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 .
Priority
Applicant’s claim for the benefit of a prior-filed application (which has PRO 63/128,146, filed 20 December 2020) under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Claim Objections
Claim 1 objected to because of the following informalities:
The phrase “contacting the sample to a separation column” should be corrected to read “contacting the sample with a separation column” for wording clarity.
Claim 10 objected to because of the following informalities:
The phrase “a concentration between 5 mM - 15 mM” at step (c) should be corrected to read “a concentration between 5 mM - 15 mM;” for missing punctuation.
Claim 19 objected to because of the following informalities:
The phrase “about between 1:5 (w/w) and 1:20 (w/w)” should be corrected to read “between about 1:5 (w/w) and about 1:20 (w/w)” for wording clarity.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION. —The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claim 6 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 6 recites the limitations “the first mobile phase” and “the second mobile phase.” There is insufficient antecedent basis for these limitations because Claim 1 recites “a first mobile phase gradient” and “a second mobile phase gradient,” but does not previously introduce “a first mobile phase” or “a second mobile phase.” As a result, the scope of Claim 6 is unclear. For examination purposes, claim 6 is interpreted as further specifying the compositions of the first and second mobile phase gradients recited in Claim 1.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS. —Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 31 rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 31 recites that the concentration of TCEP is between 20 μM and 80 μM. Claim 1 has been amended to recite “TCEP at a concentration of about 40 μM,” and a broader concentration range does not further narrow a specific concentration. Therefore, claim 31 no longer further limits the subject matter of Claim 1.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 6, 9, 10, 19, 24, 26, and 33 are rejected under 35 U.S.C. 103 as being unpatentable over LI et al. (Liquid chromatography and mass spectrometry with post-column partial reduction for the analysis of native and scrambled disulfide bonds, 2013, hereinafter LI (a)) in view of LI et al. (Disulfide bond assignment of an IgG1 monoclonal antibody by LC–MS with post-column partial reduction, 2013, hereinafter LI (b)) and NEUMANN (US20140081000A1).
Regarding Claim 1, LI (a) discloses a method for detecting both native and scrambled disulfide bonds. Nonreduced protein digests are separated using a reversed-phase C18 column, partially reduced, and analyzed by mass spectrometry (abstract).
The antibody, at a concentration of 12 mg per mL in 50 mM Tris, pH 8.0, with 6 M guanidine hydrochloride and 10 mM iodoacetamide, was incubated at 37 °C for 30 minutes. It was then diluted to 2 mg per mL using 20 mM Tris with 2 mM iodoacetamide and digested with Lys-C at a ratio of 1 to 20 Lys-C to antibody and trypsin at a ratio of 1 to 20 trypsin to antibody at 37 °C. After 16 hours, a second aliquot of trypsin was added at the same ratio of 1 to 20, and the sample was further digested for 2 hours. To promote disulfide bond scrambling, the antibody, at 12 mg per mL in 50 mM Tris and 6 M guanidine hydrochloride, was incubated at 37 °C for 1 hour and digested using the same procedure (Pg. 184, ¶ 6).
An ultra-high-performance liquid chromatography system with a C18 column, coupled to a quadrupole time-of-flight mass spectrometer, was used to analyze the samples. A volume of 10 µL of sample was injected with 98 percent mobile phase A, consisting of 0.02 percent trifluoroacetic acid in water, and 2 percent mobile phase B, consisting of 0.02 percent trifluoroacetic acid in acetonitrile, at a flow rate of 50 µL per min. Peptides were eluted using a gradient that increased mobile phase B from 2 percent to 98 percent. The column temperature was maintained at 60 °C, and the autosampler temperature at 5 °C. The mass spectrometer was operated in positive ion mode, scanning a mass-to-charge ratio range from 300 to 2000 (Pg. 185, ¶ 2).
A 260 mM stock solution of TCEP was prepared in 0.1 M ammonium bicarbonate, with the pH adjusted to 7.8 using ammonium hydroxide. Various concentrations of TCEP were prepared using freshly made 0.1 M ammonium bicarbonate, and the solutions were introduced into the column effluent through a mixing tee at a flow rate of 2 µL per min via syringe pump. Post-column reduction with 1 mM, 2 mM, and 10 mM TCEP revealed peaks corresponding to CL-1 and CL-2, with the level of reduction increasing alongside TCEP concentration. Based on peak intensity, 2 mM TCEP was selected for further experiments (Pg. 185, ¶¶ 3–5).
However, LI (a) does not explicitly disclose TFA at about 0.05% or TCEP at about 40 µM.
LI (b) discloses LC-MS as a known method for determining protein disulfide bond structures, including by comparing reduced and nonreduced peptide maps and by using partial reduction followed by MS analysis for more complicated disulfide linkages (Pg. 93, ¶¶ 1–2).
