DETAILED ACTION
The amendment filed on 08/28/2026 has been entered and fully considered. Claims 1-3, 5 and 7-11 are pending, of which claim 1 and 10-11 are amended.
Response to Amendment
In response to amendment, the examiner modifies rejection over the prior art established in the previous Office action.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1-3, 5 and 7-11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Pett et al. (Angew. Chem. Int. Ed. 2018) (Pett) in view of Morimoto et al. (Bioinformatics, 2015) (Morimoto) and Riley et al. (Nature Communications, 2019) (Riley).
Regarding claim 1, Pett teaches a mass spectrometry method comprising:
preparing a sample containing a glycan including a plurality of sialic acids (page 9322, par 1);
generating a plurality of oxonium ions by ionizing the glycan having the plurality of sialic acids modified in the performing step and dissociating the ionized glycan under the same dissociation condition, wherein the plurality of oxonium ions include a first oxonium ion derived from the first modified form and a second oxonium ion derived from the second modified form, and a mass of the first oxonium ion is different from a mass of the second oxonium ion (Fig. 1B, page 9321, par 0);
detecting, in a first mass spectrometry intensities of the first oxonium ion and the second oxonium ion included in the plurality of oxonium ions generated in the generating step (Fig. 1B, page 9321, par 0);
calculating relative values of intensities of the detected first oxonium ion and second oxonium ion (Fig. 1C, 1D, page 9321, par 0); and
calculating a ratio of a number of sialic acids modified into the first modified form corresponding to the first oxonium ion and a number of sialic acids modified into the second modified form corresponding to the second oxonium ion in the glycan contained in the sample based on the relative values (Fig. 1C, 1D, page 9321).
Pett repeatedly teaches quantitative or semi-quantitative calculations of oxonium-ion intensity ratios, including:
MS² LacNAc-to-Neu5Ac oxonium-ion intensity ratio (L/N) (page 9321, Fig. 1C, 1D);
CF-normalized ratios (Ln/Nn) used to distinguish glycopeptide isomers quantitatively (page 9321, left column);
Measured numerical Ln/Nn ratios for each glycopeptide isomer in Figures 2B and 3D–F (pages 9322–9323);
Pett explicitly describes “threshold values” and “ratio differences” between α2,3 and α2,6 isomers (page 9322, par 1-2).
These quantitative intensity ratios are directly analogous to the claimed “relative values of intensities of the plurality of oxonium ions” and are used for isomer characterization. Pett therefore teaches calculating ratios of oxonium-ion intensities, even though the article describes the use as “differentiation.” Quantitative differentiation is still quantitative analysis.
Pett also fairly suggests that sialic acids can be modified differently, in order to generate a substantial mass difference (More recent strategies utilize derivatization steps to form esters/amides of a2,6-linked sialic acids, while a2,3-linked sialic acids form lactones, which generate a substantial mass difference.[7]” (page 9320, par 2). It would have been obvious to one of ordinary skill in the art to perform a linkage-specific modification on the plurality of sialic acids included in the glycan to classify the plurality of sialic acids into at least a first group and a second group depending on linkage types thereof, wherein the first group is modified into a first modified form having a first mass and the second group is modified into a second modified form having a second mass different from the first mass as suggested by Pett, in order to obtain differential mass of ions of linkage type sialic acid for easier analysis.
The court has ruled that a reference must be considered for all that it teaches, and not limited to its preferred embodiments. Applied Materials, 692 F.3d at 1298; Merck, 874 F.2d at 807. Thus, Pett’s discussion of known prior approaches — including derivatization-based differentiation of α2,3- and α2,6-linked sialic acids — is properly considered part of the prior art and supports that detecting diagnostic ions from differently modified sialic acids and comparing their relative intensities would have been obvious to a POSITA.
Pett does not teach “searching for a plurality of glycan structure candidates based on a mass-to-charge ratio of the glycan detected in the first mass spectrometry.”
Morimoto, however, teaches a glycan database-search program for identifying N-glycan structures from mass-spectrometric data (p. 2217). Morimoto states that its algorithm calculates m/z values of fragment ions and searches glycan structural databases for structures matching measured MS/MS spectra (p. 2217). More particularly, Morimoto teaches:
“Search for matched precursor ions and diagnostic ions in the MS/MS spectrum by comparing m/z values.” (p. 2218).
Morimoto further teaches that, following the database search, “candidates for the target glycan structure are listed” (p. 2218), and Figure 1 expressly shows a plurality of predicted candidate glycan structures having associated scores. Thus, Morimoto teaches searching for a plurality of glycan structure candidates based on measured mass-to-charge information.
It would have been obvious to one of ordinary skill in the art to modify Pett's mass-spectrometric glycan analysis to employ Morimoto's glycan-structure database searching technique in order to facilitate automated identification of glycan structures from Pett's measured mass-spectrometric data. Pett and Morimoto are in the same field of endeavor of mass-spectrometric glycan characterization, and Morimoto expressly teaches that its GlycanAnalysis software was developed to support the identification of glycan structures from MS data (pp. 2217-2218).
