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 .
Status of the Claims
Claims 1, 53, 56-57, 60-69, and 75-77 were pending. Claims 54-55 were withdrawn.
Claims 53-55 are canceled.
Claims 1, 56-57, 60-69, and 75-77 are examined herein.
Withdrawn Rejections
The objection to claims 53-55 is withdrawn in view of claims cancelation.
The rejection of claims 1, 56-57, 60-69, and 75-77 under 112(b) is withdrawn in view of claim 1 amendments.
The rejection of claim 53 is withdrawn in view of claim cancelation.
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, 60-69 and 75-77 are rejected under 35 U.S.C. 103 as being unpatentable over Barnidge et al. (IDS; WO 2015/154,052), in view of Vlasak et al. (IDS; mAbs vol. 3,3 (2011): 253-63), as evidenced by Wang et al. (Analyst. 2013 May 21; 138(10):3058-65).
Regarding claim 1, Barnidge teaches “Isotyping immunoglobulins using accurate molecular mass” (Title) and “methods for detecting and quantifying heavy and light chains of immunoglobulin using mass spectrometry techniques” (Abstract). Barnidge also teaches a method of identifying immunoglobulins in a sample, the method comprising:
providing a biological sample comprising immunoglobulins – specifically, Barnidge teaches “providing a sample comprising an immunoglobulin light chain, an immunoglobulin heavy chain, or mixtures thereof” (pg. 4, lines 25-27) and the sample can be a biological sample (pg. 3, line 21);
immunopurifying IgG immunoglobulins from the biological sample – specifically, Barnidge teaches “immunopurifying the sample utilizing a single domain antibody fragment” (pg. 4, lines 27-28). The reference also teaches that immunoglobulins can
be IgG antibodies (infliximab or adalimumab, pg. 13, lines 17 and 20);
subjecting the IgG-purified sample to a mass spectrometry to obtain a mass spectrum – specifically, Barnidge teaches “subjecting the immunopurified sample to a mass spectrometry technique to obtain a mass spectrum of the sample” (pg. 4, lines 28-30). In example 11 Barnidge teaches subjecting the beads with bound immunoglobulins to 5% acetic acid (pg. 36, line 19) prior to MALDI analysis; and
analyzing the IgG heavy chain based on the multiply charged ion peaks in the spectrum – specifically, Barnidge teaches “(i) liquid chromatography electrospray ionization coupled to mass analyzer (quadrupole, time of flight or orbitrap) (ii) a microflow liquid chromatography electrospray ionization coupled to a quadrupole time-of-flight mass spectrometry (microLC-ESI-Q-TOF MS or MS/MS) technique” (pg. 5, lines 4-5) and “determining one or more of (iii) the isotype of the immunoglobulin heavy chains; (iv) the isotype of one or more of the immunoglobulin light chains and immunoglobulin heavy chains; and (v) the quantitative amount of one or more of the immunoglobulin light chains and immunoglobulin heavy chains in the sample” (pg. 4, lines 30-31 and pg. 5, lines 1-3). The reference teaches that electrospray ionization often produces multiply charged ions (pg. line 30).
Barnidge fails to teach specifically subjecting the IgG immunoglobulins to an acid to hydrolyze the IgG immunoglobulins, identifying IgG heavy chain subclass, and identifying the IgG acid heavy chain acid hydrolysis products based on the multiply charged ion peaks of IgG heavy chain acid hydrolysis products in the spectrum, wherein the ion peaks are in the range of 21,600 to 21,700 Da when the IgG heavy chains are not glycosylated and are in the range of 21,750 to 21,850 Da when the IgG heavy chains are glycosylated.
Regarding claim 1, Vlasak teaches “Fragmentation of monoclonal antibodies” (Title) and “non-enzymatic fragmentation or cleavage of the protein backbone” (pg. 253, Col. 2, 2nd paragraph). Vlasak also teaches subjecting the IgG immunoglobulins to an acid to hydrolyze the IgG immunoglobulins.
Specifically, Vlasak teaches that some peptide bonds hydrolyze under mild acidic conditions “Fragmentation occurring at the C-terminus of an Asp residue (peptide bond Asp-Xaa, where Xaa is any residue) is one of the most frequent degradation pathways of mAbs under mildly acidic conditions” (pg. 254, Col. 2, last paragraph) and “Asp-Pro bond fragmentation is usually much faster than fragmentation of other Asp-Xaa bonds” (pg. 255, Col. 1, 2nd paragraph).
Additionally, Vlasak teaches that one of the bonds undergoing rapid cleavage is the D270-P271 bond “The D270-P271 bond is subject to acid hydrolysis involving the Asp side chain, and therefore, is specifically accelerated under low pH conditions” (pg. 261, Col. 2, 4th paragraph). This bond is the exact cleavage site of the instant disclosure and the sequence PEVKFNWYVD (SEQ ID NO:5) starts with P271 amino acid residue, as evidenced by Wang. Top sequence on Fig. 1 shows amino acid sequence of an IgG1 heavy chain “Rituximab belongs to the immunoglobin G1 (IgG1) subclass of the IgG family” (pg. 3060, Col. 1, last paragraph). Line 6 of Fig. 1 shows sequence identical to SEQ ID NO:5 starting at amino acid residue P275.
