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.
Claims20
A method for determining a relative amount of Cys78-Cys88 disulfide bridged TNFR2:Fc in a sample comprising Cys74-Cys88/Cys78-Cys96 disulfide bridged TNFR2:Fc and Cys78-Cys88 disulfide bridged TNFR2:Fc, wherein the method comprises 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 under non-reducing conditions;
- (d) determining by HPLC the amount of a fragment indicative of Cys78-Cys88 disulfide bridged TNFR2:Fc in the sample resulting from step (c);
- and
- (e) determining the relative amount of the Cys78-Cys88 disulfide bridged TNFR2:Fc in the sample based on the amount of the fragment indicative of Cys78-Cys88 disulfide bridged TNFR2:Fc relative to the amount of a fragment not affected by disulfide bridging of Cys74, Cys78, Cys88 and Cys96;
- wherein the amino acid sequence of the TNFR2 part of TNFR2:Fc has at least 97% identity to the amino acids 1-235 of the amino acid sequence of SEQ ID NO:
- 1, and wherein the fragment indicative of Cys78-Cys88 disulfide bridged TNFR2:Fc consists of SEQ ID NO:4 (“T7”).
Dependencies are inferred from the claim text.