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 .
Election/Restrictions
Applicant’s election without traverse of claims 1-20 and 26-33 in the reply filed on 08/24/2026 is acknowledged.
Claims 21-25 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 08/24/2026.
Drawings
The drawings are objected to because the mass or m/z values on figures 3, 5A, and 5B are illegible. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, “The disclosure concerns,” “The disclosure defined by this invention,” “The disclosure describes,” etc. In addition, the form and legal phraseology often used in patent claims, such as “means” and “said,” should be avoided.
The abstract of the disclosure is objected to because it contains the implied phrase “The present invention generally pertains to…”; and it also appears to be too brief (currently ~45 words). A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
Claim Objections
Claim 1 objected to because of the following informalities: in line 4, the first instance of “nSEC” should be revised to “native size exclusion chromatography (SEC)” and “SEC” should be revised to “size exclusion chromatography (SEC)” for clarity. Appropriate correction is required.
Claim 17 is objected to because of the following informalities: in step (c) “dSEC” should be amended to “size exclusion chromatography under denaturing conditions (dSEC)” for clarity.
Claim 33 objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n). Accordingly, the claim has not been further treated on the merits.
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 4-5 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 4 recites the limitation “SEC eluent flow” in lines 1-2. There is insufficient antecedent basis for this limitation in the claim. In the interest of compact prosecution, Examiner interprets “SEC eluent flow” to mean “SEC eluate.”
Claim 5 recites the limitation "combined denaturing solution flow" in line 1. There is insufficient antecedent basis for this limitation in the claim. In the interest of compact prosecution, Examiner interprets “combined denaturing solution flow” to mean “denatured SEC eluate.”
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-8, 17-20, 26-27, and 30-32 are rejected under 35 USC § 101 for being directed to an abstract idea without significantly more. The claims will be analyzed below according to MPEP 2106.
Inquiry 1: Is the claim directed to a statutory category of invention (process, machine, manufacture, or composition of matter)?
Claims 1-8, 17-20, 26-27, and 30-32 are directed to processes.
Inquiry 2A Prong One: Does the claim recite an abstract idea, law of nature, or natural phenomenon?
Claim 1 recites “comparing said mass measurements to characterize said covalent cross-link,” which reads as an abstract idea that can occur in the human mind and/or with pen/paper by simply observing the mass measurements. Therefore, claim 1 is directed to a mental process, which is an abstract idea. See MPEP 2106.04(a)(2)(III).
Claims 2-8 are dependent on claim 1 and are therefore also drawn to an abstract idea.
Claim 17 recites “comparing said peptide masses to predicted masses of crosslinked peptides to identify said covalent cross-link,” which reads as an abstract idea that can occur in the human mind and/or with pen/paper by simply observing the mass measurements. Therefore, claim 1 is directed to a mental process, which is an abstract idea. See MPEP 2106.04(a)(2)(III).
Claims 18-20 are dependent on claim 17 and are therefore also drawn to an abstract idea.
Claim 26 recites “comparing said first set of peptide masses and said second set of peptide masses to identify said covalent cross-link,” which reads as an abstract idea that can occur in the human mind and/or with pen/paper by simply observing the mass measurements. Therefore, claim 1 is directed to a mental process, which is an abstract idea. See MPEP 2106.04(a)(2)(III).
Claim 27 is dependent on claim 26 and are therefore also drawn to an abstract idea.
Claim 30 recites “comparing said mass measurements to characterize said covalent cross-link” which reads as an abstract idea that can occur in the human mind and/or with pen/paper by simply observing the mass measurements. Therefore, claim 1 is directed to a mental process, which is an abstract idea. See MPEP 2106.04(a)(2)(III).
Claims 31-32 are dependent on claim 30 and are therefore also drawn to an abstract idea.
Inquiry 2A Prong Two: Does the claim recite additional elements that integrate the judicial exception into a practical application?
In each instance where a claim recites a judicial exception, there is/are no step(s) that occur afterwards, which indicates that the judicial exception is not integrated into a practical application.
Furthermore, the steps prior to the judicial exceptions amount to the insignificant extra-solution activity of mere data-gathering, which is insufficient for integrating the judicial exception into a practical application (see MPEP 2106.05(g)):
Claim 1 contains the additional steps (a)-(c), which pertain to a post-column denaturation-assisted native size exclusion chromatography mass spectrometry analysis (PCD nSEC-MS) of a covalent cross-link in a size variant of a protein of interest, but none of these steps involve/apply/integrate the judicial exception in a practical application (removing the judicial exception makes no difference on the steps prior); rather, they amount to mere data-gathering because they are meant to gather the mass data from PCD nSEC-MS.
Claims 2-8 are drawn to the steps prior to the judicial exception and therefore also amount to mere data-gathering.
Claim 17 contains the additional steps (a)-(f), which pertain to preparing a sample for LC-MS/MS and performing the analysis to identify crosslinking amino acid residues in a protein of interest, but none of these steps involve/apply/integrate the judicial exception in a practical application (removing the judicial exception makes no difference on the steps prior); rather, they amount to mere data-gathering because they are meant to gather the mass data from LC-MS/MS.
