DETAILED ACTION
The amendment filed on 12/11/2025 has been entered and fully considered. Claims 1, 3-10, 12, 15-25 and 27 are pending. Claims 22 have been withdrawn from consideration. Claims 1, 3-10, 12, 15-21, 23-25 and 27 are considered on merits, of which claim 1 and 23 are amended, and Claim 27 is newly added.
Response to Amendment
In response to amendment, the examiner withdraws rejection under 112(b), and maintains 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, 3-10, 12, 15-21, 23-25 and 27 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ohta et al. (WO 2018/034346, IDS) (Ohta) in view of Li et al. (Methods in Molecular Biology, 2018, IDS) (Li).
Regarding claim 1, Ohta teaches a method for the quantification of one or more glycoproteins in a sample, wherein each glycoprotein to be quantified comprises one or more Asn-linked glycans (abstract), said method comprising the steps of:
a. providing a sample comprising said one or more glycoproteins to be quantified (abstract),
b. adding an internal standard to said sample, wherein said internal standard comprises a variant form of each of said one or more glycoproteins to be quantified, wherein said variant form is a form containing only core Asn-linked GlcNAc moieties, each core Asn-linked GlcNAc moiety being an N-acetylglycosamine moiety that is directly attached to an Asn acceptor residue in the polypeptide chain of said variant (abstract, par [0013]); and
c. quantifying said one or more glycoproteins by comparison with the internal standard, wherein the quantifying is performed using mass spectrometry (abstract, par [0013]), and
wherein prior to step c. the sample is treated with an enzyme, wherein said enzyme is capable of fully removing Asn-linked glycans from said one or more glycoproteins, but is not capable of removing core Asn-linked GlcNAc moieties from said variant form (par [0002][0013]).
Ohta clearly establishes that enzymatic removal of N-linked glycans to leave a core GlcNAc residue is a known and routine preparatory step in glycoprotein analysis, used to facilitate accurate LC-MS comparison between glycosylated and deglycosylated forms (par [0002] [0013]). The fact that Ohta employs endoglycosidases rather than PNGase F in a specific embodiment does not amount to a teaching away from alternative enzymes capable of fully removing Asn-linked glycans while preserving the GlcNAc stub, as now recited in claim 1. Applicant’s amended claim does not require PNGase F specifically, and therefore encompasses routine enzymatic treatments well within the scope of Ohta’s disclosure.
Ohta does not specifically teach that wherein no fucose moieties are linked to said core GlcNAc moieties. However, Ohta notes that “one fucose (Fus) residue may be bound to the GlcNAc on the peptide” (par [0013]). “a glycosylation position determination step in which an MS/MS ion search or de novo sequence analysis is performed taking into account GlcNAc modification or GlcNAc-Fuc modification of the Asn residue to determine the glycan binding position on the glycopeptide” (par [0013]). Here, Ohta teaches that special care needs to be taken to deal with the heterogeneity caused by possible Core fucosylation in the spectral analysis.
Li teaches removing fucose from said core GlcNAc moieties by α-fucosidase (Fig. 1) and fucose removal is achievable and desirable to eliminate heterogeneity (albeit in the therapeutic context).
A person of skill would recognize that the same principle of reducing heterogeneity applies in the analytical context of internal standards, because homogeneity improves clarity of comparison.
Thus, it would have been obvious to one of ordinary skill in the art to remove fucose from said core GlcNAc moieties by α-fucosidase as taught by Li, in order to simplify the spectral analysis. The result is predictable.
The Supreme Court in KSR International Co. v. Teleflex Inc., 550 U.S., 82 USPQ2d 1385, 1395-97 (2007) identified a number of rationales to support a conclusion of obviousness which are consistent with the proper “functional approach” to the determination of obviousness as laid down in Graham. One of the rationales is applying a known technique to a known device (method, or product) ready for improvement to yield predictable results (see MPEP 2143). In this instance, the known technique is removing fucose from said core GlcNAc moieties by α-fucosidase as taught by Li; the known method is the quantification of one or more glycoproteins in a sample, wherein each glycoprotein to be quantified comprises one or more Asn-linked glycans (abstract) as taught by Ohta. Ready to simplify the spectral analysis, and the combination yielded nothing more than predictable results to one of ordinary skill in the art. (see KSR, 550 U.S. at 416, 82 USPQ2d at 1395) (see MPEP 2143).
Regarding claim 3, Ohta teaches that wherein said enzyme treatment is performed prior to step b (par [0002][0013]).
Regarding claim 4, Ohta fairly suggests that wherein said enzyme treatment is performed after addition of the internal standard in step b (par [0002] [0013]).
Regarding claim 5, Ohta teaches that wherein said enzyme is PNGase F (par [0002]).
Regarding claim 6, Ohta teaches that wherein the method prior to step b. comprises treating the sample with an enzyme capable of fully removing Asn-linked glycans from said one or more glycoproteins (par [0002]).
Regarding claim 7, Ohta teaches that wherein the quantification in step c. is performed using mass spectrometry (par [0002]).
