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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on May 27, 2026 has been entered.
Status of Claims
Claims 1-5, 8-10, and 15-21 were pending. Claim 1 is amended. Claim 22 is newly added.
Claims 1-5, 8-10, and 15-22 are examined herein.
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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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 1-4, 8-10, 19, and 21-22 are rejected under 35 U.S.C. 103 as being unpatentable over Haberger et al. (MAbs. 2016;8(2):331-9) in view of Xu et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Jun 1; 960:230-8).
Regarding claim 1, Haberger teaches a method for characterizing at least one high molecular weight species of a protein of interest, said method comprising:
a. obtaining a sample including said protein of interest and said at least one high molecular weight species – specifically, Haberger teaches that the protein of interest was obtained during bio-process and formulation development and it had an aggregate present (pg. 332, col. 1, par. 1);
b. contacting said sample to a native size exclusion chromatography column – specifically, Haberger teaches that the aggregates were analyzed by a size exclusion-based ultrahigh-pressure liquid chromatography method (pg. 332, col. 1, par. 1). Haberger teaches size exclusion chromatography run under native conditions using nondenaturing buffer 200 mM KH2PO4, 250 mM KCl, pH 7.0 (HPLC-SEC, pg. 338, col. 1, last par., and FastSEC, pg. 9, col. 2, par. 2);
c. washing said column to collect an eluate – specifically, Haberger teaches that the protein of interest (a monoclonal antibody CrossMAb) was separated using two size exclusion chromatography columns (Fig. 3B – HPLC-SEC and Fig. C - FastSEC). Separation on a size exclusion chromatography column necessarily requires washing the column to collect an eluate. Both size exclusion chromatography columns (HPLC-SEC and FastSEC) demonstrated separation of the monomeric CrossMab reference material from high-molecular weight species (e.g., monomer at about 16 min on Fig. 1A and B, and high molecular weight species at about 19 min in Fig. 1B).
Regarding the new limitation reciting an eluate comprising said at least one high molecular weight species, Haberger teaches that eluate includes at least one high molecular weight species – peak 1 represents the CrossMAb trimer and peaks 2 and 3 represent the CrossMAb dimers (pg. 334, col. 1, par. 1 and Fig. 3B). The trimer and dimers are high molecular weight species of the CrossMAb;
e. subjecting said mixture to a mass spectrometer to characterize said at least one
high molecular weight species – specifically, Haberger teaches “we aimed to characterize the LMW and HMW variants observed in the Fast-SEC UV-profile by coupling UHPLC-SEC to native ESI-MS” (pg. 333, col. 2, par. 1). The HMW variants are high molecular weight species of the instant disclosure.
Haberger does not specifically teach step (d) of claim 1 - adding a denaturing solution to the eluate to form a mixture.
Regarding claim 1, Xu teaches size-exclusion chromatography-mass spectrometry for characterization of size variants of monoclonal antibodies (Abstract). Xu also teaches adding a denaturing solution to the eluate to form a mixture. Specifically, Xu teaches adding denaturing solution of m-nitrobenzyl alcohol, 50% ACN, and 0.1% formic acid to the liquid chromatography column eluate (pg. 231, col. 2, last par.), meeting limitation of claim 8 reciting the denaturing solution comprises a combination of acetonitrile and formic acid.
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the step of adding a denaturing solution to the eluate in the method of Haberger by allowing a mixture to form as taught by Xu, in order to improve performance of the mass spectrometry analysis (Abstract). One having ordinary skill in the art would have been motivated to adding a denaturing solution as a post-SEC additive to improve the ionization of antibody (id.). This combination would have been desirable to those of ordinary skill in the art for the reasons mentioned above.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because both Haberger and Xu teach closely related mass spectrometry analysis of antibodies, and the denaturing solution does not have a negative effect on antibody samples.
Regarding claim 2, Haberger teaches the protein of interest is an antibody - a bi-specific antibody CrossMAb (pg. 331, col. 2, par. 2 and pg. 332, col. 1, par. 1).
Regarding claims 3 and 19, Haberger teaches that eluate includes at least one high molecular weight species – peak 1 represents the CrossMAb trimer and peaks 2 and 3 represent the CrossMAb dimers (pg. 334, col. 1, par. 1 and Fig. 3B). The trimer and dimers are high molecular weight species of the CrossMAb. Additionally, the reference teaches that “by the application of ESI-MS under denaturing conditions, only charge state signals corresponding to the CrossMAb monomer were detected, whereas using native (ESI-MS) conditions the CrossMAb dimer, trimer, and tetramer could also be verified” (pg. 333, col. 1, par. 2), meeting the limitation of claim 19 reciting at least one high molecular weight species is a noncovalent high molecular weight species.
