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 .
DETAILED ACTION
Acknowledgement is hereby made of receipt and entry of the communication filed on Jun. 17, 2026. Claims 1-4, 6-7, 12, 14, 16, 18-20, 23-25, 30-32, 34 and 40 are pending and currently examined.
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.
(Previous Rejection – Withdrawn) Claims 1-4, 6-7, 12, 14, 16, 18-20, 23-25, 30-32, 34 and 40 were rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
This rejection is withdrawn in view of applicant’s clarification that “online denaturation” and “online desalting and denaturation” refer to desalting and denaturation processes performed within an integrated workflow in which the sample is transferred directly between the system components without manual collection or reinjection.
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 of this title, 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.
(Previous Rejection – Maintained) Claims 1-4, 6-7, 12, 14, 16, 18-20, 23-25 and 30-32 are rejected under 35 U.S.C. 103 as being unpatentable over Jin et al. (US 2021/0041451 A1, published on Feb. 11, 2021; submitted in IDS filed on Mar. 7, 2023) and Daud et al. (US 2023/0204595 A1, published on Jun. 29, 2023; PCT filed on Dec. 30, 2020) in view of Shen et al. (Anal. Chem. 2015, 87, 8555−8563).
Base claim 1 is amended to recite “using an online two-dimensional liquid chromatography mass spectrometry (2DLC- MS) system, the system comprising fluidly coupled components comprising a first-dimension chromatography column for a first-dimension chromatography, a valve for peak picking, a second-dimension chromatography column for a second-dimension chromatography, and a mass spectrometer”. Base claim 19 is amended to recite “using an online two-dimensional liquid chromatography mass spectrometry (2DLC-MS) system, the system comprising fluidly coupled components comprising an anion-exchange column for a first-dimension anion-exchange chromatography, a valve for peak picking, a reverse-phase chromatography column for a second-dimension reverse- phase chromatography or a hydrophilic interaction liquid chromatography column for a second- dimension hydrophilic interaction liquid chromatography, and a mass spectrometer”.
The step (a) of both claims specifies subjecting a sample of viral particles to a (first-dimension) chromatography to separate intact viral capsid components of the sample, reading on a process for obtaining isolated/purified viral particles to be applied to the succeeding steps.
Jin teaches an invention relating to methods for determining the serotype of a virus particle and/or determining the heterogeneity of a virus particle (e.g., an AAV particle). See Abstract. Jin teaches that the invention provides a method to determine the serotype of a viral particle comprising a) denaturing the viral particle, b) subjecting the denatured viral particle to liquid chromatography/mass spectrometry (LC/MS), and c) determining the masses of one or more capsid proteins of the viral particle; wherein the specific combination of masses of the one or more capsid proteins are indicative of the virus serotype. See [0010]-[0011]. Jin teaches that the liquid chromatography can be reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography. See [0012].
Jin teaches that the invention provides a method to determine the serotype of an adeno-associated virus (AAV) particle comprising a) denaturing the AAV particle, b) subjecting the denatured AAV particle to liquid chromatography/mass spectrometry (LC/MS), and c) determining the masses of VP1, VP2 and VP3 of the AAV particle; wherein the specific combination of masses of VP1, VP2, and VP3 are indicative of the AAV serotype. In some embodiments, the calculated masses of VP1, VP2 and VP3 are compared to the theoretical masses of VP1, VP2 and VP3 of one or more AAV serotypes. See [0017].
Jin teaches that a viral particle, such as an AAV particle, may be denatured using detergent, heat, high salt, or buffering with a low or high pH. In certain embodiments, an AAV particle may be denatured using acetic acid or guanidine hydrochloride. The skilled artisan will recognize that a variety of methods useful for promoting and/or monitoring protein denaturation are available in the art and may suitably select a denaturation method compatible with liquid chromatography/mass spectrometry. For example, if heat denaturation is used, care may be applied to avoid protein precipitation and reverse phase column clogging. Similarly, high salt denaturation may be coupled with a desalting step prior to LC/MS or LC/MS/MS. In other embodiments, high pH denaturation, low pH denaturation, or denaturation using organic solvents is used. See [0099].
