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Summary for Patent: 8,163,518
|Title:||Microwave assisted deglycosylation of proteins for molecular weight determination by mass spectrometry|
|Abstract:||Methods are presented for microwave assisted, enzymatic deglycosylation of proteins. The rate at which deglycosylation is achieved and without protein degradation facilitates rapid and accurate molecular weight determination by mass spectrometry.|
|Inventor(s):||Arnott; David P. (San Mateo, CA), Lill; Jennie (Pacifica, CA), Sandoval; Wendy N. (San Francisco, CA), Vandlen; Richard L. (Hillsborough, CA)|
|Assignee:||Genetech, Inc. (South San Francisco, CA)|
|Patent Claims:||1. A method of deglycosylation of proteins comprising treating an N-linked glycosylated protein with PNGase F enzyme under microwave radiation from 1 to 20 watts power for
an hour or less, whereby a deglycosylated protein is formed.
2. The method of claim 1 further comprising the step of applying the deglycosylated protein to a desalting media.
3. The method of claim 2 wherein the deglycosylated protein is eluted from the desalting media in a solution comprising water and an organic solvent.
4. The method of claim 1 further comprising the step of analyzing the deglycosylated protein by mass spectrometry.
5. The method of claim 4 wherein the deglycosylated protein is ionized by electrospray ionization into a mass spectrometer.
6. The method of claim 4 wherein the deglycosylated protein is ionized by MALDI.
7. The method of claim 1 wherein PNGase F is a catalytic amount relative to glycosylated protein.
8. The method of claim 7 wherein the molar ratio of PNGase F to glycosylated protein is between 1:10 to 1:1000.
9. The method of claim 1 wherein the glycosylated protein is a phospho-protein.
10. The method of claim 1 wherein the glycosylated protein is an antibody.
11. The method of claim 10 wherein the antibody is an IgG.
12. The method of claim 11 wherein the antibody is trastuzumab or bevacizumab.
13. The method of claim 11 wherein the IgG is conjugated to a detection label.
14. The method of claim 10 wherein the antibody is conjugated to a metal complexing linker.
15. The method of claim 14 wherein the metal complexing linker is selected from DOTA.
16. The method of claim 10 wherein the antibody is conjugated to a drug moiety.
17. The method of claim 1 further comprising the step of analyzing a sugar moiety removed from the glycosylated protein wherein the sugar moiety is analyzed by mass spectrometry.
18. The method of claim 17 further comprising the step of isolating the sugar moiety by high pH anion-exchange chromatography.
|Applicant||Tradename||Biologic Ingredient||Dosage Form||BLA||Number||Approval Date||Patent No.||Assignee||Estimated Patent Expiration||Status||Orphan||Source|
|Genentech||HERCEPTIN||trastuzumab||VIAL; INTRAVENOUS||103792||001||1998-09-25||Start Trial||Genetech, Inc. (South San Francisco, CA)||2039-03-29||RX||Orphan||search|
|Genentech||AVASTIN||bevacizumab||VIAL; INTRAVENOUS||125085||001||2004-02-26||Start Trial||Genetech, Inc. (South San Francisco, CA)||2039-03-29||RX||search|
|Genentech||AVASTIN||bevacizumab||VIAL; INTRAVENOUS||125085||002||2004-02-26||Start Trial||Genetech, Inc. (South San Francisco, CA)||2039-03-29||RX||search|
|Genentech Inc||HERCEPTIN HYLECTA||trastuzumab; hyaluronidase-oysk||INJECTABLE;SUBCUTANEOUS||761106||001||2019-02-28||Start Trial||Genetech, Inc. (South San Francisco, CA)||2039-03-29||RX||search|
|>Applicant||>Tradename||>Biologic Ingredient||>Dosage Form||>BLA||>Number||>Approval Date||>Patent No.||>Assignee||>Estimated Patent Expiration||>Status||>Orphan||>Source|
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