Claims 18-20 are drawn to the steps prior to the judicial exception and therefore also amount to mere data-gathering.
Claim 26 contains the additional steps (a)-(d), which pertain to preparing a sample for LC-MS using two different reducing agents and performing the LC-MS analysis to identify a covalent cross-link in a protein of interest, but none of these steps involve/apply/integrate the judicial exception in a practical application (removing the judicial exception makes no difference on the steps prior); rather, they amount to mere data-gathering because they are meant to gather the mass data from LC-MS.
Claims 27 is drawn to the steps prior to the judicial exception and therefore also amount to mere data-gathering.
Claim 30 contains the additional steps (a)-(f), which pertain to preparing a sample for PCD-assisted nSEC-MS and performing the PCD-assisted nSEC-MS analysis to identify a covalent cross-link in a HMW species of a therapeutic antibody, but none of these steps involve/apply/integrate the judicial exception in a practical application (removing the judicial exception makes no difference on the steps prior); rather, they amount to mere data-gathering because they are meant to gather the mass data from MS.
Inquiry 2B: Does the claim recite additional limitations that amount to significantly more than the judicial exception?
No they do not.
Firstly, for claims 1, 17, 26, and 30, the steps prior to the judicial exceptions amount to the insignificant extra-solution activity of mere data-gathering, which is insufficient for amounting to significantly more than the judicial exception (see MPEP 2106.05(g)):
Claim 1 contains the additional steps (a)-(c), which pertain to a post-column denaturation-assisted native size exclusion chromatography mass spectrometry analysis (PCD nSEC-MS) of a covalent cross-link in a size variant of a protein of interest, but they amount to mere data-gathering because they are meant to gather the mass data from PCD nSEC-MS.
Claims 2-8 are drawn to the steps prior to the judicial exception and therefore also amount to mere data-gathering.
Claim 17 contains the additional steps (a)-(f), which pertain to preparing a sample for LC-MS/MS and performing the analysis to identify crosslinking amino acid residues in a protein of interest, but they amount to mere data-gathering because they are meant to gather the mass data from LC-MS/MS.
Claims 18-20 are drawn to the steps prior to the judicial exception and therefore also amount to mere data-gathering.
Claim 26 contains the additional steps (a)-(d), which pertain to preparing a sample for LC-MS using two different reducing agents and performing the LC-MS analysis to identify a covalent cross-link in a protein of interest, but they amount to mere data-gathering because they are meant to gather the mass data from LC-MS.
Claims 27 is drawn to the steps prior to the judicial exception and therefore also amount to mere data-gathering.
Claim 30 contains the additional steps (a)-(f), which pertain to preparing a sample for PCD-assisted nSEC-MS and performing the PCD-assisted nSEC-MS analysis to identify a covalent cross-link in a HMW species of a therapeutic antibody, but they amount to mere data-gathering because they are meant to gather the mass data from MS.
Furthermore, the concepts in claims 1-7, 17-20, 26-27, and 30-32 are well-understood, routine, and conventional in the field of endeavor: Yan et al. (J. Am. Soc. Mass Spectrom., Vol. 32, pgs. 2885-2894, 17 November 2021, As cited on the IDS submitted on 06/17/2024) teaches subjecting a protein sample to reduction/digestion/denaturation/deglycosylation and native size exclusion chromatography for the characterization of covalent cross-links in monoclonal antibodies via mass spectrometry based on mass comparisons; Xu et al. (Anal. Chem., Vol. 89, pg. 7915-7923, 21 June 2017, as submitted in the IDS submitted on 06/17/2024) teaches of preparing protein samples with reduction/alkylation/stabilization/digestion and characterizing imine covalent crosslinks (and other crosslinks involving His, Ser, and Lys) in proteins of interest via SEC-MS and RPLC-MS; She et al. (Proteomics, Vol. 12, pg. 369-379, 5 December 2011) teaches alkylating and reducing/stabilizing a protein sample prior to LC-MS/MS as well as repeating such methods using both NaBH3CN and the heavier isotope NaBD3CN as stabilizers; Paulech et al. (Biochemica et Biophysica Acta, Vol. 1834, pg. 372-379, January 2013) teaches alkylating protein samples prior to LC-MS/MS studies. See MPEP 2106.05(d).
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1-6, 9, 10-12, 15, and 28-29 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Yan et al. (J. Am. Soc. Mass Spectrom., Vol. 32, pgs. 2885-2894, 17 November 2021, As cited on the IDS submitted on 06/17/2024).
