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 01/07/2026 has been entered. Claims 1-11 remain pending in the application. Claims 3 and 7 have been amended for minor informalities. Claim 5, stated to be an original claim, has a new objection for the term “tri pie” in line 2 that was not present in the original set of claims.
Status of Objections and Rejections
The objection to claim 7 has been withdrawn in view of Applicant's amendment.
The objection to the drawings has been withdrawn in view of Applicant's replacement drawing sheets.
New grounds of objection are necessitated by the amendments.
The rejection of claims 1-11 under 35 USC 103 maintained.
Response to Arguments
Applicant's arguments, see pages 6-10, filed 01/07/2026, with respect to the rejection of claims 1-11, under 35 U.S.C. 103 have been fully considered but they are not persuasive.
Applicant argues (pp. 7-8) that the Examiner’s rejection under 35 U.S.C. 103 improperly combines fundamentally different technologies without adequate motivation or reasonable expectation of success. Specifically, Applicant contends that Stoll teaches an offline cation-exchange (CE) based workflow with SEC desalting, whereas Dai teaches an online cIEF-MS system designed for direct MS coupling that does not allow for intermediate sample preparation steps, with the additional obstacle of buffer incompatibility between cIEF and MS. Applicant emphasizes that offline cIEF methods permit the additional sample preparation steps and that the claimed invention improves conventional cIEF-MS workflows by comprehensively collecting cIEF fractions while preserving pI resolution and using SEC both for buffer exchange and size-based separation. Therefore, the Examiner has not established why one of ordinary skill in the art would be motivated to combine the teachings.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., collection of cIEF fractions offline) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Applicant’s argument improperly assumes that collecting a fraction requires the workflow to be offline. However, Stoll states that “in online 2D-LC fractions of effluent from the first dimension column are collected in loops or traps and transferred (injected) into a second dimension column for further separation” wherein a CE column can be a first dimension and SEC for the second dimension (p. 7, ll. 8-17). Additionally, Dai mobilizes resolved cIEF charge variant zones sequentially toward MS detection, meaning the system already processes discrete separated fractions (See separated bands in the Figure in the Abstract of Dai). Therefore, temporary fraction collection is not limited to an offline automated workflow, and the fractions of Dai can also be collected in the loops or traps of Stoll which are capable of receiving hydrolytic/reducing agents into the sample. Therefore, one of ordinary skill in the art would have recognized that substituting cIEF for CE would yield predictable results.
Although Applicant argues (p. 8, ll. 1-2) that the instant application relies upon SEC for both buffer exchange and desalting separation based on size, a buffer exchange is not a requirement of SEC since the separation technique does not depend on the chemistry of the column but rather on the size of the sample contents. Dai’s cIEF fraction can therefore remain in the same buffer, desalt, and then transfer to the MS for further analysis.
Applicant argues (pp. 8-10) that CE and cIEF operate under materially different principles, CE relying on elution through changing pH buffers without electric fields or focusing, and cIEF relying on stationary pH gradients, electric fields, and analyte/catholyte buffers to focus proteins at their isoelectric points. According to Applicant, the Examiner’s proposed substitution of CE with cIEF is based on hindsight because the references use incompatible buffer systems, Dai does not disclose integrating cIEF and SEC, and Dai’s online system is specifically designed to avoid intermediate offline processing steps such as SEC. Thus, Applicant asserts there was neither motivation nor reasonable expectation of success in combining Stoll and Dai.
In response to applicant's argument that CE and cIEF operate under materially different principles and the Examiner has not established why one of ordinary skill in the art would have had a reason and would know how to replace the CE in Stoll with the cIEF in Dai, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
Dai provides a list of different charge-based separation techniques that are commonly used for charge variant analysis, including ion-exchange chromatography which is taught by Stoll (cation-exchange, CE)(Dai, p. 2246, para. 2). As explained in the previous office action, Dai goes on to explain that cIEF is a later development that has become an important technique due to the production of high resolution samples for subsequent mAbs identification and characterization (p. 2246, para. 2). Likewise, the aim of Stoll is to separate and analyze mAb variants, and therefore switching to cIEF from CE would enhance the results. In the substitution, the specialized sample buffer used by Dai would still accompany the cIEF equipment, and the CE equipment and associated buffer taught by Stoll would be swapped out.