The actual position of P275 is off by 4 amino acid residues when compared to teaching by Vlasak, but that is explained by different authors using different base reference sequences. Vlasak teaches another cleavage site G236-G237 that corresponds to G240-G241 of Wang “For IgG1, this cleavage occurs in the hinge region at the linkage between (ELL)G and G(PSVFL) on the heavy chains (i.e., between Gly240/Gly241)” (Wang, pg. 3060, Col. 2, last paragraph). The difference is exactly the same 4 amino acids, confirming that P275 of Wang corresponds to P271 of Vlasak, and that SEQ ID NO:5 of the instant invention corresponds to the N-terminal end of the cleavage fragment starting with P271 of Vlasak.
The acid fragmentation method taught by Vlasak cleaves the D270-P271 peptide bond in IgG1 antibodies, that ends with the peptide sequence PEVKFNWYVD (SEQ ID NO:5) (Wang, pg. 3060, Col. 2, last paragraph). The sequence PEVKFNWYVD is unique to IgG1 subclass. Identification of PEVKFNWYVD sequence at the N-terminus of one of the IgG hydrolysis products clearly indicates the presence of IgG1 subclass in the biological sample, meeting limitation (a) of claim 1 reciting a) IgG1 when the sequence at the N-terminus of the heavy chain acid hydrolysis products is PEVKFNWYVD (SEQ ID NO:5).
Finally, Vlasak teaches that mAb fragmentation patterns can be unique to specific immunoglobulin subclasses “due to the considerable sequence similarity of mAbs, fragmentation properties are expected to be very similar for molecules belonging to the same subclass (for instance, IgG1)” (pg. 253, Col. 2, paragraph 4).
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 immuno-purification and mass spectrometry analysis of IgG antibodies of Barnidge by employing acid fragmentation of the heavy chains as taught by Vlasak, as an obvious matter of using of known technique (acid fragmentation) to improve similar method (mass spectrometry analysis of IgG antibodies) in the same way. Substituting the fragmentation method taught by Barnidge with the acid hydrolysis method taught by Vlasak would have resulted in a predictable outcome, namely providing fragments for analyzing mAb patterns. One having ordinary skill in the art would have been motivated to make such a change because acid fragmentation of the heavy chains makes them shorter and by doing so, simplifies mass spectrometry analysis of IgG heavy chains. The use of such combination would have been desirable to those of ordinary skill in the art because shorter proteins are better separated by mass spectrometry.
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 Barnidge in view of the teaching of Vlasak and Wang for IgG1 fragmentation properties as an obvious matter of combining prior art elements according to known methods to yield predictable results and use the acid IgG fragmentation for identification of IgG1 heavy chains (Vlasak, pg. 253, Col. 2, paragraph 4).
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because Barnidge teaches mass spectrometry analysis of whole, unfragmented immunoglobulin chains and Vlasak teaches fragmentation of immunoglobulin chains, which simplifies analysis by mass spectrometry.
Regarding claim 1 limitation of “identifying the IgG acid heavy chain acid hydrolysis products based on the multiply charged ion peaks in the spectrum” - as presented above, Barnidge teaches analyzing the full-length IgG heavy chains based on the multiply charged ion peaks in the spectrum using a microflow liquid chromatography electrospray ionization coupled to a quadrupole time-of-flight mass spectrometry (microLC-ESI-Q-TOF MS or MS/MS) technique” (pg. 5, lines 4-5). It is a well-known feature of mass spectrometry analysis that molecules with larger sizes are more difficult to analyze. Since IgG heavy chain acid hydrolysis products are smaller than the full-length IgG heavy chains analyzed by and Barnidge, it would have been obvious to one having ordinary skill in the art that mass spectrometry approach of Barnidge would be capable of analyzing IgG heavy chain acid hydrolysis products.
Regarding claim 1 limitation of “wherein the ion peaks are in the range of 21,600 to 21,700 Da when the IgG heavy chains are not glycosylated and are in the range of 21,750 to 21,850 Da when the IgG heavy chains are glycosylated” – the specific ranges for detection of the IgG heavy chain acid hydrolysis products are defined by the sequence of IgG molecule, its glycosylation state, and the applied hydrolysis method. Therefore, the ranges of detection are inherent to the sequence of the IgG, its glycosylation state, and the hydrolysis method. It would have been obvious to one having ordinary skill in the art to analyze the IgG heavy chain acid hydrolysis products in the recited ranges when the same IgG molecules were hydrolyzed by the same method as disclosed in the specification.