Regarding claim 1, Yan teaches a method for characterizing a covalent cross-link in a size variant of a protein of interest (pg. 2892 right column second paragraph) (more accurate mass measurement of the nondissociable HMW complexes can be obtained, and therefore, facilitate the identification of the potential crosslinks), comprising:
(a) subjecting a sample including a protein of interest and a size variant thereof to nSEC to form an SEC eluate (pg. 2887 left column second paragraph) (Native SEC chromatography was performed… to separate and elute protein size variants);
(b) contacting said SEC eluate to a denaturing solution to form a denatured SEC eluate (pg. 2887 left column second paragraph) (To enable postcolumn denaturation, a denaturing solution…mixed with the SEC eluent (1:1 mixing) using a T-mixer);
(c) subjecting said denatured SEC eluate to mass spectrometry analysis to obtain at least one mass measurement of said protein of interest and at least one mass measurement of said size variant (pg. 2887 left column second paragraph) (microflow-nanospray electrospray ionization (MnESI) source and a microfabricated monolithic multinozzle (M3) emitter… for native MS analysis); and
(d) comparing said mass measurements to characterize said covalent cross-link (pg. 2888 right column first paragraph and pg. 2889 right column bottom paragraph) (under PCD conditions, the MS signal of the dissociated HC and LC were well isolated on the m/z scale with minimal overlapping (Figure 2a) and This delta mass was proposed to correspond to a previously reported covalent cross-link that occurs between two histidine (His) residues (cross-linker mass: 13.98 Da). Subsequent peptide mapping analysis also identified several His−His cross-linked dipeptides).
Regarding claim 2, Yan teaches the method of claim 1 as rejected above, wherein said protein of interest is an antibody, a bispecific antibody, an antibody fragment, an antibody-drug conjugate, a fusion protein, or a recombinant protein (abstract) (native SECMS method that allows rapid and in-depth characterization of mAb HMW species directly from unfractionated DS samples). Note that “mAb” is a therapeutic monoclonal antibody. See also figure 5 of Yan that shows the analysis of antibody fragments (“F(ab)’2” subdomain).
Regarding claim 3, Yan teaches the method of claim 1 as rejected above, wherein said denaturing solution comprises acetonitrile and formic acid (pg. 2887 left column second paragraph) (a denaturing solution consisting of 60% ACN, 36% water, and 4% FA was delivered). Note that ACN is acetonitrile and FA is formic acid.
Regarding claim 4, Yan teaches the method of claim 1 as rejected above, wherein said denaturing solution is introduced into the SEC eluent flow using a T-mixer (pg. 2887 starting at left column bottom paragraph) (introducing a postcolumn denaturant flow (0.2 mL/min) to the nSEC flow (0.2 mL/min) via a T-mixer (Figure 1)).
Regarding claim 5, Yan teaches the method of claim 4 as rejected above, wherein said combined denaturing solution flow and SEC eluent flow is split into a low flow for mass spectrometry detection and a high flow for UV detection (pg. 2887 left column second paragraph) (the combined analytical flow (0.4 mL/min) was split into a microflow (<10 μL/min) for nanoelectrospray ionization (NSI)-MS detection and a remaining high flow for UV detection).
Regarding claim 6, Yan teaches the method of claim 1 as rejected above, wherein said mass spectrometry analysis comprises nano- electrospray ionization (pg. 2887 left column second paragraph) (microflow-nanospray electrospray ionization (MnESI) source… for native MS analysis).
Regarding claim 9, Yan teaches a method for identifying crosslinking amino acid residues in a protein of interest (pg. 2886 right column second paragraph) (this strategy… confirming the identities of the constituent subunits dissociated from the noncovalent HMW complexes… reveal both the interaction nature and interaction interfaces of mAb aggregates at subdomain levels), comprising:
(a) subjecting a sample including a protein of interest and a crosslinked variant thereof to chromatographic separation to form an enriched variant sample (pg. 2887 left column second paragraph) (Native SEC chromatography was performed… to separate and elute protein size variants);
(b) subjecting said enriched variant sample to digestion conditions to form a peptide digest (pg. 2886 right column bottom paragraph) (For subdomain analysis, an aliquot of the deglycosylated mAb3 HMW sample and mAb4 DS samples was each subjected to site-specific digestion with FabRICATOR); and
(c) subjecting said peptide digest to LC-MS analysis (pg. 2887 left column second paragraph) (microflow-nanospray electrospray ionization (MnESI) source and a microfabricated monolithic multinozzle (M3) emitter… for native MS analysis) to identify cross-linking amino acid residues (pg. 2889 right column second paragraph) (This delta mass… correspond to a previously reported covalent cross-link that occurs between two histidine (His) residues (cross-linker mass: 13.98 Da). Subsequent peptide mapping analysis also identified several His−His cross-linked dipeptides).
Regarding claim 10, Yan teaches the method of claim 9 as rejected above, wherein said crosslinked variant is a dimer (pg. 2889 right column second paragraph) (This delta mass… correspond to a previously reported covalent cross-link that occurs between two histidine (His) residues (cross-linker mass: 13.98 Da). Subsequent peptide mapping analysis also identified several His−His cross-linked dipeptides); see also figure 2b which shows the mAb2 dimer that was analyzed.
Regarding claim 11, Yan teaches the method of claim 9 as rejected above, wherein said chromatographic separation is selected from a group consisting of reverse phase liquid chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, and mixed-mode chromatography (pg. 2887 left column second paragraph) (Native SEC chromatography was performed… to separate and elute protein size variants).