Jin teaches that AAV particles may be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anionic exchange filtration; tangential flow filtration (TFF) for concentrating the AAV particles; AAV capture by apatite chromatography; heat inactivation of helper virus; AAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and AAV capture by anionic exchange chromatography, cationic exchange chromatography, or affinity chromatography. These steps may be used alone, in various combinations, or in different orders. See [0192].
Accordingly, Jin teaches/suggests a method for identifying constituents of capsid proteins of AAV (i.e., VP1, VP2, and VP3) of different serotypes, comprising: (a) subjecting a sample of viral particles (e.g., cell culture product of AAV) to a first(-dimension) chromatography (such as anionic exchange chromatography) for isolating/purifying AAV particles (i.e., intact viral capsids), (b) subjecting a (selected) portion of isolated/purified AAV particles (intact viral capsids) to denaturation (which may include desalting) to produce disassembled viral structural proteins VP1, VP2 and VP3 in a mixture, (c) subjecting the mixture of disassembled structural proteins to a second(-dimension) chromatography (which may be reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography) to separate the different viral structural proteins, and (d) determining the masses of the separated intact viral proteins to identify the viral protein constituents of the sample of viral particles by MS.
However, Jin is silent on if the process including the first chromatography (the first dimension), the second chromatography (the second dimension) and the denaturation process is performed “online”.
Daud teaches an invention relating to methods to characterize the VP1, VP2 and VP3 capsid proteins in an adeno-associated virus (AAV) particle using liquid chromatography mass spectrometry, and/or ultraviolet (UV)-visible spectroscopy. The methods generally include the steps of (a) subjecting an AAV particle to liquid chromatography to denature and then separate the VP1, VP2 and VP3 capsid proteins, and (b) subjecting the separated VP1, VP2 and VP3 capsid proteins produced in step (a) to UV and mass spectrometry to determine the ratio and masses of the VP1, VP2 and VP3 capsid proteins in the AAV particle. In another aspect, the disclosure provides an AAV composition comprising a post-translation modification. The disclosure also provides methods for characterizing the purity of AAV compositions using liquid chromatography mass spectrometry. See Abstract. Daud teaches that, in some aspects, the capsids on the AAV particle is denatured into the individual VP1, VP2 and VP3 proteins in the column of the liquid chromatography. In some aspects, the capsid proteins are separated by the liquid chromatography. See [0010].
Accordingly, Daud also teaches a process of characterizing AAV capsid proteins VP1, VP2 and VP3 by liquid chromatography and mass spectrometry (MS), including a process of obtaining individual VP1, VP2 and VP3 by denaturing the viral particles (capsids). Daud teaches that denaturing process can be done “in the column of the liquid chromatography”, indicating that it is contemplated and practiced to denature AAV particles “online” for obtaining disassembled VP proteins. See the interpretation of “online denaturation” in the 112(b) rejection above.
However, Jin and Daud are silent on performing the studies on a two-dimensional liquid chromatography-mass spectrometry (2D LC-MS) system, which is considered as an assay that combines two liquid chromatography processes and a mass spectrometry in one integrated system; and they are silent on if “a valve for peak picking” is included in the process.
Shen reports the development and validation of a robust LC-MS assay capable of quantifying therapeutic protein immunoglobulin A1 protease (IgAP) in human serum in the presence of pre-existing anti-IgAP antibodies. The procedure included sodium dodecyl sulfate (SDS) denaturation and chemical reduction of serum proteins to dissociate ADA-drug bindings, followed by tryptic digestion of protein pellets and subsequent LC-MS analysis of the surrogate IgAP peptide using stable isotope labeled peptide internal standard. Substantial enhancements in the sensitivity and selectivity were achieved by the combination of online two-dimensional reversed-phase LC (2D-LC) operated in high and low pH buffers, respectively, for efficient enrichment and quantitation of the surrogate peptide by multiple-reaction monitoring (MRM) mass spectrometry. See Abstract.