In Method Optimization, to achieve partial reduction, the pH of the reducing reagent and the TCEP concentration were optimized. Various concentrations of ammonium hydroxide were tested to raise the pH of mobile phases containing TFA and formic acid, and 10% ammonium hydroxide was chosen for further optimization of TCEP concentration. The degree of reduction of the disulfide bond in the CL domain was used to monitor performance. TCEP concentrations of 20 mM, 50 mM, and 100 mM clearly produced reduced CL-1 and CL-2 peptides, with 50 mM TCEP providing a higher degree of reduction than 20 mM TCEP, while 100 mM TCEP resulted in lower reduction efficiency, probably due to lower pH caused by the higher amount of TCEP (Pg. 95, Method Optimization).
LI (b) provides optimization guidance for achieving partial reduction by adjusting the pH of the reducing reagent and the TCEP concentration, which are the specific concentration-adjustment details not explicitly disclosed by LI (a). Because LI (a) already discloses an LC-MS workflow using TFA-containing mobile phases and post-column TCEP partial reduction for detecting native and scrambled disulfide bonds, a person skilled in the art would have had reason to apply LI (b)’s optimization guidance to LI (a)’s workflow to obtain suitable mobile-phase acidity and controlled partial reduction with predictable results.
The concentrations of TFA at about 0.05% and TCEP at about 40 µM are optimized values of known result-effective variables. LI (a) discloses TFA-containing mobile phases and post-column TCEP partial reduction, while LI (b) expressly identifies reducing-reagent pH and TCEP concentration as parameters optimized to achieve partial reduction. Optimizing such known parameters to obtain a workable or improved result would have been obvious to a person skilled in the art (In re Aller, 220 F.2d 454, 456–57 (CCPA 1955)).
However, LI (a) in view of LI (b) (hereinafter, modified LI) does not explicitly disclose including glycine at about 2 mM in the first and second mobile phase gradients.
NEUMANN discloses a method for separating polypeptide monomers, aggregates, and fragments using ion exchange chromatography, wherein a solution comprising a nonionic polymer and an additive is applied during recovery of the polypeptide from the ion exchange chromatography material (¶[0002]). The additive is selected to have low conductivity and no detectable buffering capacity, with suitable additives including amino acids such as glycine, bicine, tricine, alanine, proline, or betaine (¶¶[0100]–[0101]).
Advantageously, the glycine additive disclosed by NEUMANN provides a low-conductivity, non-buffering additive for mobile-phase conditions. Unlike charged-side-chain amino acids that may require pH adjustment and increase ionic strength, glycine is zwitterionic, does not significantly contribute to conductivity, and does not exhibit a buffering effect in aqueous solution, which avoids pH shifts in the mobile phase (¶¶[0111]–[0112]). In view of modified LI’s TFA-containing first and second mobile phase gradients, a person skilled in the art would have included the glycine additive in the first and second mobile phase gradients to maintain suitable mobile-phase conditions with predictable results.
Regarding the limitation “glycine at about 2 mM,” the concentration reflects an optimized value of a known additive concentration. NEUMANN suggests determining an additive concentration range where the intended effect is present and tolerated in the method (¶[0113]). Optimizing such a known parameter to obtain a workable or improved result would have been obvious to a person skilled in the art (In re Aller, 220 F.2d 454, 456–57 (CCPA 1955)).
Therefore, it would have been obvious to a person having ordinary skill in the art, prior to the effective filing date of the claimed invention, to incorporate the glycine additive, as disclosed by NEUMANN, into the first and second mobile phase gradients of the LC-MS method with post-column reduction for native and scrambled disulfide bond analysis in modified LI.
Regarding Claim 6, modified LI makes obvious a LC-MS method with post-column reduction for native and scrambled disulfide bond analysis of Claim 1. LI (a) discloses that a volume of 10 µL of sample was injected with 98% mobile phase A, consisting of 0.02% trifluoroacetic acid in water, and 2% mobile phase B, consisting of 0.02% trifluoroacetic acid in acetonitrile, at a flow rate of 50 µL/min. Peptides were eluted using a gradient that increased mobile phase B from 2% to 98% (Pg. 185, ¶ 2). As discussed above, modified LI makes obvious TFA at about 0.05% as an optimized mobile-phase concentration.
Regarding Claim 9, modified LI makes obvious the LC-MS method with post-column reduction for native and scrambled disulfide bond analysis of Claim 1. LI (a) discloses that an antibody was denatured and alkylated in 50 mM Tris, pH 8.0, with 6 M guanidine hydrochloride and 10 mM iodoacetamide at 37 °C, then diluted with 20 mM Tris and 2 mM iodoacetamide. Digestion was performed using Lys-C and trypsin at a ratio of 1:20 enzyme to antibody at 37 °C, followed by a second trypsin aliquot after 16 hours and an additional 2-hour digestion, yielding the sample for LC-MS analysis (Pg. 184, ¶ 6).