Regarding claim 12, Yan teaches the method of claim 9 as rejected above, wherein subjecting said enriched variant sample to digestion conditions includes contacting said enriched variant sample to at least one digestive enzyme (pg. 2886 right column bottom paragraph) (For subdomain analysis, an aliquot of the deglycosylated mAb3 HMW sample and mAb4 DS samples was each subjected to site-specific digestion with FabRICATOR). Note that FabRICATOR® is a commercially available protease.
Regarding claim 15, Yan teaches the method of claim 9 as rejected above, wherein at least one of said crosslinking amino acid residues is an oxidized variant of an amino acid residue (pg. 2889 left column first paragraph) (This mass increase can be potentially attributed to… an oxidation modification (+16 Da) within a noncovalent complex).
Regarding claim 28, method for detecting a covalently bound HMW species of a therapeutic antibody, comprising:
(a) subjecting a sample including a therapeutic antibody and at least one covalently bound HMW species of said therapeutic antibody to SEC to form at least one fraction including said at least one HMW species (pg. 2886 right column bottom paragraph) (The mAb3 enriched HMW sample was generated by fractionating the HMW species from a mAb3 DS sample using a semipreparation scale SEC column);
(b) collecting said at least one fraction to form an enriched HMW sample (pg. 2886 right column bottom paragraph) (The mAb3 enriched HMW sample was generated by fractionating the HMW species from a mAb3 DS sample using a semipreparation scale SEC column);
(c) subjecting said enriched HMW sample to deglycosylating conditions to form a deglycosylated sample (pg. 2886 right column bottom paragraph) (all mAb samples, including enriched HMW samples…, were treated with PNGase F… to remove the N-glycan chains); and
(d) subjecting said deglycosylated sample to post-column denaturation (PCD)- assisted nSEC-MS analysis to detect said covalently bound HMW species (pg. 2889 right column second paragraph) (PCD was implemented post-SEC separation).
Regarding 29, The method of claim 28, wherein subjecting said enriched HMW sample to deglycosylating conditions comprises contacting said enriched HMW sample to PNGase F (pg. 2886 right column bottom paragraph) (all mAb samples, including enriched HMW samples…, were treated with PNGase F… to remove the N-glycan chains).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 13 rejected over Yan et al. (J. Am. Soc. Mass Spectrom., Vol. 32, pgs. 2885-2894, 17 November 2021, As cited on the IDS submitted on 06/17/2024), as applied to 12 above.
Regarding claim 13, Yan teaches the method of claim 12 as rejected above. Yan teaches the use of PNGase F as a digestive enzyme (pg. 2886 right column bottom paragraph) (all mAb samples, including enriched HMW samples, individual DS samples, and coformulated DP samples, were treated with PNGase F). Yan teaches that LysC is also known in the art to be used as a digestive enzyme (2886 left column first paragraph) (Limited enzymatic digestion (e.g., IdeS digestion and limited Lys-C digestion) followed by mass spectrometry (MS) analysis has also proven effective in determining the aggregation interfaces at subdomain levels based on accurate mass measurement).
Yan does not clearly teach within a single embodiment wherein said at least one digestive enzyme is selected from a group consisting of trypsin, chymotrypsin, pepsin, Tryp-N, LysN, LysC, Asp-N, Arg- C, Glu-C, and papain.
However, it would have been obvious to a person having ordinary skill in the art before the effective filing date to substitute PNGase F with LysC because both are acceptable digestion enzymes that are used in characterization of proteins and LysC is particularly useful in elucidating aggregation interfaces at subdomain levels of monoclonal antibodies (see pg. 2886 right column bottom paragraph 2886 left column first paragraph of Yan). See MPEP 2143(I)(B).
Claims 7-8, 14 and 16 rejected over Yan et al. (J. Am. Soc. Mass Spectrom., Vol. 32, pgs. 2885-2894, 17 November 2021, as cited on the IDS submitted on 06/17/2024), as applied to claims 1 and 9 above, further in view of Xu et al. (Anal. Chem., Vol. 89, pg. 7915-7923, 21 June 2017, as submitted in the IDS submitted on 06/17/2024).
Regarding claim 7, Yan teaches the method of claim 1 as rejected above. Yan teaches of analyzing histidine-histidine covalent cross-links (pg. 2889 left column first paragraph) (This mass increase can be potentially attributed to… a covalent cross-link (e.g., 14 Da between two histidine residues)).
In the analogous art of MS-based characterization of proteins, Xu teaches that when two histidine residues on mAb form a crosslink, then the crosslink comprises an imine; see annotated graphical abstract of Xu:
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Xu also teaches that imine cross-links are of interest in mAb analysis because lysine forms imine bonds with oxidized amino acid residues (pg. 7915 right column first paragraph) (imine crosslink, which is formed by the reaction of carbonyls derived from protein oxidation with primary amines of lysine residues).
