Claims for Patent: 10,787,671

Summary for Patent: 10,787,671

Title:	Method for production of recombinant Erwinia asparaginase
Abstract:	Provided herein are methods of production of recombinant Erwinia asparaginase. Methods herein produce asparaginase having high expression levels in the periplasm or the cytoplasm of the host cell having activity comparable to commercially available asparaginase preparations.
Inventor(s):	Coleman; Russell J. (San Diego, CA), Bruck; Torben (Lakeside, CA)
Assignee:	Pfenex Inc. (San Diego, CA)
Application Number:	16/163,382
Patent Claims:	1. A method for producing a recombinant type II asparaginase, the method comprising culturing a Pseudomonadales host cell in a culture medium and expressing the recombinant type II asparaginase in the cytoplasm of the Pseudomonadales host cell from an expression construct comprising a nucleic acid encoding the recombinant type II asparaginase, wherein the nucleic acid encoding the recombinant type II asparaginase comprises a sequence at least 85% homologous to SEQ ID NO: 1, and wherein the recombinant asparaginase is produced in the cytoplasm at a yield of about 20% to about 40% total cell protein (TCP) in soluble form. 2. The method of claim 1, wherein the recombinant type II asparaginase is produced in the cytoplasm at a yield of about 10 g/L to about 25 g/L. 3. The method of claim 1, further comprising measuring the activity of an amount of the soluble recombinant type II asparaginase produced, using an activity assay. 4. The method of claim 1, wherein the recombinant type II asparaginase is an Erwinia chrysanthemi L-asparaginase type II (crisantaspase). 5. The method of claim 1, wherein the nucleic acid encoding the recombinant type II asparaginase comprises a sequence at least 85% homologous to SEQ ID NO: 2. 6. The method of claim 1, wherein the recombinant type II asparaginase has an amino acid sequence as set forth in SEQ ID NO: 1. 7. The method of claim 1, wherein the Pseudomonadales host cell is a Pseudomonas fluorescens cell. 8. The method of claim 1, wherein the host cell is deficient in the expression of one or more native asparaginases. 9. The method of claim 8, wherein the one or more deficiently expressed native asparaginase is selected from: a type I asparaginase; a type II asparaginase; and a combination thereof. 10. The method of claim 1, wherein the host cell: is deficient in the expression of one or more proteases; overexpresses one or more folding modulators; or both. 11. A method for producing a recombinant type II asparaginase, the method comprising: culturing a Pseudomonadales host cell in a culture medium and expressing the recombinant type II asparaginase in the periplasm of the host cell from an expression construct comprising a nucleic acid encoding the recombinant type II asparaginase, wherein the nucleic acid encoding the recombinant type II asparaginase comprises a sequence at least 85% homologous to SEQ ID NO: 1, and wherein the recombinant type II asparaginase is produced in the periplasm at a yield of about 20% to about 40% total cell protein (TCP) in soluble form. 12. The method of claim 11, wherein the recombinant type II asparaginase is produced in the periplasm at a yield of about 5 g/L to about 30 g/L. 13. The method of claim 11, wherein the method further comprises measuring the activity of an amount of the recombinant type II asparaginase produced, using an activity assay. 14. The method of claim 11, wherein the recombinant type II asparaginase is an Erwinia chrysanthemi L-asparaginase type II (crisantaspase). 15. The method of claim 11, wherein the nucleic acid encoding the recombinant type II asparaginase comprises a sequence at least 85% homologous to SEQ ID NO: 2. 16. The method of claim 11, wherein the recombinant type II asparaginase has an amino acid sequence as set forth in SEQ ID NO: 1. 17. The method of claim 11, wherein the Pseudomonadales host cell is a Pseudomonas fluorescens cell. 18. The method of claim 11, wherein the host cell is deficient in the expression of one or more asparaginases. 19. The method of claim 18, wherein the deficiently expressed asparaginase is selected from: a type I asparaginase, a type II asparaginase, or both. 20. The method of claim 11, wherein the expression construct comprises a secretion leader that directs transfer of the recombinant type II asparaginase produced to the periplasm of the host cell. 21. The method of claim 20, wherein the secretion leader is selected from the group consisting of FlgI, Ibps31A, PbpA20V, DsbC, 8484, and 5193. 22. The method of claim 3, further comprising comparing the measured activity of the recombinant type II asparaginase produced with a measured activity of the same amount of a control type II asparaginase using the same activity assay, wherein the measured activity of the recombinant type II asparaginase produced is comparable to the activity of the control type II asparaginase. 23. The method of claim 13, further comprising comparing the measured activity of the recombinant type II asparaginase produced with a measured activity of the same amount of a control type II asparaginase using the same activity assay, wherein the measured activity of the recombinant type II asparaginase produced is comparable to the activity of the control type II asparaginase. 24. The method of claim 1, wherein the recombinant type II asparaginase is modified to increase half-life in patients. 25. The method of claim 1, wherein the host cell: is deficient in the expression of one or more proteases; overexpresses one or more folding modulators; or both. 26. The method of claim 25, wherein the host cell: is deficient in HslUV protease, is deficient in PrtB protease; is deficient in Prc protease; is deficient in DegP protease; is deficient in AprA protease; is deficient in Lon protease; is deficient in La protease; is deficient in DegP1; is deficient in DegP2; overexpresses DegP S219A; or a combination thereof. 27. The method of claim 11, wherein the recombinant type II asparaginase is modified to increase half-life in patients. 28. The method of claim 1, wherein the recombinant type II asparaginase produced is used in the treatment of patients with acute lymphoblastic leukemia. 29. The method of claim 11, wherein the recombinant type II asparaginase produced is used in the treatment of patients with acute lymphoblastic leukemia.

Details for Patent 10,787,671

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Recordati Rare Diseases, Inc.	ELSPAR	asparaginase	For Injection	101063	January 10, 1978		2037-10-27
Jazz Pharmaceuticals, Inc.	ERWINAZE	asparaginase erwinia chrysanthemi	For Injection	125359	November 18, 2011		2037-10-27
Jazz Pharmaceuticals Ireland Limited	RYLAZE	asparaginase erwinia chrysanthemi (recombinant)-rywn	Injection	761179	June 30, 2021		2037-10-27