AN IMPROVED ASSAY METHOD FOR DETERMINING THE POTENCY OF RECOMBINANT PROTEIN
The present invention provides an improved assay method for determining the potency of recombinant proteins by culturing the immune cells such as antigen presenting cell and/or T cell in culture media comprising FBS less than 10%. Furthermore, the invention provides a method to eliminate or reduce the false estimation of potency of target recombinant protein. The assay method determines the potency of recombinant protein by evaluating the expression of inflammatory protein. More specifically the present invention describes the IL-2 inhibition assays for determination of potency of CTLA4 IgG1.
1 . An improved assay method for determining potency of recombinant protein comprising;
a) culturing suitable antigen presenting cells and/or T cells in media comprising Fetal bovine serum less than 10%;
b) incubating treated antigen presenting cells and/or T cells for suitable time and suitable temperature;
c) mixing suitable recombinant protein with suitable stimulant to stimulate T cells in the cultured antigen presenting cells and/or T cells;
d) incubating treated mixture for suitable time and suitable temperature;
e) adding reagent in the incubated multi well plate;
f) determining the inhibition of inflammatory protein;
wherein cultured antigen presenting cells and/or T cells in Fetal bovine serum less than 10% provides improved potency compared to antigen presenting cells and/or T cells cultured in 10% Fetal bovine serum.
2 . The assay as claimed in claim 1 , further improve suitability and comparability of protein mixture formulated in liquid prefilled syringe (PFS) and lyophilized vial.
3 . The improved assay method as claimed in claim 1 , wherein suitable antigen presenting cell is a Raji cell and suitable T cell is an IL-2 Luc Jurkat E6.1 cell.
4 . The improved assay method as claimed in claim 1 , wherein recombinant protein is fusion protein.
5 . The improved assay method as claimed in claim 4 , wherein fusion protein is CTLA4 IgG1.
6 . The improved assay method as claimed in claim 5 , wherein CTLA4 IgG1 is Abatacept.
7 . The improved assay method as claimed in claim 1 , wherein the suitable stimulant is selected from Phytohemagglutinin (PHA) or anti-CD3 antibody.
8 . The improved assay method as claimed in claim 1 , wherein reagent is Bright-Glo reagent.
9 . The improved assay method as claimed in claim 1 , wherein the incubation of treated antigen presenting cells and/or T cells is performed at least for about 15 minutes; preferably for about 15 minutes to about 60 minutes at about 37° C. with 5% CO 2 .
10 . The improved assay method as claimed in claim 9 , wherein the incubation of treated antigen presenting cells and/or T cells is performed for about 30 minutes to about 60 minutes at about 37° C. with 5% CO 2 .
11 . The improved assay method as claimed in claim 1 , wherein the incubation of treated mixture of step (d) is performed at least for about 2 hours; preferably for about 2 hours to about 8 hours at about 37° C. with 5% CO 2.
12 . The improved assay method as claimed in claim 11 , wherein the incubation of treated mixture of step (d) is performed for about 5 hours to about 6 hours at about 37° C. with 5% CO 2.
13 . The improved assay method as claimed in claim 1 , wherein the antigen presenting cells and/or T cells are added in multi well plate separately or simultaneously.
14 . An improved assay method of recombinant protein comprising; the cultured antigen presenting cells and/or T cells in Fetal bovine serum less than 10% provides improved potency by improving 95% confidence intervals of inflammatory protein inhibition.
15 . A process for reducing false potency estimation of recombinant protein comprising;
a) culturing suitable antigen presenting cells and/or T cells in media comprising Fetal bovine serum less than 10%;
b) incubating treated antigen presenting cells and/or T cells for suitable time and suitable temperature;
c) mixing suitable recombinant protein with suitable stimulant to stimulate T cells in the cultured antigen presenting cells and/or T cells;
d) incubating treated mixture for suitable time and suitable temperature;
e) adding reagent in the incubated multi well plate;
f) determining the inhibition of inflammatory protein;
wherein cultured antigen presenting cells and/or T cells in Fetal bovine serum less than 10% provides improved potency compared to antigen presenting cells and/or T cells cultured in 10% Fetal bovine serum.
16 . The process for reducing false potency estimation as claimed in claim 15 , wherein suitable antigen presenting cell is a Raji cell and suitable T cell is an IL-2 Luc Jurkat E6.1 cell.
17 . The process for reducing false potency estimation as claimed in claim 15 , wherein recombinant protein is Abatacept.
18 . The process for reducing false potency estimation as claimed in claim 15 , wherein the suitable stimulant is selected from Phytohemagglutinin (PHA) or anti-CD3 antibody.
19 . The process for reducing false potency estimation as claimed in claim 15 , wherein reagent is Bright-Glo reagent.
20 . The process for reducing false potency estimation as claimed in claim 15 , wherein the incubation of treated antigen presenting cells and/or T cells is performed for about 30 minutes to about 60 minutes at about 37° C. with 5% CO 2.
21 . The process for reducing false potency estimation as claimed in claim 15 , wherein the incubation of treated mixture of step (d) is performed for about 5 hours to about 6 hours at about 37° C. with 5% CO 2 .