Claims for Patent: 9,029,081

Summary for Patent: 9,029,081

Title:	Characterization of N-glycans using exoglycosidases
Abstract:	The present disclosure provides methods for analyzing structure and/or composition of N-glycans. Such methods often involve digestion of N-glycans with multiple exoglycosidases. In some embodiments, N-glycans are digested with multiple exoglycosidases simultaneously. In some embodiments, N-glycans are digested with multiple exoglycosidases sequentially. In some embodiments, methods in accordance with the present disclosure involve comparison of cleavage products of N-glycans that have been digested with multiple exoglycosidases simultaneously to N-glycans that have been digested with multiple exoglycosidases sequentially.
Inventor(s):	Parsons; Ian Christopher (Belmont, MA), Bulik; Dorota A. (Winchester, MA), Bosques; Carlos J. (Arlington, MA), Thiruneelakantapillai; Lakshmanan (Boston, MA), Collins; Brian Edward (Arlington, MA)
Assignee:	Momenta Pharmaceuticals, Inc. (Cambridge, MA)
Application Number:	13/720,192
Patent Claims:	1. A method comprising steps of: providing a glycan preparation; subjecting a first portion of the glycan preparation to a first exoglycosidase procedure, wherein the first exoglycosidase procedure comprises sequential treatment of the first portion with at least two exoglycosidases, wherein the first portion is exposed to each exoglycosidase one at a time; subjecting a second portion of the glycan preparation to a second exoglycosidase procedure, wherein the second exoglycosidase procedure comprises simultaneous treatment of the second portion with the at least two exoglycosidases, wherein the second portion is exposed to each exoglycosidase at the same time; characterizing the cleavage products of the first and second exoglycosidase procedure; comparing the characterization of the cleavage products of the first exoglycosidase procedure to the characterization of the cleavage products of the second exoglycosidase procedure; determining at least one glycosylation feature of the glycan preparation; and recording the at least one determined glycosylation feature in a quality control record. 2. A method comprising steps of: providing a glycan preparation; subjecting a first portion of the glycan preparation to a first exoglycosidase procedure, wherein the first exoglycosidase procedure comprises sequential treatment of the first portion with at least two exoglycosidases, wherein the first portion is exposed to each exoglycosidase one at a time; subjecting a second portion of the glycan preparation to a second exoglycosidase procedure, wherein the second exoglycosidase procedure comprises simultaneous treatment of the second portion with the at least two exoglycosidases, wherein the second portion is exposed to each exoglycosidase at the same time; characterizing the cleavage products of the first and second exoglycosidase procedure; comparing the characterization of the cleavage products of the first exoglycosidase procedure to the characterization of the cleavage products of the second exoglycosidase procedure; determining at least one glycosylation feature of the glycan preparation; and comparing the at least one determined glycosylation feature with that of a reference glycan preparation. 3. The method of claim 2, further comprising a step of comparing the at least one determined glycosylation feature with that of a historical record of the reference glycan preparation. 4. A method comprising steps of: providing a glycan preparation from a therapeutic glycoprotein preparation; subjecting a first portion of the glycan preparation to a first exoglycosidase procedure, wherein the first exoglycosidase procedure comprises sequential treatment of the first portion with at least two exoglycosidases, wherein the first portion is exposed to each exoglycosidase one at a time; subjecting a second portion of the glycan preparation to a second exoglycosidase procedure, wherein the second exoglycosidase procedure comprises simultaneous treatment of the second portion with the at least two exoglycosidases, wherein the second portion is exposed to each exoglycosidase at the same time; characterizing the cleavage products of the first and second exoglycosidase procedure; comparing the characterization of the cleavage products of the first exoglycosidase procedure to the characterization cleavage products of the second exoglycosidase procedure; and determining at least one glycosylation feature of the glycan preparation. 5. The method of claim 4, wherein the therapeutic glycoprotein preparation is obtained from a culture of cells producing a therapeutic glycoprotein. 6. The method of claim 5, further comprising a step of comparing the at least one determined glycosylation feature with that of a reference sample. 7. The method of claim 6, wherein the reference sample is a glycan preparation from a different batch of cells producing the therapeutic glycoprotein. 8. The method of claim 6, wherein the reference sample is a glycan preparation from the culture of cells at a different time than the therapeutic glycoprotein preparation. 9. The method of claim 6, further comprising a step of recording the result of the comparing in a quality control record for the therapeutic glycoprotein preparation. 10. The method of claim 6, wherein the at least one determined glycosylation feature is compared with a historical record of the reference sample. 11. The method of claim 4, wherein the therapeutic glycoprotein preparation is an antibody preparation. 12. The method of claim 11, wherein the antibody preparation is a preparation of alemtuzumab, etanercept, adalimumab, abatacept, infliximab, bevacizumab, rituximab, natalizumab, or cetuximab. 