Regarding claim 8, Ohta teaches that wherein the quantification in step c. is performed using a combination of mass spectrometry and a non-mass-based separation technique (par [0002]).
Regarding claim 9, Ohta teaches that wherein the quantification in step c. is performed using liquid chromatography-mass spectrometry (LC-MS) (par [0002]).
Regarding claim 10, it is conventional for a method does not comprise a step of proteolytically digesting, or otherwise fragmenting, the amino acid chain of the glycoproteins to be quantified.
Regarding claim 12, Ohta teaches that wherein the one or more glycoproteins to be quantified are antibodies (par [0012]).
Regarding claim 15, Ohta teaches that wherein at least one of the one or more glycoproteins to be quantified contains fucose on core Asn-linked GlcNAc moieties (par [0013]).
Regarding claim 16, Ohta teaches that wherein the sample is a cell culture sample (par [0064]).
Regarding claim 17, it is conventional that the sample comprises purified recombinantly produced glycoproteins.
Regarding claim 18, The Courts have held that to provide a mechanical or automatic means to replace manual activity, which accomplishes the same result, is within the ambit of a person of ordinary skill in the art. (See In re Venner, 120 USPQ 192 CCPA 1958) (see MPEP section 2144.04). Therefore, it would have been obvious to one of ordinary skill in the art to automate the procedure, wherein steps b. and c. of the method are performed in an automated manner, preferably wherein steps a., b. and c. are performed in an automated manner.
Regarding claim 19, Ohta teaches that wherein the sample is a blood sample, plasma sample or serum sample (par [0104]).
Regarding claim 20, Ohta teaches a process for monitoring production of glycoproteins in a cell culture, said process comprising culturing host cells producing said glycoproteins (par [0064]) and performing the method according to claim 16 (par [0064]).
Regarding claim 21, Ohta teaches a process for the quality control of purified cell culture produced glycoproteins (par [0064]), said process comprising performing the method according to claim 16 (par [0064]). it is conventional that the sample comprises purified recombinantly produced glycoproteins.
Regarding claim 23, Ohta teaches a method for the quantification of one or more glycoproteins in a sample (abstract), wherein each glycoprotein to be quantified comprises one or more Asn-linked glycans (abstract), said method comprising the steps of:
a. preparing an internal standard by treating said one or more glycoproteins with one or more enzymes (endo-β-N-acetylglucosaminidase) to obtain a variant form of each of said one or more glycoproteins, wherein said variant form is a form containing only core Asn-linked GlcNAc moieties, each core Asn-linked GlcNAc moiety being an N-acetylglycosamine moiety that is directly attached to an Asn acceptor residue in the polypeptide chain of said variant (par [0013]),
b. providing a sample comprising said one or more glycoproteins to be quantified (par [0013]),
c. adding said internal standard to said sample (par [0013]), and
d. quantifying said one or more glycoproteins by comparison with the internal standard, wherein the quantifying is performed using mass spectrometry (par [0013]),
wherein prior to step d. the sample is treated with an enzyme (PNGase F), wherein said enzyme is capable of fully removing Asn-linked glycans from said one or more glycoproteins, but is not capable of removing core Asn-linked GlcNAc moieties from said variant form (par [0002][0013]).
Ohta clearly establishes that enzymatic removal of N-linked glycans to leave a core GlcNAc residue is a known and routine preparatory step in glycoprotein analysis, used to facilitate accurate LC-MS comparison between glycosylated and deglycosylated forms (par [0002] [0005]). The fact that Ohta employs endoglycosidases rather than PNGase F in a specific embodiment does not amount to a teaching away from alternative enzymes capable of fully removing Asn-linked glycans while preserving the GlcNAc stub, as now recited in claim 23. Applicant’s amended claim does not require PNGase F specifically, and therefore encompasses routine enzymatic treatments well within the scope of Ohta’s disclosure.
Ohta does not specifically teach that wherein no fucose moieties are linked to said core GlcNAc moieties. However, Ohta notes that “one fucose (Fus) residue may be bound to the GlcNAc on the peptide” (par [0013]). “a glycosylation position determination step in which an MS/MS ion search or de novo sequence analysis is performed taking into account GlcNAc modification or GlcNAc-Fuc modification of the Asn residue to determine the glycan binding position on the glycopeptide” (par [0013]). Here, Ohta teaches that special care needs to be taken to deal with the heterogeneity caused by Core fucosylation in the spectral analysis.
Li teaches removing fucose from said core GlcNAc moieties by α-fucosidase (Fig. 1). Thus, it would have been obvious to one of ordinary skill in the art to remove fucose from said core GlcNAc moieties by α-fucosidase, in order to simplify the spectral analysis. The result is predictable.