Regarding claim 4, Haberger teaches using a SEC-UV/MS mass spectrometer comprising of a SEC column, a Dionex UltiMate 3000 chromatography system equipped with UV detection at 280 nm (pg. 338, col. 1, par. 3), and a Q-TOF mass spectrometer (pg. 337, col. 2, par. 3).
Regarding claims 9 and 10, Haberger and Xu teach the denaturing solution comprising 50% ACN, and 0.1% formic acid (Xu, pg. 231, col. 2, last par.). Since Applicant has not disclosed that the specific concentrations of acetonitrile and formic acid recited in instant claims are for any particular purpose or solve any stated problem and the prior art teaches that the concentrations of the denaturing components often vary according to the sample being analyzed, absent unexpected results, it would have been obvious for one of ordinary skill to discover the optimum workable ranges by optimization procedures known in the art.
Regarding claim 21, Haberger teaches that the mass spectrometric analysis of the CrossMAb size variants by native SEC-UV/MS suggested a high HMW species consisting of a modified CrossMAb non covalently associated with a LC-Dimer. In order to confirm this non-covalent interaction, 10% acetonitrile was added to the chromatographic eluent. Fig. 4A shows the result of Fast-SEC separation with UV detection of a CrossMAb stability sample with and without addition of acetonitrile to the eluent (pg. 334, col. 2, par. 2), meeting the limitation of claim 21 reciting comparing at least one peak from a mass spectra obtained using (e) with a mass spectra obtained by carrying out an online size exclusion chromatography-mass spectrometry of said sample of (a).
Regarding claim 22, Haberger teaches size-exclusion chromatography directly coupled to mass spectrometer (pg. 338, col. 1, par. 3). Specifically, the outlet of the RSLC system (an HPLC system) was directly coupled to the NanoMate direct infusion system. The RSLC flow was split into 2 parts, 4 ml/min were online infused to the MS system. The NanoMate system was installed on a modified ‘High Mass QTOF’ mass spectrometer (id.).
Claims 5 and 15-18 are rejected under 35 U.S.C. 103 as being unpatentable over Haberger in view of Xu, as applied to claim 1 above, and further in view of Yan et al. (J Am Soc Mass Spectrom. 2020 Oct 7;31(10):2171-2179. Epub 2020 Sep 11) as evidenced by Sanders et al. (Anal Methods. 2020 Sep 28;12(36):4404-4417) and Newomics (M3 emitters, 2025).
The teachings of Haberger and Xu have been set forth above.
Haberger and Xu fail to specifically teach mass spectrometer is a nano-electrospray ionization mass spectrometer; a flow of said mixture of (d) in said mass spectrometer is less than 10 μL/min; mixture of (d) split into said mass spectrometer and ultraviolet detector; a desolvation gas is added to said mixture of (d) prior to subjecting it to mass spectrometer; and a multi-nozzle emitter is used to add said desolvation gas with said mixture of (d).
Regarding claims 5 and 15-18, Yan teaches an LC−MS method for intact mass analysis of protein drugs (Abstract).
Regarding claim 5, Yan also teaches a mass spectrometer is a nano-electrospray ionization mass spectrometer. Specifically, a Thermo Q Exactive UHMR or Exactive Plus EMR mass spectrometer equipped with a Nanospray Flex Ion Source was used for native MS analysis (pg. 2173, col. 1, par. 1) with a multinozzle emitter used for nanoelectrospray ionization (pg. 2172, col. 2, par. 1).
Regarding claim 15, Yan also teaches a flow of said mixture of (d) in said mass spectrometer is less than 10 μL/min. Specifically, Yan teaches a postcolumn splitting strategy was applied to reduce the flow rates to a range (<10 μL/min) that can be readily accommodated by a
multinozzle emitter (M3) for NSI (pg. 2173, col. 2, par. 1).
Regarding claim 16, Yan also teaches a method wherein mixture of (d) split into said mass spectrometer and ultraviolet detector. Specifically, Yan teaches that eluate exiting a SEC column is split into two flow paths, one leading to a UV detector and another leading to a mass spectrometer (Fig. 1).
Regarding claim 17, Yan also teaches a desolvation gas is added to said mixture of (d) prior to subjecting it to mass spectrometer. Specifically, Yan teaches that a desolvation nitrogen gas was applied to assist nanoelectrospray (pg. 2173, col. 1, par. 1 and Fig. 1).
Regarding claim 18, Yan also teaches a multi-nozzle emitter is used to add said desolvation gas with said mixture of (d). Specifically, Yan teaches using a multi-nozzle emitter for nanoelectrospray ionization, wherein it is connected to a bottle with desolvation gas (pg. 2172, col. 2, par. 1 and Fig. 1).