Shen teaches that an automated two-dimensional reversed-phase LC system (2D-LC) operated at high and low pH, respectively, was employed for enhanced separation of the surrogate peptide from matrix interference. The 2D-LC configuration (Figure 1) consisted of a Waters UPLC as the first dimension (Milford, MA) connected to a Shimadzu Nexera UHPLC as the second dimension (Columbia, MD,USA) through a six-port, two-position valve (Valco Inc., Austin, TX). See page 8557, left column, last para. Shen teaches that the 2D-LC system was coupled to an AB Sciex API 5500 Qtrap mass spectrometer equipped with a TurboIonspray source (Concord, Ontario, Canada) for peptide quantitation. The column eluent was ionized in the positive ionization and analyzed by the Qtrap analyzers in the MRM mode. See page 8557, right column, last para. Fig. 1 of Shen is shown below:
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Regarding the newly added limitation that the 2D-chromatography system comprises a valve for peak-picking, Shen teaches that valves are included in the 2D-LC platform. See Legend of Figure 1 above. Shen teaches that column switch was achieved by the six-port valve, which was placed initially in position 1 during the sample injection and gradient elution of the first LC at which the column eluent was diverted to waste (Figure 1); that the LC fraction containing the surrogate peptide and IS, eluting between 10.1 and 10.6 min from the first column, was directed to the second column by switching the valve to position 2 at 10.1 min; and that shortly after complete elution of the analyte from the first column and transfer to the second column, the valve was switched back to position 1 at 10.6 min, and gradient elution of the trapped peptides for the second column began. See page 8557, right column, para 2. Shen further teaches that analyst software (Version 1.5.2) was used for raw MRM data acquisition and chromatogram processing, as well as data regression using peak area ratios of the surrogate peptide to the IS. Calibration curves were constructed using peak area ratios of the calibration standards by applying a linear, 1/x weighted, least-squares regression algorithm. All calibration standards and QCs were then back calculated from their peak area ratios against the calibration line. Mean accuracy and precision statistics for QC samples were calculated using Microsoft Excel. See page 8558, left column, para 2.
Accordingly, Shen teaches the concept and practice of combining two liquid chromatography processes and mass spectrometry into one interconnected system for automation. Teachings of Shen also indicate that a valves system is included in the 2D-LC system to control the flow of the analytes in the sample in chromatography process and peaks of analytes are among the data analyzed by a specific software. Such diversion of unwanted fluid to waste and keeping fluid containing analytes (peaks) in the chromatography system by the valves is considered as peak-picking (see PGPub [0104] of the instant specification for interpretation of the phrase “a valve for peak picking”).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the current invention to combine the teachings of Jin, Daud and Shen to arrive at the invention as claimed. One would have been motivated to do so, e.g., to combine the processes of AAV particle denaturation, chromatography and mass spectrometry in one integrated system to streamline and/or automate the overall process. Since a valve system is often included in a 2D-LC platform, as shown in Shen, it would have also been obvious for one of skill in the art to include it in an invention as claimed, so that unwanted and wanted components may be separated by the valves in the LC/MS system.
Regarding claims 4 and 23, Jin teaches that non-limiting examples of AAV capsid proteins of the invention include VP proteins of any of the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrhSR, AAV9, etc. See [0177].
Regarding claims 6, 16 and 20, Jin teaches that AAV particle assembly may be measured using various assays known in the art, including without limitation, measuring particle production amount and/or rate, quantifying capsid production (e.g., after purification using any of the methods described herein), assaying production of complete vectors vs. empty capsids, measuring transduction efficiency, imaging or structural analysis to observe particle formation (e.g., using electron microscopy), production of AAV capsid proteins (e.g., as assayed by Western blotting), and the like. See [0164].
Regarding claims 12, 13, 24 and 25, Jin teaches that the accurate masses of VP1, VP2 and VP3 of each AAV serotype are unique and can be used to identify or differentiate AAV capsid serotypes. These methods are based in part on the discovery described herein that direct LC/MS of different types of AAVs after denaturation may be used to monitor the protein sequence and post-translational modifications with accurate mass measurement in the intact protein level. Further, acetylations of N-termini of VP1 and VP3 may also be identified and/or monitored in different AAV serotypes. See [0098].