The Supreme Court in KSR International Co. v. Teleflex Inc., 550 U.S., 82 USPQ2d 1385, 1395-97 (2007) identified a number of rationales to support a conclusion of obviousness which are consistent with the proper “functional approach” to the determination of obviousness as laid down in Graham. One of the rationales is applying a known technique to a known device (method, or product) ready for improvement to yield predictable results (see MPEP 2143). In this instance, the known technique is removing fucose from said core GlcNAc moieties by α-fucosidase as taught by Li; the known method is the quantification of one or more glycoproteins in a sample, wherein each glycoprotein to be quantified comprises one or more Asn-linked glycans (abstract) as taught by Ohta. Ready to simplify the spectral analysis, and the combination yielded nothing more than predictable results to one of ordinary skill in the art. (see KSR, 550 U.S. at 416, 82 USPQ2d at 1395) (see MPEP 2143).
Li teaches enzymatic removal of core fucose from GlcNAc residues using α-fucosidase and establishes that defucosylation produces a more homogeneous glycoprotein population. Although Li is discussed in a therapeutic antibody context, the teaching that core fucose can be selectively removed from GlcNAc moieties is directly applicable to analytical internal standards. A person of ordinary skill in the art would have recognized that reducing glycan heterogeneity is equally beneficial in analytical mass spectrometry, where homogeneity improves clarity and reproducibility of quantitative comparisons.
Thus, Li provides a clear motivation and enabling teaching to modify Ohta’s GlcNAc-stubbed internal standards so that no fucose moieties are linked to the core GlcNAc residues, as now recited in amended claim 1
Regarding claim 24, Ohta teaches that wherein said treatment with one or more enzymes in step a. comprises treatment with an endo-β-N-acetylglucosaminidase, such as EndoS2 or EndoS (par [0013]).
Regarding claim 25, Ohta teaches that wherein said treatment with one or more enzymes in step a. comprises treatment with an endo-β-N-acetylglucosaminidase, such as EndoS2 or EndoS (par [0013]) and Li teaches a fucosidase, capable of hydrolyzing alpha-1,6-linkage from an Asn-linked fucose-alpha-1,6-GlcNAc moiety (Fig. 1).
Regarding claim 27, Ohta teaches that wherein the quantification in step c. is performed using reverse-phase-based LC-MS (par [0075]).
Response to Arguments
Applicant's arguments filed12/11/2025 have been fully considered but they are not persuasive.
Applicant amended claim 1 to recite that (i) quantification is performed using mass spectrometry and (ii) prior to quantification the sample is treated with an enzyme capable of fully removing Asn-linked glycans from the glycoproteins, but not capable of removing core Asn-linked GlcNAc moieties from the variant form.
These limitations do not overcome the obviousness rejection.
Ohta teaches generating glycoprotein variants in which N-linked glycans are enzymatically cleaved to leave a single GlcNAc residue attached to Asn, and explicitly teaches using such sugar-chain-cleaved glycoproteins as internal standards for LC-MS-based quantitative analysis (par [0013]).
With respect to enzymatic treatment, Applicant argues that Ohta does not teach or suggest treating the sample with an enzyme capable of fully removing Asn-linked glycans and that PNGase F treatment would render Ohta’s method inoperable for its intended purpose. This argument is not persuasive.
Ohta clearly establishes that enzymatic removal of N-linked glycans to leave a core GlcNAc residue is a known and routine preparatory step in glycoprotein analysis, used to facilitate accurate LC-MS comparison between glycosylated and deglycosylated forms (par [0002][0013]). The fact that Ohta employs endoglycosidases rather than PNGase F in a specific embodiment does not amount to a teaching away from alternative enzymes capable of fully removing Asn-linked glycans while preserving the GlcNAc stub, as now recited in claim 1 (par [0002]).
Applicant contends that modifying Ohta to include full deglycosylation of the sample would render Ohta’s method inoperable, relying on In re Gordon. The Examiner disagrees.
The pending claims are not limited to Ohta’s two-stage “preliminary analysis / main analysis” workflow. Rather, claim 1 broadly recites adding an internal standard and performing mass-spectrometric quantification after enzymatic treatment. Ohta’s disclosure of sugar-chain cleavage to obtain GlcNAc-stubbed glycoproteins directly supports this approach. The proposed combination does not negate the function of the method, but instead represents a predictable variation of enzymatic deglycosylation strategies long used in glycoprotein mass spectrometry.
Accordingly, the modification would not render the prior art unsatisfactory for its intended purpose, but would instead yield the predictable result of simplified and comparable mass spectra.
Applicant further argues that Li does not cure the alleged deficiencies of Ohta. This argument is not persuasive.
Li teaches enzymatic removal of core fucose from GlcNAc residues using α-fucosidase and establishes that defucosylation produces a more homogeneous glycoprotein population. Although Li is discussed in a therapeutic antibody context, the teaching that core fucose can be selectively removed from GlcNAc moieties is directly applicable to analytical internal standards. A person of ordinary skill in the art would have recognized that reducing glycan heterogeneity is equally beneficial in analytical mass spectrometry, where homogeneity improves clarity and reproducibility of quantitative comparisons.
Thus, Li provides a clear motivation and enabling teaching to modify Ohta’s GlcNAc-stubbed internal standards so that no fucose moieties are linked to the core GlcNAc residues, as now recited in amended claim 1
Conclusion
THIS ACTION IS MADE FINAL. 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