DETAILED 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 .
Response to Amendment
The Amendment filed 07/30/2026 has been entered. Claims 1, 7, and 16 have been amended. Claim 15 is cancelled. Claims 1-14, 16-18 remain pending in the application and are examined herein.
Status of Objections and Rejections
The objections to the claims have been withdrawn in view of Applicant's amendment.
The rejections of claims 1, 7, and 16 under 35 USC 112(b) are withdrawn in view of Applicant's amendment. The rejection of claims 2-6, 8-14, and 17-18 are withdrawn based on their dependency of all of the limitations of claim 1.
The rejection of claims 1-10,13-14, 16-18 under 35 U.S.C. 103 as being unpatentable over Wang, Birdsall, and Waters is withdrawn in view of Applicant's amendment. The rejection of claims 11-12 are withdrawn based on their dependency of all of the limitations of claim 1.
New grounds of rejection under 35 U.S.C. 103 are necessitated by the amendments.
Response to Arguments
Applicant’s arguments, see pages 4-9, filed 07/30/2026, with respect to claim(s) 1-10,13-14, 16-18 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-10,13-14, 16-18 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (US 20190234964 A1), in view of Amenta (US 20140038906 A1), as evidenced by Wolff et al. (“Distinguishing Glucuronic from Iduronic Acid in Glycosaminoglycan Tetrasaccharides by using Electron Detachment Dissociation”) and Ibanez et al. (“Analytical method for the determination of organic acids in dilute acid pretreated biomass hydrolysate by liquid chromatography-time-of-flight mass spectrometry”), and Birdsall (US 20210255196 A1, entitled to the EFD of 20201013 by provisional application, US 63091169), as evidenced by supporting reference Waters (“ACQUITY UPLC Peptide CSH C18, 130 Å, 1.7 μm and XP 2.5 μm Columns and ACQUITY PREMIER Peptide CSH C18, 130 Å, 1.7 μm Columns”; October 2020).
Regarding claim 1, Wang teaches a method for determining the relative distribution of glucuronylation of a population of polypeptides (“a method for identifying glucuronylation of a protein drug product,” wherein the drug product is a monoclonal antibody which is a population of polypeptides including Fc and Fab2 polypeptide fragments; [0007]; See relative abundances in Figs. 4B-7D which show a distribution of glucuronylation), the method comprising:
a) contacting the population of polypeptides with a protease to generate peptides (“The dried and denatured acidic fractions are alkylated and then digested with trypsin to form a sample,” which would naturally generate peptides from Fc and Fab2 polypeptide fragments; [0007]);
b) contacting the peptides to a chromatographic support comprising a hydrophobic stationary phase (“samples are then subjected to reverse phase liquid chromatography,” which naturally requires a contacting the sample to a chromatographic support comprising a hydrophobic stationary phase; [0007];[0050])(Wang also discusses use of a SCX column prior to RPLC which is a chromatographic support comprising a stationary phase; See para. [0016],[0050]).
d) analyzing the separated peptides to determine the relative distribution of glucuronylation of the peptides (samples are then subjected to reverse phase liquid chromatography/mass spectroscopy analysis to identify glucuronylation of the protein drug product; [0007])(See relative abundances in Figs. 4B-7D which show a distribution of glucuronylation).
Wang is silent to teaching determining the relative distribution of glucuronidation (Emphasis Added), iduronidation, and galacturonidation,
a chromatographic support comprising a positive surface charge (Emphasis Added), and
c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides, wherein the weak acid mobile phase gradient has pH>3.
Wang instead teaches that glucuronylation (direct glucuronic acid addition) results in glucuronic acid attachment on the Lys residues and that it has been reported that glucuronidation (enzymatic addition) results in glucuronic acid attachment on the Ser and Thr residues ([0052]). However, Wang states that both modifications exhibit an accurate mass addition of 176.03 Da and are therefore believed to have the same elemental composition (C.sub.6H.sub.8O.sub.6)([0052]). Likewise, paragraphs [0004] and [0019] of the instant specification (US 20230314389 A1) teaches modification of the antibody through forced glucuronidation which would result in modification of lysine residues as taught by Wang. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method taught by Wang by substituting glucuronidation for glucuronylation since both modifications result in the covalent attachment of glucuronic acid to a molecule thereby yielding the predictable results of confirming the identity of the modification (See MPEP 2143(I)(B)).