Dai does not disclose integrating cIEF and SEC, however, Dai is interested in desalting the sample and performs this cleanup step before cIEF (p. 2248, para. 5). Desalting again via Stoll’s SEC column after cIEF would be a duplicate step for further sample cleanup resulting in increased resolution before reaching the mass spectrometer (See MPEP 2144.04(VI)(B)). Combining SEC with cIEF, collecting cIEF fractions offline/online via traps, and the addition of hydrolytic/reducing agents to a cIEF fractionated sample online are well-known in the art; additionally, it has also been proven that even increasing the number of strong CE fractions produced less peak resolution than that of cIEF fractions, and applying these techniques would therefore be expected to yield predictable results (See Abstract of supporting reference, Xu (“Automated Capillary Isoelectric Focusing-Tandem Mass Spectrometry for Qualitative and Quantitative Top-Down Proteomics”; 2018))(See pp. 37-38, p. 77, para. 2, ll. 1-7, and p. 82 para. 3, ll. 5-8 of supporting reference Wang (“Capillary isoelectric focusing-based multidimensional peptide/protein separations for proteomics analysis”; 2005)).
Drawings
The drawings were received on 01/07/2026. These drawings are acceptable.
Claim Objections
Claim 5 is objected to because of the following informalities:
Regarding claim 5, l. 3 recites “tri pie”. Applicant may amend the claim to recite “triple”.
Appropriate correction is required.
Claim Interpretation
The claims contain limitations which are directed to intended uses or capabilities of the claimed invention. These limitations are only given patentable weight to the extent which effects the structure of the claimed invention. Please see MPEP 2114. Note that functional limitations are emphasized in italics herein.
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 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.
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 are rejected under 35 U.S.C. 103 as being unpatentable over Stoll (“Evolution of Coupling Ion-Exchange Separations and Mass Spectrometry”; 2019), in view of Dai (“Capillary Isoelectric Focusing-Mass Spectrometry Method for the Separation and Online Characterization of Intact Monoclonal Antibody Charge Variants”; 2018).
Regarding claim 1, Stoll teaches a method for characterizing charge variants of a protein of interest (Direct coupling of ion-exchange separations to mass spectrometric detection is increasingly being used for analyses of molecules ranging from organic acids to proteins. These approaches leverage both the exquisite selectivity of the ion-exchange mode for charge-based separation, and the tremendous power of mass spectrometry for identification of unknowns and trace-level quantitation.; page 1, lines 5-12) comprising:
(a) Separating charge variants of said protein of interest (“elute mAbs from a first dimension cation-exchange column,” by using “charge-based separation”; page 6, lines 29-30; page 1, lines 9-10),
(b) collecting fractions from step (a) (online 2D-LC fractions of effluent from the first dimension column; page 6, lines 17-18),
(c) subjecting said fractions to desalting size exclusion chromatography (fractions of effluent from the first dimension column are collected in loops or traps and transferred (injected) into a second dimension column for further separation, wherein “the second dimension separation can be a short, fast desalting step using reversed-phase or SEC conditions”; page 6, lines 17-20; page 6, lines 20-22),
(d) subjecting the eluate from step (c) to mass spectrometry (Stoll describes flowing the sample through the first and second dimension columns, “prior to MS detection,”; page 6, line 32) to characterize said charge variants of said protein of interest (The MS is used to identify “subunit variants of the mAb”; page 6, line 35; See Fig. 2).
Stoll fails to teach subjecting a sample including a protein of interest to capillary isolelectric focusing.
Stoll instead teaches the use of subjected a sample to a cation-exchange column.
Dai teaches subjecting a sample including a protein of interest to capillary isolelectric focusing (the charge variant profiles of trastuzumab, bevacizumab, infliximab, and cetuximab, obtained using this CIEF-MS method, were corroborated by imaged CIEF; Abstract).
Dai is considered to be analogous to the claimed invention because it is in the same field of endeavor for a cIEF-MS method in the separation and online characterization of intact mAb charge variants (Title). Dai lists a number of charge-based separation techniques including Stoll’s ion-exchange chromatography (e.g. cation-exchange) and later mentions that “(iCIEF),17−19 has become an important technique for charge variant analysis and been applied routinely,” for the benefit of increased sample resolution (page 2246, col. 1-2). 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 substituted the cation-exchange column taught by Stoll with the cIEF equipment taught by Dai because increased sample resolution using cIEF would improve Stoll’s entire goal of separating mAb variants, and this involves the substitution of one known charge-based separation method for another to obtain predictable results (see MPEP 2143(I)(B)).
Regarding claim 2, Modified Stoll teaches the method of claim 1, wherein said protein is an antibody, a bispecific antibody, a monoclonal antibody (mAb; Stoll, page 6, lines 29-30), a fusion protein, an antibody-drug conjugate, an antibody fragment, or a protein pharmaceutical product
Regarding claim 3, Modified Stoll teaches the method of claim 1, wherein said capillary isoelectric focusing is imaged capillary isoelectric focusing (by imaged CIEF; Dai, Abstract)
Regarding claim 4, Modified Stoll teaches the method of claim 1, wherein said desalting size exclusion chromatography system is coupled to said mass spectrometer (“Coupling of Ion-exchange and MS Using 2D-LC,” wherein “the second dimension separation can be a short, fast desalting step using reversed-phase or SEC conditions”; Stoll, page 6, lines 4-5, )(Under broadest reasonable interpretation, the Examiner understands that a teaching of either online or offline coupling satisfies the limitation).