Regarding claims 18 and 30, Jin teaches that AAV particle stability may be measured using various assays known in the art, including without limitation differential scanning fluorescence (DSF), differential scanning calorimetry (DSC), other thermal denaturation assays, susceptibility to proteolysis, imaging or structural analysis to observe denaturation (e.g., using electron microscopy), transduction efficiency or another functional assay on AAV particle compositions kept for a designated time interval at a particular temperature (e.g., room temperature, or 4° C., for thermal stability) or treated at a particular pH (e.g., pH stability), and the like. See [0163].
Regarding claim 31, Jin teaches that mass spectrometry (e.g., used in LC/MS as described herein) may refer to electrospray ionization mass spectrometry (ESI-MS). See [0102].
(Previous Rejection - Maintained) Claims 34 and 40 are rejected under 35 U.S.C. 103 as being unpatentable over Jin et al. (US 2021/0041451 A1, published on Feb. 11, 2021; submitted in IDS filed on Mar. 7, 2023) and Daud et al. (US 2023/0204595 A1, published on Jun. 29, 2023; PCT filed on Dec. 30, 2020) in view of Shen et al. (Anal. Chem. 2015, 87, 8555−8563), as applied above, and further in view of Wang et al. (Molecular Therapy: Methods & Clinical Development, 2019, Vol. 15, pp 257-263; submitted in IDS filed on Mar. 7, 2023).
Claims 34 and 40 further specify that the intact viral capsid components of the sample subjected to anion-exchange chromatography are separated by a mobile phase comprising bis-tris-propane (BTP), tetramethylammonium chloride (TMAC), and magnesium chloride at certain ranges of concentrations and time durations.
Relevance of Jin, Daud and Shen is set forth above. Jin further teaches that AAV particles may be isolated/purified by anionic exchange chromatography. See [0192]. However, Jin and Daud are silent on mobile phase conditions as claimed.
Wang teaches that the authors used AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full capsid percentages. The reported assay requires several microliters of material with a minimum titer of 5x1011 vg/mL, and it can detect the presence of as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples. See Abstract. Wang teaches that the separation was accomplished on a CIMac AAV full/empty-0.1-mL column (BIA Separations, Ajdovscina, Slovenia) using a salt gradient, delivered from four channels of the quaternary pump. The mobile phases were A, water; B, 1 M tetramethylammonium chloride; C, 20 mM MgCl2; and D, 250 mM Bis-tris propane (pH 9.0). A linear gradient was set as 62%A/10%C/28%D at t0, 37%A/25%B/10%C/28%D at 25 min, and 72%B/28%D at 30 min. The column was equilibrated at the initial condition for 6 min between injections. The separation was monitored by photodiode array detector at 260 and 280 nm and fluorescence detector with 280-nm excitation and 340-nm emission. See para spanning pages 261 and 262.
Accordingly, Wang teaches a method of separating AAV particles by anionic exchange chromatography and mobile phase conditions using the agents as claimed.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the current invention to combine the teachings of Jin, Daud, Shen and Wang to arrive at the invention as claimed. One would have been motivated to do so to use the mobile phases taught in Wang in the AAV particle separation in the studies taught in Jin and Daud. As to the claimed concentration ranges of BTP, TMAC and MgCl2 and ranges of time, one of skill in the art would have found it obvious to obtain them through routine experimental optimization based on settings and requirements of each specific study unless there is evidence that the claimed conditions are critical.
Response to Applicant’s Arguments
Applicant’s arguments filed on Jun. 17, 2026 have been fully considered. Arguments regarding withdrawn rejections are moot. Applicant’s arguments related to the current rejections are addressed as follows.
To the 103 rejections, Applicant argues that Jin does not teach an online denaturation step and that Daud does not remedy Jin's deficiencies. Applicant argues that Paragraph [0010] of Daud teaches subjecting AAV particles to liquid chromatography at temperatures of about 70°C to about 90°C is performed after the first-dimension Jin does not teach an online denaturation step. Daud does not remedy Jin's deficiencies. Paragraph [0010] of Daud teaches subjecting AAV particles to liquid chromatography at temperatures of about 70°C to about 90°C, and that therefore, a person of ordinary skill in the art would understand Daud to teach denaturation of the viral capsids as part of, and occurring during, the liquid chromatographic separation of the viral capsids. Applicant argues that under the conditions of Duan, the AAV capsids undergo denaturation and dissociation into their constituent proteins, VP1, VP2 and VP3, during the chromatographic separation.