Wang fails to teach determining the relative distribution of iduronidation and galacturonidation, a chromatographic support comprising a positive surface charge (Emphasis Added), and c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides, wherein the weak acid mobile phase gradient has pH>3.
Amenta teaches determining the relative distribution of glucuronic acid, iduronic acid, and galacturonic acid in polypeptides (See Table 2 which shows the mole % and masses of all three acids in the biglycan glycoforms which are associated with the biglycan polypeptide; See para. [0020]).
Amenta is considered to be analogous to the claimed invention because it is in the same field of endeavor for characterizing PTMs in proteins using analytical methods. Both Wang and Amenta are concerned with the characterization of therapeutic proteins that include post translational modifications such as glycosylation, with Wang acknowledging a need to “further characterize and discover PTMs that alter mAb activity and stability” ([0005]). Likewise, Amenta explains that “cells that can be used for producing a biglycan therepeutic can further be modified to increase the level and/or activity of an enzyme that catalyzes posttranslational modifications, e.g., glycosylations or sulfonations” ([0144]). Amenta then establishes that carbohydrate modifications associated with a therapeutic polypeptide can contain multiple uronic acid species including GlcA, IdoA, and GalA. Thus, in view of Wang’s objective of characterizing glucuronylation of a glycosylated therapeutic protein, Amenta would have provided reason to investigate and characterize the additional related uronic acid species rather than limiting the characterization to GlcA alone.
Furthermore, determining the relative quantities of each uronic acid would prove beneficial because it is well known in the art that these acids may interfere with one another during analysis, resulting in inaccurate perceived outputs. Reference Wolff states that “Distinguishing the epimers iduronic acid (IdoA) versus glucuronic acid (GlcA) has been a longstanding challenge for the mass spectrometry analysis of glycosaminoglycan oligogosaccharides” (Abstract); Wolff also expresses that “the pattern of modification (sulfation, acetylation, GlcA vs. IdoA) affects their biological activity” (p. 2, ll. 11-12). Likewise, reference Ibanez teaches the need to chromatographically distinguish and separately quantify GlcA and GalA because they are isobaric and cannot be distinguished by accurate mass alone (See p. 3, ll. 47-52; See same m/z of 193.0427 in Fig. 1; See separate concentrations in Table 2). Wang identifies glucuronylation based in part on a mass shift ([0052]) which alone would not distinguish it from the other uronic acid species having the same mass. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the peptide separation method taught by Wang by extending the analysis to determine the claimed relative distribution of iduronic acid and galacturonic acid (which is indicative of iduronidation and galacturonidation) as taught by Amenta, Wolff and Ibanez because it would provide a more complete characterization of the therapeutic protein while reducing the possibility of misidentification/false contributions among the structurally related uronic acid species, and this involves combining prior art elements according to known methods to yield predictable results (See MPEP 2143(I)(A)).
Modified Wang fails to teach a chromatographic support comprising a positive surface charge (Emphasis Added), and c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides, wherein the weak acid mobile phase gradient has pH>3.
Wang instead uses an SCX column to separate the Fab and Fc fragments followed by formic acid quenching ([0053]) and then subjects the fractions to RPLC which necessarily requires a hydrophobic stationary phase ([0007][0053]).
Birdsall teaches a chromatographic support comprising a hydrophobic stationary phase and a positive surface charge (Emphasis Added) and c) contacting the chromatographic support with a weak acid mobile phase gradient to separate the peptides, wherein the weak acid mobile phase gradient has pH>3 (“A commercially available lyophilized NIST mAb digest was used for all experiments. Samples were separated on a C18 reversed phase chemistry (CSH C18 columns) using a 50-min gradient at 0.68 %B/min with 0.1% formic acid as a mobile phase additive,” wherein Table 5 in paragraph [0182] shows a mobile phase of Acetonitrile, 0.1% Formic acid which has a pH of around 3.5 (>3))(Coated ACQUITY Premier CSH C18 columns are positively charged when pH≤6, which is satisfied when the mobile phase is Acetonitrile, 0.1% Formic acid; [0182]; See supporting reference, Waters (“ACQUITY UPLC and ACQUITY Premier Peptide CSH C18 , 130 Å, 1.7 µm and XSelect XP and XSelect Premier 2.5 µm Columns”; February 2020) at p. 4, col. 1, last 3 ll.).