Regarding claim 5, it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Haberger and Xu by employing a nano-electrospray ionization mass spectrometer as taught by Yan, to provide a mass spectrometry method with improved performance as evidenced by Sanders (Abstract). One having ordinary skill in the art would have been motivated to choose nano electrospray mass spectrometer because nano format of liquid chromatography-mass spectrometry has improved chromatographic efficiency and increased sensitivity of the electrospray process (id.). This combination would have been desirable to those of ordinary skill in the art for the reasons mentioned above.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because nano electrospray mass spectrometers are commercially available instruments designed for protein analysis.
Regarding claim 15, it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Haberger and Xu by employing a flow rate of less than 10 μL/min as taught by Yan, to provide a nanoelectrospray mass spectrometer with a required sample flow rate. One having ordinary skill in the art would have been motivated to reduce the flow rates to a range (<10 μL/min) that can be readily accommodated by a multinozzle emitter (M3) for NSI (pg. 2173, col. 2, par. 1). This combination would have been desirable to those of ordinary skill in the art because it is a mass spectrometer requirement.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because nano electrospray mass spectrometers are commercially available instruments designed for protein analysis and Haberger used Waters Q-TOF ESI mass spectrometer.
Regarding claim 16, it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Haberger and Xu by splitting the postcolumn eluate flow into separate UV and mass spectrometry paths as taught by Yan, in order to reduce the flow rates to a range that could be accommodated by a multinozzle emitter for nanoelectrospray ionization (Yan, pg. 2172, col. 2, par. 1). One having ordinary skill in the art would have been motivated to split the postcolumn flow to match higher column flow rate with a slower flow rate of the mass spectrometer. This combination would have been desirable to those of ordinary skill in the art to ensure proper functioning the mass spectrometer.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because electrospray mass spectrometers of Haberger and Yan are very similar instruments designed for mass spectrometry analysis.
Regarding claim 17, it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Haberger and Xu by adding a desolvation gas prior to mass spectrometer input as taught by Yan, in order to achieve charge-reduction native MS (Yan, pg. 2173, col. 1, par. 1). One having ordinary skill in the art would have been motivated to use a desolvation gas because it is a standard practice in the art of mass spectrometry. This combination would have been desirable to those of ordinary skill in the art for the reasons mentioned above.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because the use a desolvation gas is a standard practice in the art of ESI mass spectrometry.
Regarding claim 18, it would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Haberger and Xu by employing a multinozzle emitter as taught by Yan, in order to deliver the postcolumn sample flow to the mass spectrometer. One having ordinary skill in the art would have been motivated to use the multinozzle emitter because it dramatically enhancing the ionization efficiency to achieve unprecedented sensitivity, robustness, and throughput as evidenced by Newomics (pg. 1). This combination would have been desirable to those of ordinary skill in the art for the reasons mentioned above.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because the multinozzle emitter of Yan is commercially produced for electrospray mass spectrometers as taught Haberger.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Haberger in view of Xu, as applied to claim 1 above, and further in view of Remmele et al. (J Pharm Sci. 2006 Jan;95(1):126-45).
The teachings of Haberger and Xu have been set forth above.
Regarding claim 20, Haberger teaches that eluate includes at least one high molecular weight species – peak 1 represents the CrossMAb trimer and peaks 2 and 3 represent the CrossMAb dimers (pg. 334, col. 1, par. 1 and Fig. 3B). The detection of trimer and dimers as separate peaks by Haberger indicates the ability of the native mass spectrometry to work in this high molecular weight range.
Haberger and Xu fail to specifically teach at least one high molecular weight species is a non-dissociable high molecular weight species of said protein of interest.
Regarding claim 20, Remmele teaches characterization of dimers of epratuzumab - a recombinant antihuman-CD22 monoclonal antibody (Abstract and pg. 127, col. 2, par. 2). Remmele also teaches non-dissociable high molecular weight species of epratuzumab. Specifically, Remmele teaches that epratuzumab dimer has about 70% covalent forms (Abstract) and the dimer has two intact monomers (where a monomer is defined as the mass of two light chains and two heavy chains covalently attached via two inter-heavy chain disulfide bonds in the Fc hinge region) with an apparent molecular mass of ~300 kDa (pg. 127, col. 2, par. 2).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Haberger and Xu by applying it for characterizing covalently linked high molecular weight species of monoclonal antibodies as taught by Remmele, as an obvious matter of using of known technique to analyze similar products in the same way.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because Haberger teaches that native mass spectrometry can separate antibody trimers and dimers, and Haberger, Xu, and Remmele are similarly drawn to analysis of antibody molecules.