Applicant argues that, even if Duan were combined with Jin, the resulting combination would not teach or suggest the particular method steps recited in claim 1, including the required order in which those steps are performed. Applicant argues that the first-dimension chromatography cannot be performed under denaturing conditions that cause the viral capsid to dissociate into its constituent proteins, and rather, the claimed method requires that intact viral capsid components first undergo chromatographic separation in the first dimension, followed by online denaturation of the separated capsid components into individual viral proteins, and then second-dimension chromatography of the resulting individual viral proteins. Applicant argues neither Jin nor Daud teaches or suggests this specific sequence of steps.
Applicant argues that Shen does not remedy the deficiencies of Jin and Daud, that Shen teaches offline denaturation of proteins using SDS and trypsin treatment during sample preparation prior to chromatographic separation. Applicant argues that Shen teaches offline denaturation as part of pre-treatment of the sample before LC-MS analysis, rather than online denaturation performed between first- and second-dimension chromatographic separation. Applicant argues that even if Shen were combined with Jin and Daud, the resulting combination would still fail to teach or suggest: separating intact viral capsid components in a first-dimension chromatography column; subsequently subjecting the separated capsid components to online denaturation; and thereafter separating the resulting intact viral proteins in a second-dimension chromatography column. Applicant argues that he cited references therefore do not teach or suggest the claimed method as a whole.
Applicant argues that as set forth in paragraph [0147] of the original specification, the method of claim 1 enables high-throughput, multi-attribute AAV characterization within a single system and that the advantages demonstrated by the claimed method are neither disclosed nor would they have been expected, because Jin, Daud, and Shen, whether considered individually or in any combination, fail to teach or suggest the recited method steps or their required order.
Applicant’s arguments are not persuasive. As indicated in the 103 rejection above, Jin teaches/suggests a method for identifying constituents of capsid proteins of AAV (i.e., VP1, VP2, and VP3) of different serotypes, comprising: (a) subjecting a sample of viral particles (e.g., cell culture product of AAV) to a first(-dimension) chromatography (such as anionic exchange chromatography) for isolating/purifying AAV particles (i.e., intact viral capsids), (b) subjecting a (selected) portion of isolated/purified AAV particles (intact viral capsids) to denaturation (which may include desalting) to produce disassembled viral structural proteins VP1, VP2 and VP3 in a mixture, (c) subjecting the mixture of disassembled structural proteins to a second(-dimension) chromatography (which may be reverse phase liquid chromatography, size exclusion chromatography, hydrophilic interaction liquid chromatography, or cation exchange chromatography) to separate the different viral structural proteins, and (d) determining the masses of the separated intact viral proteins to identify the viral protein constituents of the sample of viral particles by MS.
Jin already teaches all of the claimed elements except for integrating them in an “online” system. As indicated in Applicant’s clarification of the term “online” as referring processes being performed within an integrated workflow in which the sample is transferred directly between the system components without manual collection or reinjection.
Duan and Shen are cited for the concept and practice of incorporating liquid chromatography, sample denaturation, and MS analysis in an “integrated” “online” system. The rejections do not require combination of the specific denaturation method (e.g., the heat denaturation of AAV capsids in Duan, or SDS and trypsin treatment of proteins in Shen) with the teachings of Jin. Rather, the rejections only rely on the teachings of Duan and Shen that the steps of chromatography separations, denaturation and MS analysis can be integrated in an “online” system which is a known at the time of invention. One of skill in the art would have been motivated to integrate the steps “online” for automation. Moreover, one of skill in the art would have readily expected such an integration would improve the efficiency of the overall process by minimizing sample handling and reducing sample loss.
Conclusion
No claims are allowed.
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 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 NIANXIANG (NICK) ZOU whose telephone number is (571)272-2850. The examiner can normally be reached on Monday - Friday, 8:30 am - 5:00 pm, EST. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, MICHAEL ALLEN, on (571) 270-3497, can be reached. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/NIANXIANG ZOU/
Primary Examiner, Art Unit 1671
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