Birdsall is considered to be analogous to the claimed invention because it is in the same field of endeavor for detecting post translational modifications (PTMs) in mAbs using a CSH C18 column within an LC-MS-MS system. Birdsall compares the quality of separation between uncoated and coated ACQUITY CSH C18 columns using a PENNYK Fc peptide sample that is susceptible to deamidation, a PTM of mAbs that is correlated with drug efficacy (Table 5; [0156]-[0157]). Use of the uncoated column showed that the detection of the deamidation products (impurities) was prevented due to coelution with the starting residue (tailing). Conversely, use of the coated column greatly reduced tailing and assay variability while increasing detector response, analyte recovery, and peak shape. Wang encounters this same hurdle when describing an inability to achieve accurate results due to a glycation modification in the sample that eluted at the same time as the antibody variant ([0044]). Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the peptide separation method taught by Wang in view of Amenta, Wolff and Ibanez by substituting Birdsall’s coated ACQUITY CSH C18 column with a weak acid mobile phase gradient for Wang’s RPLC stationary phase because it would improve separation during sample characterization, and this involves the simple substitution of one known element for another to obtain predictable results (See MPEP 2143(I)(B)).
Regarding claim 2, Modified Wang teaches the method of claim 1, wherein the protease is trypsin (trypsin; Wang, [0007]), lys-C, Glu-C, or Asp-N.
Regarding claim 3, Modified Wang teaches the method of claim 1, wherein the chromatographic support comprises a support material (130 Å particles…; Waters, p. 4, col. 1, Important Note, l. 2) with a positively charged surface modifier (…that undergo a surface modification by the addition of a low concentration of weakly basic ionizable silanes…provide frequently desired positive surface charge benefits; Waters, p. 4, col. 1, Important Note).
Regarding claim 4, Modified Wang teaches the method of claim 3, wherein the hydrophobic stationary phase (C18; Waters, p. 4, col. 1, Important Note) is linked to the surface modifier (130 Å particles that undergo a surface modification by the addition of a low concentration of weakly basic ionizable silanes, followed by C18 bonding and end capping; Waters, p. 4, col. 1, Important Note).
Regarding claim 5, Modified Wang teaches the method claim 4, wherein the hydrophobic stationary phase comprises an alkyl (C18; Waters, p. 4, col. 1, Important Note), alkenyl, alkynyl or aryl functional group.
Regarding claim 6, Modified Wang teaches the method of claim 5, wherein the hydrophobic stationary phase comprises a C18 functional group (C18; Waters, p. 4, col. 1, Important Note).
Regarding claim 7, Modified Wang teaches the method of claim 1, wherein the chromatographic support comprises a C18 stationary phase (ACQUITY ™ PREMIER Peptide CSH 130 Å C18 column (2.1 × 100 mm, 1.7 μm p/n 186009461; Birdsall, Table 5) comprising a hybrid material with a charged surface at a pH range of 1 to 11 (the ACQUITY UPLC Peptide CSH C18, 130 Å particles can tolerate a pH range from 1 to 11; Waters, p. 4, col. 1, last 3 ll.).
Regarding claim 8, Modified Wang teaches the method of claim 1, wherein the separated peptides are analyzed by using mass spectrometry (subjected to reverse phase liquid chromatography/mass spectroscopy analysis to identify glucuronylation of the protein drug product; Wang, [0007]).
Regarding claim 9, Modified Wang teaches the method of claim 1, comprising the identification of glucuronidated peptides (Paragraphs [0050]-[0054] explain that mAb samples were subjected to peptide fragmentation followed by peptide mapping of the sequence. The results showed an unknown modification on Lys residues of the sequence suspected to be from glucuronidation. To confirm, forced glucuronylation was performed on the same mAb samples and confirmed the unknown modification to be from a glucuronidated peptide).