Regarding claim 5, Modified Stoll teaches the method of claim 1,
Modified Stoll is silent to teaching said mass spectrometer is an electrospray ionization mass spectrometer, nano-electrospray ionization mass spectrometer, or a triple quadrupole mass spectrometer.
Dai teaches a nano-electrospray ionization mass spectrometer. (The regular ESI ion source on the 6224 TOF was modified to accommodate nanospray by replacing the ESI spray shield with a nanospray shield and a drying gas diverter; page 2248, paragraph 3, lines 1-3)
Dai is considered to be analogous to the claimed invention because it is in the same field of endeavor for capillary isoelectric focusing-mass spectrometry method for the separation and online characterization of intact monoclonal antibody charge variants (Title). 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 specified the generic mass spectrometry detection taught by Stoll in view of Dai to be nanoESI-MS taught by Dai in order to facilitate “efficient introduction of analytes into the MS” (page 2248, column 2, paragraph e, lines 9-10).
Regarding claim 6, Modified Stoll teaches the method of claim 1, said mass spectrometry analysis comprises intact mass analysis ( intact monoclonal antibodies (mAbs); Stoll, page 2, line 5) or reduced peptide mapping analysis.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Stoll (“Evolution of Coupling Ion-Exchange Separations and Mass Spectrometry”; 2019), in view of Dai (“Capillary Isoelectric Focusing-Mass Spectrometry Method for the Separation and Online Characterization of Intact Monoclonal Antibody Charge Variants”; 2018) in further view of Xu (“Automated Capillary Isoelectric Focusing-Tandem Mass Spectrometry for Qualitative and Quantitative Top-Down Proteomics”; 2020).
Regarding claim 7, The method of claim 1.
Modified Stoll fails to teach said mass spectrometer is capable of performing multiple reaction monitoring or parallel reaction monitoring.
Xu teaches the use of a mass spectrometer capable performing multiple reaction monitoring (tandem MS (MS/MS); Abstract).
Xu is considered to be analogous to the claimed invention because it is in the same field of endeavor of coupling isoelectric focusing-based fractionation with mass spectrometry analysis for protein analysis. 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 specified the generic mass spectrometry detection taught by Stoll in view of Dai to be a mass spectrometer that is capable performing a multiple reaction monitoring in order to maximize proteome identification. Xu states that “Previous cIEF-MS studies concentrated on measuring the protein’s mass without MS/MS, impeding the confident proteoform identification in complex samples and the accurate localization of post-translational modifications on proteoforms” (Abstract). Since MS/MS analysis is commonly known in the art for proteomic analysis, and useful for the same purpose of MS, one of ordinary skill in the art would expect that carrying out this substitution of MS for MS/MS would enable identification of a greater magnitude of proteins by “ large-scale delineation of proteoforms in complex proteomes” (Xu, Abstract).
Claims 8-10 are rejected under 35 U.S.C. 103 as being unpatentable over Stoll (“Evolution of Coupling Ion-Exchange Separations and Mass Spectrometry”; 2019), in view of Dai (“Capillary Isoelectric Focusing-Mass Spectrometry Method for the Separation and Online Characterization of Intact Monoclonal Antibody Charge Variants”; 2018), as applied to claim 1 above, and in further view of Leitner (“Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography”; 2012).
Regarding claim 8, Modified Stoll teaches the method of claim 1,
Modified Stoll fails to teach a step wherein said fractions are contacted to at least one hydrolyzing agent prior to desalting size exclusion chromatography. (mention teachings of Dai about future use of proteases
Leitner teaches contacting the sample to at least one hydrolyzing agent prior to desalting size exclusion chromatography (the sample was diluted… and trypsin…was added; ; page 3, column 1, paragraph 5, lines 1-3)(The Experimental Procedures Section on page 3 describes the progression of steps from digestion (hydrolysis) to SEC separation).
Leitner is considered to be analogous to the claimed invention because it is in the same field of endeavor of coupling SEC with mass spectrometry analysis for protein analysis (Abstract). Reference Dai, of claim 1, expresses that combining CIEF-MS analysis with a hydrolysis step (enzymatic cleavage treatment) will further aid in reduced sample complexity for understanding of protein structures (page 2253, column 1, lines 11-17). Leitner evidences this in that the “nonredundant cross-link identifications in each SEC fraction for the five proteases [was] investigated. The use of several proteases resulted in a further increase of more than 70% on the level of nonredundant cross-linking sites by using four other proteases, and more than 45% using Asp-N alone, for model proteins”. 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 fractions taught by Stoll in view of Dai to incorporate the teachings of Leitner by hydrolyzing the fractions prior to desalting size exclusion chromatography because increasing the number and quality of detectable peptides from a protein sample would aid in the analysis of separated mAb variants and this involves combining prior art elements according to known methods to yield predictable results (See MPEP 2143(I)(A)).
