Claims for Patent: 9,964,545

Summary for Patent: 9,964,545

Title:	Methods for quantifying polypeptides using mass spectrometry
Abstract:	A method for identifying a polypeptide a specimen can include (i) treating a specimen suspected of including an insulin with a base; (ii) extracting a first fraction of the treated specimen by solid phase extraction using a mixed mode or polymeric reversed-phase media and a first solvent including an acid; (iii) separating a component of the first fraction by liquid chromatography using a chromatographic surface including a hydrophobic surface group and one or more ionizable modifiers, and a second solvent including an acid; and (iv) analyzing the component of the first fraction by mass spectroscopy, thereby identifying the polypeptide, if present, using a signal corresponding to a sequence fragment ion from the polypeptide. The signal can correspond to an intact multiply charged precursor fragment selected in a first quadrupole and its corresponding sequence fragment ion selected in a final quadrupole.
Inventor(s):	Chambers; Erin E. (North Brookfield, MA), Stapels; Martha (Bellingham, MA), Mather; Joanne (Millis, MA)
Assignee:	WATERS TECHNOLOGIES CORPORATION (Milford, MA)
Application Number:	15/447,838
Patent Claims:	1. A method for identifying a polypeptide in a specimen, the method comprising: treating a specimen comprising an amount of a polypeptide with a base to form a treated specimen; extracting a first fraction of the treated specimen by solid phase extraction using a mixed mode or reversed-phase media and a first solvent comprising an acid; separating a component of the first fraction by liquid chromatography using a chromatographic surface material comprising a hydrophobic surface group and one or more ionizable modifiers, and a second solvent comprising an acid, wherein the component of the first fraction comprises undigested polypeptide; and analyzing the component of the first fraction by mass spectroscopy, thereby identifying the polypeptide-using a signal corresponding to a sequence fragment ion from the polypeptide. 2. The method of claim 1, further comprising quantifying the amount of the polypeptide using the signal corresponding to the sequence fragment ion from the polypeptide, wherein the signal corresponds to an intact multiply charged precursor fragment selected in a first quadrupole and its corresponding sequence fragment ion selected in a final quadrupole, and wherein the sequence fragment ion exhibits an m/z>800. 3. The method of claim 1, wherein the hydrophobic surface group comprises a carbon bonded phase. 4. The method of claim 1, wherein the hydrophobic surface group comprises an embedded polar group. 5. The method of claim 1, wherein liquid chromatography uses a chromatographic core material in addition to the chromatographic surface material. 6. The method of claim 5, wherein the chromatographic core material comprises a silica material. 7. The method of claim 5, wherein the chromatographic core material comprises a hybrid inorganic/organic material. 8. The method of claim 5, wherein the chromatographic core material comprises a superficially porous material. 9. The method of claim 5, wherein the chromatographic core material comprises a monolith. 10. The method of claim 1, wherein the base comprises 2-amino-2-hydroxymethyl-propane-1,3-diol. 11. The method of claim 1, wherein treating the specimen further comprises an organic precipitation. 12. The method of claim 1, wherein the mixed mode media comprises ion exchange moieties and reverse phase moieties. 13. The method of claim 1, wherein the first solvent comprises acetic acid. 14. The method of claim 1, wherein the second solvent comprises formic acid. 15. The method of claim 1, wherein the component of the first fraction is analyzed by triple quadrupole mass spectrometry. 16. The method of claim 15, wherein the component of the first fraction is analyzed by triple quadrupole mass spectrometry carried out in positive electrospray ionization mode. 17. The method of claim 1, wherein the polypeptide has a detection limit of 0.25 ng/mL or less. 18. The method of claim 1, wherein the signal corresponds to an intact multiply charged precursor fragment selected in a first quadrupole and its corresponding sequence fragment ion selected in a final quadrupole. 19. The method of claim 1, wherein analyzing the component of the first fraction by mass spectroscopy can resolve any two or more of insulin glargine, insulin detemir, insulin aspart, insulin glulisine, human insulin, or a derivative or analog thereof. 20. The method of claim 1, wherein analyzing the component of the first fraction by mass spectroscopy can resolve any two or more of exenatide, hepcidin, teriparatide, enfuvirtide, calcitonin, brain natriuretic peptide (BNP), amyloid beta peptides, GLP-1, glucagon, bombesin, or a derivative or analog thereof. 21. A method for assessing the bioequivalence of a first polypeptide and a second polypeptide, the method comprising: obtaining a specimen from a bioequivalence assay for a first polypeptide; treating the specimen with a base to form a treated specimen; extracting a first fraction of the treated specimen by solid phase extraction using a mixed mode or reversed-phase media and a first solvent comprising an acid; separating a component of the first fraction by liquid chromatography using a chromatographic surface material comprising a hydrophobic surface group and one or more ionizable modifiers, and a second solvent comprising an acid, wherein the component of the first fraction comprises undigested polypeptide; analyzing the component of the first fraction by mass spectroscopy, thereby quantifying the first polypeptide using a signal corresponding to a sequence fragment ion from the first polypeptide; and comparing the quantity of the first polypeptide to a quantity of the second polypeptide expected in a bioequivalence assay for a first polypeptide, thereby determining if the first polypeptide and second polypeptide are bioequivalent. 22. The method of claim 21, wherein bioequivalence is determined based upon an absence of a significant difference in rate and extent to which the first polypeptide and second polypeptide become available at a site of drug action when administered at an identical molar dose under similar conditions in the bioequivalence assay. 23. The method of claim 21, wherein the bioequivalence assay comprises a pharmacokinetic study. 24. The method of claim 21, wherein the bioequivalence assay comprises a pharmacodynamics study. 25. The method of claim 21, wherein the bioequivalence assay comprises a clinical trial. 26. The method of claim 21, wherein the bioequivalence assay comprises an in vitro test. 27. The method of claim 21, wherein the first polypeptide and the second polypeptide are each independently insulin glargine, insulin detemir, insulin aspart, insulin glulisine, human insulin, or a derivative or analog thereof. 28. The method of claim 21, wherein the first polypeptide and the second polypeptide are each independently exenatide, hepcidin, teriparatide, enfuvirtide, calcitonin, brain natriuretic peptide (BNP), amyloid beta peptides, GLP-1, glucagon, bombesin, or a derivative or analog thereof. 29. The method of claim 21, wherein liquid chromatography uses a chromatographic core material in addition to the chromatographic surface material. 30. The method of claim 29, wherein the chromatographic core material comprises a hybrid inorganic/organic material.

Details for Patent 9,964,545

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Novo Nordisk Inc.	NOVOLOG	insulin aspart	Injection	020986	06/07/2000		2032-04-18
Novo Nordisk Inc.	NOVOLOG	insulin aspart	Injection	020986	01/19/2001		2032-04-18
Novo Nordisk Inc.	NOVOLOG	insulin aspart	Injection	020986	04/23/2004		2032-04-18