Modified Wang fails to teach the identification of iduronidated, and galacturonidated peptides. However, it is known that “the modification of polypeptides by glucuronic acid, iduronic acid, or galacturonic acid has the potential to impact the safety and/or efficacy of such recombinantly-expressed products “ ([0004] of the instant specification). A supporting reference, Liang (“Quantitation of protein post-translational modifications using isobaric tandem mass tags”; 2015) also states that post-translationally modified peptides contain uronic acids due to glycosylation. Furthermore, Wang acknowledges a need to “further characterize and discover PTMs that alter mAb activity and stability” ([0005]). Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have tried to apply the same forced glycuronidation and peptide mapping analysis steps taught by Wang to identify the other known uronic acids responsible for the PTMs of mAbs including iduronic and galacturonic acid (See MPEP 2143(I)(E)).
Regarding claim 10, Modified Wang teaches the method of claim 8, wherein the peptides are identified by their respective chromatographic retention time and mass spectra (an acidic shoulder peak was detected and attributed to a variant of the antibody with a mass increase of approximately 176 Da; Wang, [0044]; [0016]-[0019]); Figs. 5A, 6A, and 7A of Wang show 3 different samples with peaks at the same retention times as when the respective sample has undergone forced glucuronidation (See Figs. 5B, 6B, and 7B).
Regarding claim 13, Modified Wang teaches the method of claim 1, wherein the weak acid is formic acid (Birdsall, [0090]; Table 5) or acetic acid.
Regarding claim 14, Modified Wang teaches the method of claim 1, wherein the acidic mobile phase is at a pH where greater than 50% of the carboxylic acid moieties associated with glucuronidation, iduronidation, or galacturonidation are in a deprotonated state (Acetonitrile, 0.1% Formic acid which has a pH of around 3.5 which is at a pH greater than 50% of the carboxylic acid moieties associated with glucuronidation, iduronidation, or galacturonidation; Birdsall, Table 5).
Regarding claim 16, Modified Wang teaches the method of claim 1, wherein the weak acid mobile phase gradient has a pKa value of 4.5 (Acetonitrile, 0.1% Formic acid; Birdsall, Table 5)(Formic acid has a pKa of around 3.75 as taught by Waters (Table 2) and the pKa of the gradient naturally increases with increasing amounts of acetonitrile. A pKa of 4.5 is therefore reached during the mobile phase gradient between 0.5-40% FA/ACN).
Regarding claim 17, Modified Wang teaches the method of claim 1.
Modified Wang fails to teach the mass spectrometry is electrospray ionization tandem mass spectrometry (ESI-MS/MS).
Modified Wang instead uses HCD to run the MS/MS experiments (Wang, [0050]).
Birdsall teaches mass spectrometry is electrospray ionization tandem mass spectrometry (ESI-MS/MS(Further Evaluation Including MS/MS; [0104])(Mass spectrometric (MS) data was also obtained for the three-peptide mixture as separated using the conditioned columns and performed with electrospray ionization and high sensitivity quadrupole time-of-flight instrumentation; [0134]).
Birdsall is considered to be analogous to the claimed invention because it is in the same field of endeavor for detecting post translational modifications (PTMs) in mAbs using a CSH C18 column within an LC-MS-MS system. It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the peptide separation method taught by Wang in view of Amenta, Wolff, Ibanez, and Birdsall by further substituting Wang’s HCD MS/MS technique with Birdsall’s electrospray ionization tandem mass spectrometry (ESI-MS/MS) technique in order to preserve fragile modifications and improve identification accuracy. ESI-MS/MS is well-known and routine in analysis instrumentation and the substitution would yield the predictable results of optimized sample result accuracy (See MPEP 2143(I)(B)).
Regarding claim 18, Modified Wang teaches the method of claim 1, wherein the polypeptide is an antibody (monoclonal antibody; Wang, [0007]).
Claims 11-12 are rejected under 35 U.S.C. 103 as being unpatentable over Wang (US 20190234964 A1), in view of Amenta (US 20140038906 A1), as evidenced by Wolff et al. (“Distinguishing Glucuronic from Iduronic Acid in Glycosaminoglycan Tetrasaccharides by using Electron Detachment Dissociation”) and Ibanez et al. (“Analytical method for the determination of organic acids in dilute acid pretreated biomass hydrolysate by liquid chromatography-time-of-flight mass spectrometry”), and Birdsall (US 20210255196 A1, entitled to the EFD of 20201013 by provisional application, US 63091169), as evidenced by supporting reference Waters (“ACQUITY UPLC Peptide CSH C18, 130 Å, 1.7 μm and XP 2.5 μm Columns and ACQUITY PREMIER Peptide CSH C18, 130 Å, 1.7 μm Columns”; October 2020), as applied to claim 1 above, and in further view of Xu (US 20190237157 A1).