Regarding Claim 10, modified LI makes obvious the LC-MS method with post-column reduction for native and scrambled disulfide bond analysis of Claim 9. LI (a) discloses denaturing the antibody in 50 mM Tris, pH 8.0, with 6 M guanidine hydrochloride and 10 mM iodoacetamide at 37 °C for 30 minutes, followed by dilution to 2 mg/mL using 20 mM Tris with 2 mM iodoacetamide. The diluted sample was digested using Lys-C at a ratio of 1:20 (w/w) Lys-C to antibody, which reads upon the claimed “(i) ...a ratio... between 1:5 (w/w) and 1:20 (w/w)” (Pg. 184, ¶ 6).
Regarding Claim 19, modified LI makes obvious the LC-MS method with post-column reduction for native and scrambled disulfide bond analysis of Claim 9. LI (a) discloses that the diluted antibody sample was digested with trypsin at a ratio of 1:20 (w/w) trypsin to antibody, followed by a second trypsin aliquot after 16 hours at the same ratio, resulting in a total trypsin-to-antibody ratio of 1:10, which reads upon the claimed “(c) ...a ratio of trypsin protease to the predigested denatured alkylated biomolecule is between 1:2 (w/w) and 1:10 (w/w)” (Pg. 184, ¶ 6).
Regarding Claim 24, modified LI makes obvious the LC-MS method with post-column reduction for native and scrambled disulfide bond analysis of Claim 1. LI (a) discloses using a recombinant human IgG1 antibody as a model protein, where reduction was optimized using disulfide-linked peptides CL-1 and CL-2 from the monoclonal antibody. Without post-column reduction, only the disulfide-linked CL-1 and CL-2 peptides were detected. Upon applying post-column reduction with 1 mM, 2 mM, and 10 mM TCEP, peaks corresponding to CL-1 and CL-2 were revealed, with the level of reduction increasing alongside TCEP concentration. Based on relative peak intensity, 2 mM TCEP was determined optimal for further experiments, which reads upon the claimed partially reduced eluted sample components including one or more disulfide peptides and corresponding reduced partner peptides (Pg. 185, ¶¶ 4–5).
Regarding Claim 26, modified LI makes obvious the LC-MS method with post-column reduction for native and scrambled disulfide bond analysis of Claim 24. LI (a) discloses confirming disulfide bonds by matching retention times of cysteine-containing peptides and corresponding disulfide-linked peptides, such that the corresponding peptides are detected at the same retention time after post-column partial reduction. Four scrambled disulfide bonds were identified from retention-aligned peak pairs of cysteine-containing peptides not involved in native linkages, with molecular-weight confirmation. Full-scan mass spectra and tandem MS/MS were used to confirm disulfide-bond assignments, which reads upon obtaining MS1 and MS2 spectra (Pg. 185, ¶ 6; Pg. 186, ¶ 7).
Regarding Claim 33, modified LI makes obvious the LC-MS method with post-column reduction for native and scrambled disulfide bond analysis of Claim 1. LI (a) discloses applying the method to a recombinant human IgG1 monoclonal antibody and detecting four scrambled disulfide bonds in the IgG1 sample (Pg. 185, ¶ 6).
Response to Arguments
The Applicant’s arguments, see Remarks filed January 27, 2026, have been fully considered but are not persuasive. The rejection under 35 U.S.C. § 103 is updated and maintained.
The Applicant argues that LI discusses TCEP in LC-MS, while NEUMANN discusses glycine in liquid chromatography, and disputes that chromatographic principles would naturally extend to LC-MS workflows. The rejection relies on LI for the LC-MS workflow and NEUMANN for glycine as a suitable chromatography additive having low conductivity and no detectable buffering capacity, not for an LC-MS signal-enhancement mechanism or identical first and second mobile phase gradient structure. Therefore, a person skilled in the art would have been motivated by the benefit of maintaining suitable mobile-phase conditions to apply NEUMANN’s glycine-additive teaching to the first and second mobile phase gradients of modified LI with a reasonable expectation of success.
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 TAK L. CHIU whose telephone number is (703)756-1059. The examiner can normally be reached M-F: 9:00am - 6:00pm (CST).
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, PREM C. SINGH can be reached at (571)272-6381. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/TAK L. CHIU/
Examiner, Art Unit 1777
/KRISHNAN S MENON/Primary Examiner, Art Unit 1777
Prosecution Insights