It would have been obvious to a person having ordinary skill in the art to modify the analysis of Histidine-Histidine crosslinking of Yan to search for imine bonds as suggested by Xu because it would lead to detecting a larger variety of crosslinks involving additional amino acids such as lysine (see pg. 2889 left column first paragraph of Yan; pg. 7915 right column first paragraph and graphical abstract of Xu).
Regarding claim 8, Yan teaches the method of claim 7 as rejected above. Yan teaches of analyzing histidine-histidine covalent cross-links (pg. 2889 left column first paragraph) (This mass increase can be potentially attributed to… a covalent cross-link (e.g., 14 Da between two histidine residues)).
In the analogous art of MS-based characterization of proteins, Xu teaches that when two histidine residues on mAb form a crosslink, then the crosslink comprises an imine; see annotated graphical abstract of Xu:
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Xu also teaches that imine cross-links are of interest in mAb analysis because lysine forms imine bonds with oxidized amino acid residues (pg. 7915 right column first paragraph) (imine crosslink, which is formed by the reaction of carbonyls derived from protein oxidation with primary amines of lysine residues).
It would have been obvious to a person having ordinary skill in the art to modify the Histidine-Histidine crosslinking analysis of Yan to characterize imine bonds as suggested by Xu because it would lead to detecting a larger variety of crosslinks involving additional amino acids such as lysine with a reasonable expectation of success (see pg. 2889 left column first paragraph of Yan; pg. 7915 right column first paragraph and graphical abstract of Xu).
Regarding claim 14, Yan teaches the method of claim 9 as rejected above. Yan teaches of analyzing reduced antibodies using SEC-MS (pg. 2886 right column bottom paragraph) (Prior to desalting SEC-MS analysis, limited reduction was performed by treating mAb1 with 2 mM DTT in 50 mM Tris-HCl (pH 7.5) at 37 °C for 30 min to only reduce interchain disulfide bonds).
Yan does not clearly teach wherein said LC-MS analysis comprises RPLC-MS/MS analysis.
In the analogous art of MS-based characterization of proteins, Xu teaches of analyzing a protein sample comprising a monoclonal antibody using RPLC-MS (pg. 7916 right column second paragraph) (Reversed Phase LC-MS (RPLC-MS) of the Deglycosylated and Reduced mAb… TSKgel Phenyl-5PW HPLC column). Xu teaches that both SEC-MS and RPLC-MS may both be used to analyze reduced antibodies (pg. 7918 left column second paragraph) (analyses using RPLC-MS of deglycosylated and reduced antibody and SEC-MS of IdeS digested and reduced antibody).
It would have been obvious to a person having ordinary skill in the art to substitute the SEC-MS analytical method of Yan with the RPLC-MS analytical method of Xu because both techniques are known in the art to be effective at analyzing protein samples comprising reduced antibodies (pg. 2886 right column bottom paragraph of Yan; pg. 7916 right column second paragraph, pg. 7918 left column second paragraph of Xu).
Regarding claim 16, Yan teaches the method of claim 9 as rejected above. Yan teaches of analyzing crosslinking histidine residues (pg. 2889 right column bottom paragraph) (peptide mapping analysis also identified several His−His cross-linked dipeptides).
Yan does not clearly teach within a single embodiment wherein said crosslinking amino acid residues are selected from a group consisting of lysine, serine, threonine, and modified variants thereof.
In the analogous art of MS-based characterization of proteins, Xu teaches analyzing crosslinking lysine residues (abstract) (Novel cross-links between an oxidized histidine and intact histidine, lysine, or cysteine residues were discovered… of an IgG1 monoclonal antibody (mAb)… by size-exclusion chromatography coupled with mass spectrometry (SEC-MS)). Xu teaches that the analysis of lysine because it forms various cross-links in monoclonal antibodies (abstract of Xu) (histidine−cysteine (His−Cys) and histidine−lysine (His−Lys) in addition to histidine−histidine (His−His) cross-links were discovered in monoclonal antibody HMW species).
It would have been obvious to a person having ordinary skill in the art to combine the analysis of histidine crosslinks as taught by Yan with lysine crosslinks as taught by Xu because histidine and lysine are both capable of forming crosslinks in monoclonal antibodies (pg. 2889 right column bottom paragraph of Yan; abstract of Xu).
Claims 17-20 and 26-27 are rejected under 35 USC 103 as being unpatentable over Yan et al. (J. Am. Soc. Mass Spectrom., Vol. 32, pgs. 2885-2894, 17 November 2021, As cited on the IDS submitted on 06/17/2024) further in view of She et al. (Proteomics, Vol. 12, pg. 369-379, 5 December 2011).