Regarding claim 9, Modified Stoll teaches the method of claim 8, wherein said at least one hydrolyzing agent is chosen from a group consisting of trypsin (the sample was diluted… and trypsin…was added; ; page 3, column 1, paragraph 5, lines 1-3), chymotrypsin, LysC, LysN, AspN, GluC and ArgC.
Regarding claim 10, Modified Stoll teaches the method of claim 1,
Modified Stoll fails to teach a step wherein said fractions are contacted to at least one reducing agent prior to desalting size-exclusion chromatography.
Leitner teaches contacting samples with at least one reducing agent prior to desalting size-exclusion chromatography (tris(carboxyethyl) phosphine stock solution in water were added [to] the samples; page 3, column 1, paragraph 4, lines 4-5)(The Experimental Procedures Section on page 3 describes the progression of steps from reduction to SEC separation)
Leitner is considered to be analogous to the claimed invention because it is in the same field of endeavor of coupling SEC with mass spectrometry analysis for protein analysis (Abstract). Reference Dai, of claim 1, expresses that combining CIEF-MS analysis with a hydrolysis step (enzymatic cleavage treatment) will further aid in reduced sample complexity for understanding of protein structures (page 2253, column 1, lines 11-17). Leitner evidences this in that the “nonredundant cross-link identifications in each SEC fraction for the five proteases [was] investigated. The use of several proteases resulted in a further increase of more than 70% on the level of nonredundant cross-linking sites by using four other proteases, and more than 45% using Asp-N alone, for model proteins”. 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 fractions taught by Stoll in view of Dai to incorporate the teachings of Leitner by hydrolyzing the fractions prior to desalting size exclusion chromatography because increasing the number and quality of detectable peptides from a protein sample would aid in the analysis of separated mAb variants and this involves combining prior art elements according to known methods to yield predictable results (See MPEP 2143(I)(A)).
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Stoll (“Evolution of Coupling Ion-Exchange Separations and Mass Spectrometry”; 2019), in view of Dai (“Capillary Isoelectric Focusing-Mass Spectrometry Method for the Separation and Online Characterization of Intact Monoclonal Antibody Charge Variants”; 2018), as applied to claim 1 above, and in further view of Kirkwood (“Characterization of Native Protein Complexes and Protein Isoform Variation Using Sizefractionation-based Quantitative Proteomics”; 2012).
Regarding claim 11, Modified Stoll teaches the method of claim 1.
Modified Stoll fails to teach said desalting size exclusion chromatography is performed under native conditions.
Kirkwood teaches desalting size exclusion chromatography is performed under native conditions (the use of the native SEC approach to profile co-fractionation of proteins at a system-wide level and thus to predict the existence of distinct forms of protein complexes; page 3862, column 2, paragraph 3, lines 4-5).
Kirkwood is considered to be analogous to the claimed invention because it is in the same field of endeavor of coupling SEC with mass spectrometry analysis for protein analysis (Abstract). Kirkwood states that native SEC was combined with high-throughput proteomic analysis to characterize soluble protein complexes (Abstract). Stoll teaches analysis of mAbs (page 6, lines 29-30) which are also soluble proteins. Performing SEC under native conditions does not change the fundamental role of SEC as a size-based, desalting step, but simply preserves the proteins in their folded or complexed states. 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 desalting SEC conditions taught by Stoll in view of Dai with the teachings of Kirkwood by performing SEC under native conditions because preserving the structure of native proteins reveals the intact mass for confirmation of the specific variant, and this involves the use of a known technique to improve a similar method in the same way (See MPEP 2143(I)(C)).
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Xu et al., 2020 (instant PTO-892) teaches SEC-CIEF.
Wang et al, 2005 (instant PTO-892) teaches collection of cIEF fractions offline/online via traps, the addition of hydrolytic/reducing agents to a cIEF fractionated sample online/offline, and proof that cIEF fractions produce higher resolution compared to CE fractions.
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 extension fee 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 VALERIE SIMMONS whose telephone number is (703)756-1361. The examiner can normally be reached M-F 7:30-4:00.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Maris Kessel can be reached on 571-270-7698. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/V.S./Examiner, Art Unit 1758
/MARIS R KESSEL/Supervisory Patent Examiner, Art Unit 1758
Prosecution Insights