Quantification of misfolded TNFR2:Fc
AssigneeSandoz Ltd.
InventorsAlfred Rupprechter, Michael Fuchs, Johann Holzmann, William Lamanna, Christoph Posch, Hansjorg Toll, Robert Mayer
Life of this patent
Estimating the term…
Abstract
The present invention is directed to methods for determining the relative amount of wrongly disulphide bridged TNFR2:Fc in a sample of TNFR2:Fc, a fusion protein which is used in a variety of therapeutic applications. In addition, the invention pertains to a method for purifying TNFR2:Fc using said method for determining the percentage of wrongly disulphide bridged TNFR2:Fc, and to TNFR2:Fc compositions obtained thereby.
Claims30
A composition comprising Cys74-Cys88/Cys78-Cys96 disulfide bridged TNFR2:Fc and Cys78-Cys88 disulfide bridged TNFR2:Fc;
- wherein the relative amount of Cys78-Cys88 disulfide bridged TNFR2:Fc is less than 2.2% of the total TNFR2:Fc in the composition when determined according to formula (1);
- x
-
- (
- in
-
-
- %
- )
- =
- [
- T
-
-
- 7
-
-
- area
- ]
- [
- T
-
-
- 7
-
-
- area
- ]
- +
- [
- T
-
-
- 27
-
-
- area
- ]
- ⨯
- 100
- (
- 1
- )
- wherein [T7 area] and [T27 area] are obtained by a method comprising the steps of:
- (a) providing a sample comprising a mixture of Cys78-Cys88 disulfide bridged TNFR2:Fc and Cys74-Cys88/Cys78-Cys96 disulfide bridged TNFR2:Fc;
- (b) denaturing and alkylating the sample of step (a);
- (c) subjecting the sample resulting from step (b) to tryptic digestion;
- (d) subjecting the sample resulting from step (c) to HPLC, thereby separating fragments indicative of Cys78-Cys88 disulfide bridged TNFR2:Fc;
- and
- (e) conducting a peak integration for the peak indicative of Cys78-Cys88 disulfide bridged TNFR2:Fc and for a peak indicative of the total TNFR:Fc in the sample, as obtained from step (d), wherein the fragments indicative of Cys78-Cys88 disulfide bridged TNFR2:Fc comprise the amino acid sequence shown in SEQ ID NO:
- 4 (“T7”), and wherein the fragments indicative of total TNFR2:Fc comprise the amino acid sequence shown in SEQ ID NO:
- 5 (“T27”);
- wherein the amino acid sequence of the TNFR2:Fc has at least 97% identity to the amino acid sequence of SEQ ID NO:
- 3.
A method of producing a composition comprising Cys74-Cys88/Cys78-Cys96 disulfide bridged TNFR2:Fc and Cys78-Cys88 disulfide bridged TNFR2:Fc, wherein the relative amount of Cys78-Cys88 disulfide bridged TNFR2:Fc is less than 2.2% of the total TNFR2:Fc in the composition, and wherein the amino acid sequence of the TNFR2:Fc has at least 97% identity to the amino acid sequence of SEQ ID NO:
- 3;
- which method comprises the steps of:
- (a) providing a sample comprising Cys74-Cys88/Cys78-Cys96 disulfide bridged TNFR2:Fc and Cys78-Cys88 disulfide bridged TNFR2:Fc;
- and
- (b) subjecting the sample of step (a) to at least one chromatographic step, wherein the at least one chromatographic step comprises a hydrophobic interaction chromatography (HIC);
- and
- (c) separating one or more fractions comprising the Cys74-Cys88/Cys78-Cys96 disulfide bridged TNFR2:Fc, said one or more fractions having a relative amount of Cys78-Cys88 disulfide bridged TNFR2:Fc of less than 2.2% of the total TNFR2:Fc when determined according to formula (1);
- x
-
- (
- in
-
-
- %
- )
- =
- [
- T
-
-
- 7
-
-
- area
- ]
- [
- T
-
-
- 7
-
-
- area
- ]
- +
- [
- T
-
-
- 27
-
-
- area
- ]
- ⨯
- 100
- (
- 1
- )
- wherein [T7 area] and [T27 area] are obtained by a method comprising the steps of:
- (i) providing a sample comprising a mixture of Cys78-Cys88 disulfide bridged TNFR2:Fc and Cys74-Cys88/Cys78-Cys96 disulfide bridged TNFR2:Fc;
- (ii) denaturing and alkylating the sample of step (i);
- (iii) subjecting the sample resulting from step (ii) to tryptic digestion;
- (iv) subjecting the sample resulting from step (iii) to HPLC, thereby separating fragments indicative of Cys78-Cys88 disulfide bridged TNFR2:Fc;
- and
- (v) conducting a peak integration for the peak indicative of Cys78-Cys88 disulfide bridged TNFR2:Fc and for a peak indicative of the total TNFR:Fc in the sample, as obtained from step (iv), wherein the fragments indicative of Cys78-Cys88 disulfide bridged TNFR2:Fc comprise the amino acid sequence shown in SEQ ID NO:
- 4 (“T7”), and wherein the fragments indicative of total TNFR2:Fc comprise the amino acid sequence shown in SEQ ID NO:
- 5 (“T27”).
Dependencies are inferred from the claim text.