Regarding claim 17, Yan teaches a method for identifying a covalent cross-link in a HMW species of a therapeutic antibody (pg. 2889 right column bottom paragraph) (peptide mapping analysis also identified several His−His cross-linked dipeptides), comprising:
(a) subjecting a sample including a therapeutic antibody and at least one HMW species of said therapeutic antibody to SEC to form an enriched HMW sample (pg. 2887 left column second paragraph) (Native SEC chromatography was performed… to separate and elute protein size variants);
(b) subjecting said enriched HMW sample to complete or partial reduction to form a reduced HMW sample (pg. 2886 right column bottom paragraph) (Prior to desalting SEC-MS analysis, limited reduction was performed by treating mAb1 with 2 mM DTT in 50 mM Tris-HCl (pH 7.5) at 37 °C for 30 min to only reduce interchain disulfide bonds);
(c) subjecting said reduced HMW sample to dSEC to form a very enriched HMW sample (pg. 2887 right column second paragraph) (mAb1 (IgG4 subclass) was partially reduced (interchain disulfide bonds disrupted) and subjected to PCD-assisted nSEC-MS analysis using a short SEC guard column);
(d) subjecting said very enriched HMW sample to digestive conditions to form a peptide digest (pg. 2886 right column bottom paragraph) (all mAb samples… were treated with PNGase F);
(f) subjecting said stabilized peptide digest to LC-MS analysis to obtain peptide masses (pg. 2887 right column second paragraph) (mAb1 (IgG4 subclass)… PCD-assisted nSEC-MS analysis); and
(g) comparing said peptide masses to predicted masses of crosslinked peptides to identify said covalent cross-link (pg. 2889 right column bottom paragraph) (This delta mass was proposed to correspond to a previously reported covalent cross-link that occurs between two histidine (His) residues (cross-linker mass: 13.98 Da)… peptide mapping analysis also identified several His−His cross-linked dipeptides).
Yan teaches that lysine is a residue of interest in the context of characterization of proteins (pg. 2886 left column first paragraph) (Limited enzymatic digestion… Lys-C digestion… has also proven effective in determining the aggregation interfaces at subdomain levels). Lys-C is a commercially available enzyme that specifically targets lysine.
Yan does not clearly teach (b) subjecting said enriched HMW sample to alkylation to form a reduced HMW sample (e) subjecting said peptide digest to stabilizing conditions to form a stabilized peptide digest nor (f) subjecting said stabilized peptide digest to LC-MS/MS analysis to obtain peptide masses.
In the analogous art of MS-based characterization of proteins, She teaches subjecting peptide samples to alkylation (pg. 371 left column second paragraph) (protein mixture… incubated in 55 mM iodoacetamide) and stabilizing conditions (pg. 371 left column second paragraph) (Equal volumes of 0.6 M NaCNBH3 (light, L) or NaCNBD3 (heavy, H) and 4% CH2O or 4%CD2O were added in sequence and protein solution), then subjecting that sample to LC-MS/MS (pg. 371 right column first paragraph) (LC MS/MS analysis was performed on a Nano-Acquity ultra-performance liquid chromatography system). She teaches that the alkalizing and stabilizing protein samples prior to LC-MS/MS is advantageous because it is highly efficient at reacting with lysine sites and it causes all the lysine residues on a protein of interest to be detectable (starting at pg. 372 right column bottom paragraph) (all lysine-containing peptides were modified by dimethylation… The high efficient labeling of intact proteins by reductive dimethylation… attributed to the small size of the chemical reagents (formaldehyde, sodium cyanoborohydride)… resulting in a full coverage of all lysine sites).
It would have been obvious to a person having ordinary skill in the art to combine the analysis method of Yan as described above with the stabilization and LC-MS/MS method of She as described above to provide: subject a sample including a therapeutic antibody and at least one HMW species of said therapeutic antibody to SEC to form an enriched HMW sample; reduce and alkylate an enriched HMW sample; subjecting said very enriched HMW sample to digestive conditions to form a peptide digest; subjecting said peptide digest to stabilizing conditions to form a stabilized peptide digest; subjecting said stabilized peptide digest to LC-MS/MS analysis to obtain peptide masses; comparing said peptide masses to predicted masses of crosslinked peptides to identify said covalent cross-link. Doing so would lead to the highly efficient and thorough characterization of lysine sites so as to better understand aggregation interfaces at the subdomain level of therapeutic antibodies with a reasonable expectation of success (see pg. 2886-2887 and pg. 2889 right column bottom paragraph of Yan; pg. 371 right column first paragraph of She).
Regarding claim 18, Modified Yan (Yan in view of She) teaches the method of claim 17 as rejected above. Modified Yan teaches wherein said partial reduction disrupts the inter-chain disulfide bonds of said therapeutic antibody (pg. 2886 right column bottom paragraph of Yan) (Prior to desalting SEC-MS analysis, limited reduction was performed by treating mAb1 with 2 mM DTT in 50 mM Tris-HCl (pH 7.5) at 37 °C for 30 min to only reduce interchain disulfide bonds).
Regarding claim 19, Modified Yan (Yan in view of She) teaches the method of claim 17 as rejected above, wherein subjecting said enriched HMW sample to complete or partial reduction comprises contacting said enriched HMW sample to a reducing agent selected from the group consisting of dithiothreitol, 3-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, and tris(2-carboxyethyl)phosphine hydrochloride (pg. 2886 right column bottom paragraph of Yan) (limited reduction was performed by treating mAb1 with 2 mM DTT). Note that “DTT” stands for dithiothreitol.
