cell cultures to provide data for the reference assay cell culture and generating the reference dose response curve from the data, to provide the bioidentity of the insulin or insulin analog in the sample.

20 - 28 . (canceled)

29 . A recombinant cell capable of gluconeogenesis comprising a nucleic acid molecule encoding a reporter molecule operably linked to the promoter for the glucose-6-phosphate catalytic-subunit-encoding gene C (G6PC).

30 . The recombinant cell of claim 29 , wherein the recombinant cells are H4IIE rat hepatoma cells or HepG2 human hepatoma cells.

31 - 33 . (canceled)

34 . A cell culture comprising a medium and a recombinant cell capable of gluconeogenesis comprising a nucleic acid molecule encoding a reporter molecule operably linked to the promoter for the glucose-6-phosphate catalytic-subunit-encoding gene C (G6PC).

35 . The cell culture of claim 34 , wherein the recombinant cells are H4IIE rat hepatoma cells or HepG2 human hepatoma cells.

36 - 41 . (canceled)

42 . A nucleic acid molecule encoding a reporter molecule operably linked to the promoter for the glucose-6-phosphate catalytic-subunit-encoding gene C (G6PC).

43 - 45 . (canceled)

46 . A vector comprising the nucleic acid molecule of claim 42 .

47 . The vector of claim 46 , wherein the vector is a plasmid or recombinant virus.

48 . The vector of claim 46 , wherein the recombinant virus is a lentivirus.