Regarding “identifying a structure of the glycan by narrowing down the plurality of glycan structure candidates using the ratio of the number of sialic acids as a constraint condition,” Morimoto teaches narrowing candidate glycan structures using diagnostic MS information. Morimoto teaches that when diagnostic D-ions are detected, the corresponding glycan structure is added to the result list, matching ions are counted, and a score is calculated (p. 2218). Morimoto further teaches that its retrieval process could be improved by incorporating “the intensity of peaks and its related data” (p. 2218).
Riley provides further express motivation to use an oxonium-ion intensity ratio as such a constraint. Riley teaches that applying a threshold to a Neu5Ac/HexNAc oxonium-ion ratio eliminated 97% and 99% of AI-ETD and HCD spectra, respectively, that had been incorrectly assigned identifications lacking Neu5Ac, while retaining 83% and 88% of identifications containing sialylated glycans (p. 3). Riley expressly concludes: “Such a calculation could be considered in future glycopeptide-centric search algorithms” (p. 3).
Therefore, it would have been obvious to one of ordinary skill in the art to use Pett's calculated linkage-specific sialic-acid ratio as an additional constraint for narrowing Morimoto's plurality of candidate glycan structures. Riley expressly teaches using an oxonium-ion intensity ratio to filter identifications and expressly suggests incorporating such a calculation into search algorithms. The modification would have been motivated to reduce erroneous glycan identifications and improve the reliability of glycan structure identification, as demonstrated by Riley (p. 3).
Regarding claim 2, Pett fairly suggests that wherein the plurality of sialic acids are amide-modified (page 9320, par 2).
Regarding claim 3, Pett teaches that wherein the first mass spectrometry is performed by tandem mass spectrometry (HCD-MS2) in two or more stages (page 9321, par 0).
Regarding claim 5, Pett fairly suggests that wherein a2,3-sialic acid, a2,8-sialic acid or a2,9-sialic acid, and a2,6-sialic acid are each modified differently (page 9320, par 2).
Regarding claim 7, Pett teaches that the mass spectrometry method comprising:
performing chromatography of the sample before the first mass spectrometry (C18 nano-LC-MS) (page 9321, par 0).
Regarding claim 8, Pett teaches that the mass spectrometry method comprising:
outputting an extracted ion chromatogram including a peak corresponding to at least one of the plurality of oxonium ions (Fig. 3, page 9322, par 2).
Regarding claim 9, Pett teaches that the mass spectrometry method comprising:
performing mass separation of ions generated by ionization of the sample based on scanned m/z, performing dissociation of the mass-separated ions, and performing second mass spectrometry for detecting oxonium ions from the ions generated by the dissociation (Fig. 1-3); and
obtaining at least one of a time during which a molecule containing a glycan from which the detected oxonium ion is derived is eluted in the chromatography and a mass of the molecule, based on a result of the second mass spectrometry (Fig. 3).
Regarding claim 10, Pett discloses a mass spectrometry apparatus comprising:
a measurement unit configured to generate a plurality of oxonium ions by ionizing a glycan having a plurality of sialic acids each in a linkage type-specific manner and dissociating the ionized glycan under the same dissociation condition, wherein the plurality of oxonium ions include a first oxonium ion derived from a first form having a first mass and a second oxonium ion derived from a second form having a second mass different from the first mass, and a mass of the first oxonium ion is different from a mass of the second oxomum ion (page 9321, par 0);
a data acquisition portion configured to acquire data obtained by detecting, in a first mass spectrometry intensities of the first oxonium ion and the second oxonium ion included in the plurality of oxonium ions (page 9321, par 0); and
a calculation portion configured to calculate relative values of intensities of the detected first oxonium ion and second oxonium ion based on the data (page 9321, par 0), and calculate a ratio of a number of sialic acids in the first form corresponding to the first oxonium ion and a number of sialic acids in the second form corresponding to the second oxonium ion in the glycan based on the relative values (Fig. 1, page 9321, par 0).
Pett repeatedly teaches quantitative or semi-quantitative calculations of oxonium-ion intensity ratios, including:
MS² LacNAc-to-Neu5Ac oxonium-ion intensity ratio (L/N) (page 9321, Fig. 1C, 1D);
CF-normalized ratios (Ln/Nn) used to distinguish glycopeptide isomers quantitatively (page 9321, left column);
Measured numerical Ln/Nn ratios for each glycopeptide isomer in Figures 2B and 3D–F (pages 9322–9323);
Pett explicitly describes “threshold values” and “ratio differences” between α2,3 and α2,6 isomers (page 9322, par 1-2).
These quantitative intensity ratios are directly analogous to the claimed “relative values of intensities of the plurality of oxonium ions” and are used for isomer characterization. Pett therefore teaches calculating ratios of oxonium-ion intensities, even though the article describes the use as “differentiation.” Quantitative differentiation is still quantitative analysis.