13. A method of characterizing a glycan preparation comprising a plurality of protein-linked N-glycans, so that one or more features of the glycan preparation is determined, the method comprising steps of: a) providing a glycan preparation comprising a plurality of glycoproteins, each glycoprotein comprising at least one N-glycan, which N-glycan is linked to a glycoprotein of the plurality; wherein the glycosylation pattern of at least one member of the plurality differs from the glycosylation pattern of at least one other member of the plurality; b) removing at least two N-glycans from at least one glycoprotein of the plurality; c) optionally, isolating removed N-glycans; d) subjecting the removed N-glycans to multiple separate exoglycosidase enzyme treatments, which multiple separate exoglycosidase treatments include: 1) a sequential exoglycosidase enzyme treatment comprising sequentially exposing the preparation-glycans to each of a first and a second exoglycosidase to obtain sequential digestion results; 2) a simultaneous exoglycosidase enzyme treatment comprising simultaneously exposing the preparation-glycans to both of the first and second exoglycosidases to obtain simultaneous digestion results; e) characterizing the cleavage products from each exoglycosidase treatment; and f) optionally, comparing the characterization of the cleavage products to a reference sample. 14. The method of claim 13, further comprising a step of recording the characterized cleavage products in a quality control record. 15. A method for comparing batches of a glycoprotein, the method comprising steps of: a) providing a first glycan preparation from a first batch of the glycoprotein; b) providing a second glycan preparation from a second batch of the glycoprotein; c) subjecting each of the first and second glycan preparations to multiple separate exoglycosidase enzyme treatments, which multiple separate exoglycosidase treatments include: 1) a sequential exoglycosidase enzyme treatment comprising sequentially exposing the glycan preparation to each of a first and a second exoglycosidase to obtain sequential digestion results; and 2) a simultaneous exoglycosidase enzyme treatment comprising simultaneously exposing the glycan preparation to both of the first and second exoglycosidases to obtain simultaneous digestion results; and d) comparing the sequential digestion results with the simultaneous digestion results from the first and second glycan preparations, so that differences are revealed, thereby determining the consistency of the two batches. 16. The method of claim 15, further comprising a step of recording the comparison in a quality control record. 17. A method for monitoring production of a glycoprotein, the method comprising steps of: a) during production of a glycoprotein, removing at least first and second glycan-containing samples from a production system; b) subjecting each of the first and second glycan-containing samples to multiple separate exoglycosidase enzyme treatments, which multiple separate exoglycosidase treatments include: (1) a sequential exoglycosidase enzyme treatment comprising sequentially exposing the glycan-containing sample to each of a first and a second exoglycosidase to obtain sequential digestion results; and (2) a simultaneous exoglycosidase enzyme treatment comprising simultaneously exposing the glycan-containing sample to both of the first and second exoglycosidases to obtain simultaneous digestion results; and c) comparing the sequential digestion results with the simultaneous digestion results from the first and second glycan-containing samples, so that differences are revealed and therefore progress of glycoprotein production is monitored. 18. The method of claim 17, further comprising a step of recording the comparison in a quality control record. 19. A method comprising steps of: providing a glycan preparation; subjecting a first portion of the glycan preparation to a first exoglycosidase procedure, wherein the first exoglycosidase procedure comprises sequential treatment of the first portion with at least two exoglycosidases, wherein the first portion is exposed to each exoglycosidase one at a time; subjecting a second portion of the glycan preparation to a second exoglycosidase procedure, wherein the second exoglycosidase procedure comprises simultaneous treatment of the second portion with the at least two exoglycosidases, wherein the second portion is exposed to each exoglycosidase at the same time; characterizing one or more cleavage products of the first and second exoglycosidase procedure; comparing the characterization of the one or more cleavage products of the first exoglycosidase procedure to the characterization of the one or more cleavage products of the second exoglycosidase procedure; and determining at least one glycosylation feature of the glycan preparation. 20. The method of claim 19, wherein the glycan preparation is from a therapeutic glycoprotein preparation. 21. The method of claim 20, wherein the therapeutic glycoprotein preparation is obtained from a culture of cells producing a therapeutic glycoprotein. 22. The method of claim 20, further comprising a step of comparing the at least one determined glycosylation feature with that of a reference sample. 23. The method of claim 20, further comprising comparing the at least one determined glycosylation feature with a historical record of a reference sample. 24. The method of claim 22, wherein the reference sample is a glycan preparation from a different batch of cells producing the therapeutic glycoprotein. 25. The method of claim 22, wherein the reference sample is a glycan preparation from the culture of cells at a different time than the therapeutic glycoprotein preparation. 26. The method of claim 22, further comprising a step of recording the result of the comparing in a quality control record for the therapeutic glycoprotein preparation. 27. The method of claim 20, wherein the therapeutic glycoprotein preparation is an antibody preparation. 28. The method of claim 27, wherein the antibody preparation is a preparation of alemtuzumab, etanercept, adalimumab, abatacept, infliximab, bevacizumab, rituximab, natalizumab, or cetuximab.

Details for Patent 9,029,081

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Genentech, Inc.	RITUXAN	rituximab	Injection	103705	11/26/1997		2027-04-16
Idec Pharmaceuticals Corp.	RITUXAN	rituximab	Injection	103737	02/19/2002		2027-04-16
Janssen Biotech, Inc.	REMICADE	infliximab	For Injection	103772	08/24/1998		2027-04-16
Immunex Corporation	ENBREL	etanercept	For Injection	103795	11/02/1998		2027-04-16
Immunex Corporation	ENBREL	etanercept	For Injection	103795	05/27/1999		2027-04-16