Regarding claim 20, Modified Yan (Yan in view of She) teaches the method of claim 17 as rejected above, wherein subjecting said peptide digest to stabilizing conditions comprises contacting said peptide digest to NaBH3CN (pg. 371 left column second paragraph of She) (Equal volumes of 0.6 M NaCNBH3 (light, L) or NaCNBD3 (heavy, H) and 4% CH2O or 4%CD2O were added in sequence and protein solution).
Regarding claim 26, Yan teaches a method for identifying a covalent cross-link in a crosslinked variant of a protein of interest, comprising:
(a) contacting a sample including a crosslinked variant of a protein of interest to a first reducing agent to form a reduced sample (pg. 2886 right column bottom paragraph) (Prior to desalting SEC-MS analysis, limited reduction was performed by treating mAb1 with 2 mM DTT in 50 mM Tris-HCl (pH 7.5) at 37 °C for 30 min to only reduce interchain disulfide bonds);
(b) subjecting said reduced sample to digestion conditions to form a peptide digest (pg. 2886 right column bottom paragraph) (all mAb samples… were treated with PNGase F);
(c) subjecting said peptide digest to LC-MS analysis to obtain a first set of peptide masses (pg. 2887 left column second paragraph) (Native SEC chromatography was performed… to separate and elute protein size variants);
(e) comparing peptide masses to identify said covalent cross-link (pg. 2888 right column first paragraph and pg. 2889 right column bottom paragraph) (under PCD conditions, the MS signal of the dissociated HC and LC were well isolated on the m/z scale with minimal overlapping (Figure 2a) and This delta mass was proposed to correspond to a previously reported covalent cross-link that occurs between two histidine (His) residues (cross-linker mass: 13.98 Da). Subsequent peptide mapping analysis also identified several His−His cross-linked dipeptides).
Yan does not teach: (d) repeating steps (a)-(c) using a second reducing agent to obtain a second set of peptide masses, wherein said second reducing agent includes a heavy isotope that is incorporated into said covalent cross-link; (e) comparing said first set of peptide masses and said second set of peptide masses to identify said covalent cross-link.
In the analogous art of MS-based characterization of proteins, She teaches:
(d) repeating steps (a)-(c) using a second reducing agent to obtain a second set of peptide masses, wherein said second reducing agent includes a heavy isotope that is incorporated into said covalent cross-link (pg. 371 left column second paragraph of She) (Equal volumes of 0.6 M NaCNBH3 (light, L) or NaCNBD3 (heavy, H) and 4% CH2O or 4%CD2O were added in sequence and protein solution) (Note that NaCNBD3 is a heavy isotope of NaCNBH3, and both are reducing agents that reduce amino acid residues and incorporate hydrogen/deuterium into a cross-link); and
(e) comparing said first set of peptide masses and said second set of peptide masses (pg. 372 left column third paragraph) (The identified peptides from MASCOT MS/MS search were directly imported into MASCOT Distiller to generate a quantification report of heavy/light (H/L) ratios).
She teaches that using a second reducing agent to obtain a second set of peptide masses, wherein said second reducing agent includes a heavy isotope, is advantageous because it introduces slight differences in the retention times of the analyzed peptide samples, which improves resolution and allows for the quantitative characterization of relevant residues (pg. 373 left column second paragraph) (a slight shift in peak retention times (RTs) between the light and heavy pairs of multiple lysine-containing peptides was detected (Fig. 1B), yielding differences of the H/L ratios).
It would have been obvious to a person having ordinary skill in the art to combine the method of Yan as described above with the method of She to provide: (a) contacting a sample including a crosslinked variant of a protein of interest to a first reducing agent to form a reduced sample; (b) subjecting said reduced sample to digestion conditions to form a peptide digest; (c) subjecting said peptide digest to LC-MS analysis to obtain a first set of peptide masses; (d) repeating steps (a)-(c) using a second reducing agent to obtain a second set of peptide masses, wherein said second reducing agent includes a heavy isotope that is incorporated into said covalent cross-link; and (e) comparing said first set of peptide masses and said second set of peptide masses to identify said covalent cross-link. Doing so would improve the resolution of peptide separations and allow for the quantitative characterization of relevant residues with a reasonable expectation of success (pg. 2886 right column bottom paragraph, pg. 2887 left column second paragraph, pg. 2888 right column first paragraph, and pg. 2889 right column bottom paragraph of Yan; pg. 371 left column second paragraph, pg. 372 left column third paragraph, pg. 373 left column second paragraph of She).
Regarding claim 27, Modified Yan (Yan in view of She) teaches the method of claim 26 as rejected above, wherein said first reducing agent is NaBH3CN and said second reducing agent is NaBD3CN (pg. 371 left column second paragraph) (NaCNBH3 (light, L) or NaCNBD3 (heavy, H) … were added in sequence and protein solution).