Pett also fairly suggests that sialic acids can be modified in a linkage type-specific manner, in order to generate a substantial mass difference (More recent strategies utilize derivatization steps to form esters/amides of a2,6-linked sialic acids, while a2,3-linked sialic acids form lactones, which generate a substantial mass difference.[7]” (page 9320, par 2). It would have been obvious to one of ordinary skill in the art to modify a2,6-linked sialic acids and a2,3-linked sialic acids in a linkage type-specific manner, in order to obtain differential mass of ions of linkage type sialic acid for easier analysis.
Pett does not teach that the calculation portion is further configured to search for a plurality of glycan structure candidates based on a mass-to-charge ratio of the glycan and identify a structure of the glycan by narrowing down the plurality of glycan structure candidates using the ratio of the number of sialic acids as a constraint condition, as presently recited in claim 10.
Morimoto teaches computer-implemented software that reads measured mass-spectrometric raw data, detects peaks and intensities, searches for matched precursor and diagnostic ions by comparing m/z values, and returns candidate glycan structures (p. 2218).
It would have been obvious to configure the calculation portion of Pett's mass-spectrometry apparatus to implement Morimoto's database-search functionality in order to automate identification of glycan structures from the acquired mass-spectrometric data.
It would further have been obvious to configure that calculation portion to use Pett's sialic-acid ratio as a constraint in narrowing the candidates in view of Riley, because Riley teaches using an oxonium-ion intensity-ratio threshold to eliminate erroneous glycan identifications and expressly suggests incorporating the ratio calculation into glycopeptide search algorithms (p. 3).
Regarding claim 11, Pett discloses a non-transitory computer readable medium containing a program for making a processor perform
a generating process of generating a plurality of oxonium ions by ionizing a glycan having a plurality of sialic acids each modified in a linkage type-specific manner and dissociating the ionized glycan under the same dissociation condition, wherein the plurality of oxonium ions include a first oxonium ion derived from a first modified form having a first mass and a second oxonium ion derived from a second modified form having a second mass different from the first mass, and a mass of the first oxonium ion is different from a mass of the second oxomum ion (page 9321, par 0);
a data acquisition process of acquiring data obtained by detecting intensities of the first oxonium ion and the second oxonium ion included in the plurality of oxonium ions generated in the generating process (Fig. 1B, page 9321, par 0); and
a calculation process of calculating relative values of intensities of detected first oxonium ion and second oxonium ion based on the data, and calculating a ratio of a number of sialic acids in the first form corresponding to the first oxonium ion and a number of sialic acids in the second form corresponding to the second oxonium ion in the glycan based on the relative values (Fig. 1C, 1D, page 9321, par 0).
Pett also fairly suggests that sialic acids can be modified in a linkage type-specific manner, in order to generate a substantial mass difference (More recent strategies utilize derivatization steps to form esters/amides of a2,6-linked sialic acids, while a2,3-linked sialic acids form lactones, which generate a substantial mass difference.[7]” (page 9320, par 2). It would have been obvious to one of ordinary skill in the art to modify a2,6-linked sialic acids and a2,3-linked sialic acids in a linkage type-specific manner, in order to obtain differential mass of ions of linkage type sialic acid for easier analysis.
Morimoto teaches implementing glycan-structure identification as computer software. Morimoto teaches that GlycanAnalysis operates as a plug-in for the Mass++ mass-spectrum analysis program (pp. 2217-2218) and specifically states that “GlycanAnalysis is implemented in C++” (p. 2218). Morimoto's program calculates m/z information, processes measured MS data, performs a database search, and outputs candidate glycan structures (pp. 2217-2218).
It would have been obvious to one of ordinary skill in the art to implement the combined Pett-Morimoto analytical method as processor-executable instructions stored on a non-transitory computer-readable medium, because Morimoto expressly implements the glycan-search method as executable computer software, and storage of such executable instructions on a non-transitory computer-readable medium would have been a conventional and predictable implementation of the disclosed software.
For the reasons discussed regarding claim 1, it would have been obvious for the program to calculate the relative oxonium-ion intensities and corresponding ratio of the differently modified sialic acids, search for a plurality of candidate glycan structures based on measured m/z information using Morimoto's database-search technique, and identify the glycan structure by narrowing those candidates using the calculated sialic-acid ratio as a constraint. Claim 11 recites these functions in the presently amended claim.
Riley provides an express reason for the latter modification because Riley demonstrates that an oxonium-ion intensity-ratio threshold substantially eliminates erroneous identifications and expressly teaches that the ratio calculation could be incorporated into glycopeptide-centric search algorithms (p. 3).
Accordingly, Pett in view of Morimoto and Riley renders claim 11 obvious.
Response to Arguments
Applicant’s arguments with respect to claim(s) 1 have been considered but are moot in view of new ground of rejection.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to XIAOYUN R XU, Ph. D. whose telephone number is (571)270-5560. The examiner can normally be reached M-F 8am-5pm.
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/XIAOYUN R XU, Ph.D./ Primary Examiner, Art Unit 1797
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