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Last Updated: March 29, 2024

Claims for Patent: 9,598,718


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Summary for Patent: 9,598,718
Title:Quantification of misfolded TNFR2:Fc
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.
Inventor(s): Rupprechter; Alfred (Kundl, AT), Fuchs; Michael (Kundl, AT), Holzmann; Johann (Kundl, AT), Lamanna; William (Kundl, AT), Posch; Christoph (Kundl, AT), Toll; Hansjorg (Kundl, AT), Mayer; Robert (Kundl, AT)
Assignee: Sandoz AG (Basel, CH)
Application Number:14/455,183
Patent Claims:1. A method for determining a relative amount of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc in a sample comprising Cys.sub.74-Cys.sub.88/Cys.sub.78-Cys.sub.96 disulfide bridged TNFR2:Fc and Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc, wherein the method comprises the steps of: (a) providing a sample comprising a mixture of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc and Cys.sub.74-Cys.sub.88/Cys.sub.78-Cys.sub.96 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 Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc in the sample resulting from step (c); and (e) determining the relative amount of the Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc in the sample based on the amount of the fragment indicative of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc relative to the amount of a fragment not affected by disulfide bridging of Cys.sub.74, Cys.sub.78, Cys.sub.88 and Cys.sub.96; 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 Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc consists of SEQ ID NO:4 ("T7").

2. The method of claim 1, wherein the amino acid sequence of the TNFR2:Fc applied to step (a) has at least 97% identity to the amino acid sequence of SEQ ID NO: 3 (etanercept).

3. The method of claim 1, wherein the peak not affected by disulfide bridging of Cys.sub.74, Cys.sub.78, Cys.sub.88 and Cys.sub.96 is not affected by disulfide bridging at all and is indicative of the total TNFR2:Fc in the sample.

4. The method of claim 3, wherein the fragment indicative of total TNFR2:Fc comprises the amino acid sequence shown in SEQ ID NO: 5 ("T27").

5. The method of claim 4, wherein the fragment indicative of total TNFR2:Fc consists of the amino acid sequence shown in SEQ ID NO: 5 ("T27").

6. The method of claim 4, wherein the relative amount of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc is determined by (i) determining the peak areas in the HPLC chromatogram indicative of Cys.sub.78-Cys.sub.88 disulfide bridged TNFR2:Fc ("T7 area") and indicative of total TNFR2:Fc ("T27 area"); and (ii) calculating the relative amount according to formula (1) .function..times..times..times..times..times..times..times..times..times.- .times..times..times..times..times..times..times..times. ##EQU00005##

7. The method of claim 1, wherein step (b) is carried out in a buffer having a pH in the range of 7 to 9, and wherein the buffer comprises at least one of: 10-100 mM TRIS, 0.5-1.5 M iodoacetamide, and 0.02%-0.5% of a cleavable surfactant.

8. The method of claim 7, wherein step (b) is carried out in a buffer having a pH in the range of 7.5 to 8.5, and wherein the buffer comprises at least one of: 20-80 mM TRIS, 0.9-1.2 M iodoacetamide, and 0.1%-0.2% of a cleavable surfactant.

9. The method of claim 8, wherein the buffer comprises a cleavable surfactant which is selected from the group consisting of: sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate, sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, and sodium 3-(4-(1,1-bis(hexyloxy)ethyl)pyridinium-1-yl)propane-1-sulfonate.

10. The method of claim 9, wherein the cleavable surfactant is sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate.

11. The method of claim 1, wherein step (b) is carried out at 40 to 70.degree. C. for 30 to 60 min.

12. The method of claim 1, wherein step (b) is carried out at 50 to 60.degree. C. for 30 to 45 min.

13. The method of claim 1, wherein step (c) is carried out in a digestion buffer having a pH in the range of 5 to 7; and wherein the digestion buffer comprises: MES as the buffering agent; or 0.02%-0.5% of a cleavable surfactant; or MES as the buffering agent and 0.02%-0.5% of a cleavable surfactant.

14. The method of claim 13, wherein step (c) is carried out in a digestion buffer having a pH in the range of 5.5 to 6.5; and wherein the digestion buffer comprises: 10-100 mM MES as the buffering agent; or 0.1%-0.2% of a cleavable surfactant; or 10-100 mM MES as the buffering agent and 0.1%-0.2% of a cleavable surfactant.

15. The method of claim 13, wherein the buffer comprises a cleavable surfactant which is selected from the group consisting of: sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate, sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, and sodium 3-(4-(1,1-bis(hexyloxy)ethyl)pyridinium-1-yl)propane-1-sulfonate.

16. The method of claim 15, wherein the cleavable surfactant is sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate.

17. The method of claim 1, wherein step (c) is carried out using an effective amount of trypsin for 1-24 h; and at 32-38.degree. C.

18. The method of claim 17, wherein step (c) is carried out using an effective amount of trypsin for 6-18 h; and at 36-37.degree. C.

19. The method of claim 1, wherein step (d) is carried out in a mobile phase comprising 0.05%-0.5% TFA in water.

20. The method of claim 19, wherein step (d) is carried out in a mobile phase comprising 0.1%-0.2% TFA in water.

Details for Patent 9,598,718

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Immunex Corporation ENBREL etanercept For Injection 103795 11/02/1998 ⤷  Try a Trial 2034-07-18
Immunex Corporation ENBREL etanercept For Injection 103795 05/27/1999 ⤷  Try a Trial 2034-07-18
Immunex Corporation ENBREL etanercept Injection 103795 09/27/2004 ⤷  Try a Trial 2034-07-18
Immunex Corporation ENBREL etanercept Injection 103795 02/01/2007 ⤷  Try a Trial 2034-07-18
Immunex Corporation ENBREL MINI etanercept Injection 103795 09/14/2017 ⤷  Try a Trial 2034-07-18
Immunex Corporation ENBREL etanercept Injection 103795 ⤷  Try a Trial 2034-07-18
Immunex Corporation ENBREL etanercept Injection 103795 03/05/2020 ⤷  Try a Trial 2034-07-18
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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