Regarding claim 11, Modified Wang teaches the method of claim 1 and monitored the liquid chromatography by UV absorbance (simultaneous UV detection; Wang, [0042]).
Modified Wang fails to teach the relative distribution of glucuronidation, iduronidation, and galacturonidation is determined by integrating the respective peaks of liquid chromatograms corresponding to the separated peptides.
Xu teaches determining the relative distribution of the PTMs in mAbs by integrating respective peaks of chromatograms corresponding to the separated peptides (spectra of peptide identification (FIG. 2A) and example extracted ion chromatograms for peptide peak integration and PTM quantitation; ([0030]; Figs. 2A-B; Abstract).
Xu is considered to be analogous to the claimed invention because it is in the same field of endeavor for detecting and quantitating post translational modifications (PTMs) in mAbs using LC-MS/MS. It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the peptide separation method taught by Wang in view of Amenta, Wolff, Ibanez, and Birdsall by integrating the peaks associated with PTMs of liquid chromatograms as taught by Xu since this is a routine and well-known technique to quantify analyte abundance. Wang already determines a relative distribution of glucuronidation through of liquid chromatograms showing defined peaks (See Figs. 5A, 6A, and 7A)(simultaneous UV detection; [0042]). Associating a true value with the modification would aide in Wang’s suggested implementation of removing glucuronylated proteins from a protein drug product to produce a purified protein drug product ([0009]). Knowing the peak concentrations associated with the modifications would help to determine the extent of removal needed to achieve this goal, yielding predictable results (See MPEP 2143(I)(A)).
Regarding claim 12, Modified Wang teaches the method of claim 1, wherein
Modified Wang fails to teach the relative distribution of glucuronidation, iduronidation, and galacturonidation is determined by integrating the respective peaks of ion chromatograms extracted from the mass spectrometry analysis.
Xu teaches determining the relative distribution of the PTMs in mAbs by integrating respective peaks of ion chromatograms extracted from the mass spectrometry analysis (MS/MS spectra of peptide identification (FIG. 2A) and example extracted ion chromatograms for peptide peak integration and PTM quantitation; ([0030]; Figs. 2A-B; Abstract).
Xu is considered to be analogous to the claimed invention because it is in the same field of endeavor for detecting and quantitating post translational modifications (PTMs) in mAbs using LC-MS/MS. It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the peptide separation method taught by Wang in view of Amenta, Wolff, Ibanez, and Birdsall by integrating the peaks associated with PTMs as taught by Xu since this is a routine and well-known technique to quantify analyte abundance. Wang already determines a relative distribution of glucuronidation through mass spectra showing defined peaks (See Figs. 5C-D, 6C-D, and 7C-D). Associating a true value with the modification would aide in Wang’s suggested implementation of removing glucuronylated proteins from a protein drug product to produce a purified protein drug product ([0009]). Knowing the peak concentrations associated with the modifications would help to determine the extent of removal needed to achieve this goal, yielding predictable results (See MPEP 2143(I)(A)).
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Franco, 2019 (instant PTO-892) teaches identifying glucuronic acid, iduronic acid, and galacturonic acid and determining their relative proportions.
Padro, 2012 (instant PTO-892), Table 2, shows the increasing pKa of a formic acid/ACN gradient with increasing ACN at the same temperature (60 ◦C) as reference Birdsall.
Mourier, 2004 (instant PTO-892) teaches the use of a coated C18 stationary phase to separate oligosaccharide mixtures containing glucuronic and iduronic acid.
Alekseeva, 2013 (instant PTO-892) teaches the integration of peak volumes for all three uronic acids within a heparin sample using a positively charged C18 column.
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.
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/V.S./Examiner, Art Unit 1758
/MATTHEW D KRCHA/Primary Examiner, Art Unit 1796
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