Response to Arguments
Applicant's arguments filed May 27, 2026 have been fully considered.
Claims 1-4, 8-10, 19, and 21 rejected as being unpatentable over Haberger in view of Xu (Remarks, pg. 4, par. 4).
Applicant disagrees with this rejection because claim 1 was amended and step (c) now recites washing said column to collect an eluate comprising said at least one high molecular weight species” ((Remarks, pg. 4, par. 5).
Regarding the new limitation of claim 1, Haberger teaches that eluate includes at least one high molecular weight species – peak 1 represents the CrossMAb trimer and peaks 2 and 3 represent the CrossMAb dimers (pg. 334, col. 1, par. 1 and Fig. 3B). The trimer and dimers are high molecular weight species of the CrossMAb.
Applicant argues that “This method provides significant advantages over the methods described in both Haberger and Xu” (pg. 5, par. 2) because:
(a) “the method of claim 1 includes a step of denaturing the SEC-resolved HMW species prior to MS detection” (pg. 5, par. 4) - the argument is not persuasive because the combination of Haberger and Xu does teach the same advantage – the SEC-resolved HMW species are denatured prior to MS detection. Specifically, Xu teaches adding denaturing solution of m-nitrobenzyl alcohol, 50% ACN, and 0.1% formic acid to the liquid chromatography column eluate (Xu, pg. 231, col. 2, last par.);
(b) “Haberger instead teaches a native SEC-MS (nSEC-MS) method for characterizing HMW species of proteins” (pg. 5, last par.) - the argument is not persuasive because for the purposes of the final OA (January 26, 2026) Haberger was cited of its teaching of a method for characterizing at least one high molecular weight species of a protein of interest, said method comprising: a. obtaining a sample including said protein of interest and said at least one high molecular weight species; b. contacting said sample to a native size exclusion chromatography column; c. washing said column to collect an eluate; and e. subjecting said mixture to a mass spectrometer to characterize said at least one high molecular weight species. While Haberger does not teach MS under denatured conditions, it is not relied upon for that since it is taught by Xu (Xu, pg. 231, col. 2, last par.);
(c) “Xu teaches adding a solution comprising m-nitrobenzyl alcohol, 50% acetonitrile, and 0.1 % formic acid to the liquid chromatography column eluate, this solution is not used to denature the proteins because they are already denatured” (pg. 6, par. 3) - the argument is not persuasive because Xu teaches exactly what is recited in step (d) of claim 1 – “adding a denaturing solution to the eluate to form a mixture” (Xu, pg. 231, col. 2, last par.).
Applicant argues that “the advantages of the method of claim 1 would not have been
expected from the teachings of Haberger and Xu” (pg. 6, last par. – pg. 7, par. 2). 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., elucidating HMW species directly from unfractionated drug substance (DS) samples (pg. 7, par. 2) or meaningful differentiation between non-covalently bound and covalently bound HMW complexes (pg. 7, par. 3)) 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).
Claims 1-4, 8-10, 19, and 21 remain rejected as being unpatentable over Haberger in view of Xu.
Claims 5 and 15-18 were rejected under 35 U.S.C. § 103 as being unpatentable over Haberger in view of Xu, and further in view of Yan, as evidenced by Sanders and Newomics (Remarks, pg. 7, last par.).
Applicant argues that “Yan, Sanders, and Newomics, whether considered individually or in combination, do not remedy the deficiencies of Haberger and Xu discussed above. Accordingly, claims 5 and 15-18 are patentable over the cited references at least by their dependency from claim 1” (pg. 8, par. 2). The argument is not persuasive because as discussed above there are no deficiencies of Haberger and Xu that Yan, Sanders, and Newomics need to remedy; therefore, the rejections of claims 5 and 15-18 stay.
Claim 20 is rejected under 35 U.S.C. § 103 as being unpatentable over Haberger in view of Xu, and further in view of Remmele (Remarks, pg. 8, par. 4).
Applicant argues that “Remmele does not remedy the deficiencies of Haberger and Xu. Therefore, claim 20 is patentable over the cited references at least by virtue of its dependency from claim 1. The argument is not persuasive because the rejection of claim 1 stays as discussed in details above.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Alexander Volkov whose telephone number is (571) 272-1899. The examiner can normally be reached M-F 9:00AM-5:00PM (EST).
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bao-Thuy Nguyen can be reached on (571) 272-0824. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/ALEXANDER ALEXANDROVIC VOLKOV/
Examiner, Art Unit 1677
/REBECCA M GIERE/Primary Examiner, Art Unit 1677
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