Claims 30-32 rejected in view of Yan et al. (J. Am. Soc. Mass Spectrom., Vol. 32, pgs. 2885-2894, 17 November 2021, As cited on the IDS submitted on 06/17/2024) further in view of Paulech et al. (Biochemica et Biophysica Acta, Vol. 1834, pg. 372-379, January 2013).
Regarding claim 30, Yan teaches a method for characterizing a covalent cross-link in a HMW species of a therapeutic antibody, comprising:
(a) subjecting a sample including a therapeutic antibody and at least one HMW species of said therapeutic antibody to SEC to form at least one fraction including said at least one HMW species (pg. 2886 right column bottom paragraph) (The mAb3 enriched HMW sample was generated by fractionating the HMW species from a mAb3 DS sample using a semipreparation scale SEC column);
(b) collecting said at least one fraction to form an enriched HMW sample (pg. 2886 right column bottom paragraph) (The mAb3 enriched HMW sample was generated by fractionating the HMW species from a mAb3 DS sample using a semipreparation scale SEC column);
(c) subjecting said enriched HMW sample to deglycosylating conditions to form a deglycosylated sample (pg. 2886 right column bottom paragraph) (all mAb samples, including enriched HMW samples…, were treated with PNGase F… to remove the N-glycan chains);
(d) subjecting said deglycosylated sample to digestive conditions to form a fragmented sample (pg. 2886 right column bottom paragraph) (For subdomain analysis, an aliquot of the deglycosylated mAb3 HMW sample and mAb4 DS samples was each subjected to site-specific digestion with FabRICATOR) (FabRICATOR is a commercially available protease);
(e) subjecting said fragmented sample to complete or partial reduction to form a reduced sample (pg. 2886 right column bottom paragraph) (Prior to desalting SEC-MS analysis, limited reduction was performed by treating mAb1 with 2 mM DTT);
(f) subjecting said reduced sample to PCD-assisted nSEC-MS analysis to obtain at least one mass measurement of at least one subunit dimer of said HMW species and at least one mass measurement of each of the component subunits of said at least one subunit dimer (pg. 2889 right column second paragraph) (PCD was implemented post-SEC separation… under PCD conditions distinctive dissociation behaviors were observed for the Fc dimer species); and
(g) comparing said mass measurements to characterize said covalent cross-link (pg. 2889 right column second paragraph) (the nondissociable Fc/2 dimer also showed a mass increase of approximately 14 Da compared to that of a noncovalent Fc/2 dimer).
Yan does not teach subjecting said fragmented sample to alkylation to form a reduced sample.
In the analogous art of characterizing digested peptides, Paulech teaches subjecting a fragmented sample to alkylation to form a reduced sample (pg. 373 left column bottom paragraph) (Protein solutions were alkylated with NEM). Paulech teaches that alkylating reduced protein samples is effective because it simplifies peptide identification via MS and prevents unwanted side-reactions (pg. 376 right column second paragraph) (Alkylation of Cys to a thioether simplifies peptide identification in MS and MS/MS by narrowing the range of chemical states Cys may inhabit. Following reduction/alkylation, Cys may no longer form or reform disulfides, thiosulfinates/onates, oxyacids or β-eliminated products).
It would have been obvious to a person having ordinary skill in the art to modify the reduction step (e) of Yan to include an alkylation step as taught by Paulech because doing so would simplify peptide identification via mass spectrometry and prevent the cysteine residues from undergoing unwanted side reactions with a reasonable expectation of success (see pg. 2886 right column bottom paragraph of Yan; pg. 373 left column bottom paragraph and pg. 376 right column second paragraph of Paulech).
Regarding claim 31, Modified Yan (Yan in view of Paulech) teaches the method of claim 30 as rejected above, wherein subjecting said deglycosylated sample to digestive conditions comprises contacting said deglycosylated sample to IdeS or a variant thereof (pg. 2888 right column second paragraph of Yan) (Limited enzymatic digestion (e.g., IdeS digestion) followed by intact mass analysis is frequently performed on the enriched HMW).
Regarding claim 32, Modified Yan (Yan in view of Paulech) teaches the method of claim 30 as rejected above, wherein said fragmented sample comprises antibody subunits, wherein said subunits are selected from a group consisting of Fab fragments, Fab' fragments, Fab2 fragments, F(ab')2 fragments, Fc fragments, Fc/2 fragments, Fv fragments, Fd fragments, Fd' fragments, and combinations thereof (pg. 2889 left column first paragraph of Yan and pg. 2889 right column second paragraph of Yan) (SEC-UV analysis of the digested HMW sample (Figure 3, middle) exhibited… F(ab)′2 and Fc and Fc/2 dimer also showed a mass increase of approximately 14 Da compared to that of a noncovalent Fc/2 dimer).
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Gygi et al. (US 20040259164 A1) teaches methods of modifying proteins via chemical modifying agents and then analyzing them via MS, including heavy isotope labels and other labels.
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/H.D.C./Examiner, Art Unit 1758
/MARIS R KESSEL/Supervisory Patent Examiner, Art Unit 1758
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