Claims for Patent: 10,760,112

Summary for Patent: 10,760,112

Title:	Enzymatic activity assays for glucocerebrosidase
Abstract:	The present invention provides, among other things, methods and compositions for determining enzyme kinetic parameters (e.g., Vmax, Km, and specific activity, etc.) indicative of clinically relevant properties of glucocerebrosidase using a physiologically relevant substrate, in particular, a substrate that is representative of substrates that typically accumulate in patients suffering from Gaucher disease such as glucosylceramide. Thus, the present invention is particularly useful to measure a kinetic parameter relating to the activity of glucocerebrosidase in a drug substance, drug product, and stability sample for enzyme replacement therapy.
Inventor(s):	Bernhardt; Peter (Lexington, MA), Yen; Chen-Chung Willy (Lexington, MA), Chhajlani; Vijay (Arlington, MA)
Assignee:	Shire Human Genetic Therapies, Inc. (Lexington, MA)
Application Number:	15/537,296
Patent Claims:	1. A method of determining potency of glucocerebrosidase, comprising the steps of: contacting a sample comprising a glucocerebrosidase with a C18:1 .beta.-D-Glucosylceramide substrate under conditions that permit the glucocerebrosidase to catalyze hydrolysis of the C18:1 .beta.-D-Glucosylceramide substrate to release glucose; and measuring the amount of glucose released to determine one or more kinetic parameters of the glucocerebrosidase, wherein the one or more kinetic parameters are selected from the group consisting of V.sub.max, k.sub.cat, K.sub.m, and specific activity, wherein the one or more kinetic parameters are indicative of the potency of the glucocerebrosidase. 2. The method of claim 1, wherein the glucocerebrosidase is a recombinant glucocerebrosidase. 3. The method of claim 2, wherein the recombinant glucocerebrosidase is velaglucerase alfa. 4. The method of claim 1, wherein the step of measuring the amount of glucose released comprises performing chromatography. 5. The method of claim 4, wherein the chromatography comprises high-performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UPLC). 6. The method of claim 4, wherein said chromatography includes anion exchange chromatography. 7. The method of claim 4, wherein the step of measuring the amount of glucose released comprises performing high-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD). 8. The method of claim 1, wherein the step of measuring the amount of glucose released comprises determining the amount of glucose as compared to a control. 9. The method of claim 8, wherein the control is a predetermined amount of glucose. 10. The method of claim 8, wherein the control is a standard curve. 11. The method of claim 1, wherein the step of measuring the amount of glucose released comprises determining the rate of glucose formation. 12. The method of claim 1, wherein the one or more kinetic parameters are determined by fitting the amount of glucose released to the Michaelis-Menten model or other kinetic model suitable to determine kinetic parameters. 13. The method of claim 1, wherein the substrate is sonicated prior to said contacting. 14. The method of claim 13, wherein the substrate is sonicated for about 1 to 10 minutes. 15. The method of claim 1, wherein the sample is a drug substance, a drug product, or a stability sample of drug substance and drug product. 16. The method of claim 1, wherein the conditions that permit the glucocerebrosidase to catalyze the substrate to release glucose comprise incubation at about 37.degree. C. for about 20 minutes. 17. The method of claim 1, wherein the conditions that permit the glucocerebrosidase to catalyze hydrolysis of the substrate to release glucose comprise taurocholic acid and/or oleic acid. 18. The method of claim 17, wherein the taurocholic acid is included. 19. The method of claim 18, wherein the taurocholic acid is included at a concentration of about 0.5 to 100 mM. 20. The method of claim 18, wherein the taurocholic acid is included at a concentration of about 2 to 10 mM. 21. The method of claim 18, wherein the taurocholic acid is included at a concentration of 2 to 7 mM. 22. The method of claim 17, wherein the oleic acid is included. 23. The method of claim 22, wherein the oleic acid is included at a concentration of about 0.001% to 5% v/v. 24. The method of claim 22, wherein the oleic acid is included at a concentration of about 0.1% to 0.5% v/v. 25. The method of claim 22, wherein the oleic acid is included at a concentration of about 0.1% to 0.3% v/v. 26. The method of claim 1, wherein the method further comprises a step of inactivation of the glucocerebrosidase. 27. The method of claim 1, wherein the method comprises a step of removing lipids from an enzyme sample prior to measuring the amount of glucose released. 28. The method of claim 27, wherein the step of removing lipids comprises in-line desalting.

Details for Patent 10,760,112

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Takeda Pharmaceuticals U.s.a., Inc.	VPRIV	velaglucerase alfa	For Injection	022575	02/26/2010		2034-12-18