Regarding claim 60, Barnidge teaches biological sample is a biological fluid selected from the group consisting of blood, serum, plasma, urine, lachrymal fluid, and saliva. Specifically, Barnidge teaches “the biological sample can be a whole blood sample, a serum sample, a plasma sample, a urine sample, or a cerebral spinal fluid sample” (pg. 3, lines 21-23).
Regarding claims 61 and 75, Barnidge teaches immunopurifying comprises using an anti-human IgG kappa or an anti-human IgG lambda antibody: “an antibody selected from the group consisting of an anti-human IgG antibody, an anti-human IgA antibody, an anti-human IgM antibody, an anti-human IgD antibody, an anti-human IgE antibody, an anti-human kappa antibody, an anti-human lambda antibody, and combinations thereof” (pg. 2, lines 18-21).
Regarding claims 62-63 and 76-77, Barnidge teaches anti-human IgG kappa or lambda antibody is a non-human antibody selected from the group consisting of a camelid antibody, a cartilaginous fish antibody, llama, sheep, goat, rabbit, and a mouse antibody; and a single domain antibody fragment: “the single domain antibody fragment is derived from a camelid antibody, a cartilaginous fish antibody, llama, a mouse antibody, sheep, goat, or a human antibody” (pg. 2, lines 28-30) and “single domain antibody fragment (SDAF) can be selected from the group consisting of an anti-human IgG SDAF” (pg. 2, lines 25-26).
Regarding claim 64, Barnidge teaches “(i) liquid chromatography electrospray ionization coupled to mass analyzer (quadrupole, time of flight or orbitrap) (ii) a microflow liquid chromatography electrospray ionization coupled to a quadrupole time-of-flight mass spectrometry (microLC-ESI-Q-TOF MS or MS/MS) technique” (pg. 5, lines 4-5). MicroLC-ESI-Q-TOF MS is a liquid chromatography-mass spectrometry (LC-MS) technique.
Regarding claim 65, Barnidge teaches mass spectrometry technique is electrospray ionization mass spectrometry (ESI-MS), and wherein the ESI-MS technique comprises a quadrupole time-of-flight (TOF) mass spectrometer “mass spectrometry technique can include a microflow liquid chromatography electrospray ionization coupled to a quadrupole time-of-flight mass spectrometry (microLC-ESI-Q-TOF MS) technique” (pg. 3, lines 26-28).
Regarding claim 66, Barnidge teaches mass spectrometry technique is a top-down mass spectrometry technique “the mass spectrometry technique includes a matrix assisted laser adsorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technique” (pg. 3, lines 30-31). MALDI-TOF MS is a top-down mass spectrometry method.
Regarding claim 67, Barnidge teaches immunoglobulins are not fragmented during the mass spectrometry technique “translational modification is a natural process performed by the B cell but the extent of the glycosylation added by the cell is different for each Ig isotype and therefore should give an additional means of identifying the isotype without performing additional MS/MS fragmentation” (pg. 16, lines 10-13).
Regarding claims 68-69, Barnidge teaches contacting the sample with a reducing agent prior to subjecting the sample to the mass spectrometry technique, wherein the reducing agent is tris (2- carboxyethyl) phosphine (TCEP) “disulfide bonds can be cleaved using a reducing agent capable of reducing the disulfide bonds. For example, the reducing agent can be selected from the group consisting of: DTT (2,3 dihydroxybutane-1,4-dithiol), DTE (2,3 dihydroxybutane-1,4-dithiol), thioglycolate, cysteine, sulfites, bisulfites, sulfides, bisulfides, TCEP (tris(2-carboxyethyl) phosphine), and salt forms thereof” (pg. 7, lines 8-12).
Claims 56-57 are rejected under 35 U.S.C. 103 as being unpatentable over Barnidge, in view of Vlasak, and as evidenced by Wang, and further in view of Mills et al. (Methods. 2015 Jun 15; 81:56-65).
Regarding claims 56-57, Barnidge, Vlasak and Wang teach mass spectrometry analysis of acid hydrolyzed IgG heavy chains. Barnidge, Vlasak and Wang fail to teach acid is 5% acetic acid.
Vlasak teaches that hydrolysis of the D270-P271 bond occurs under mild acidic conditions (pg. 254 Col. 2 last paragraph). Mills teaches “Detecting monoclonal immunoglobulins in human serum using mass spectrometry” (Title). Mills also teaches immune-purification of antibodies using 5% acetic acid. Specifically, Mills teaches that after binding antibodies to an affinity support “beads were then washed 2x with PBS and with HPLC grade water and then resuspended in a solution of 5% acetic acid with 50 mM TCEP” (pg. 60, Col. 2, 2nd paragraph). This experimental step is an exact match to example 1 of the instant specification (pg. 21).
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 Barnidge, Vlasak and Wang by employing fragmentation of the heavy chains using 5% acetic acid as taught by Mills, in order to provide a method for identifying and quantifying heavy chains of antibodies, as an obvious matter to try, namely choosing from a finite list of suitable, art recognized/known mild acid alternatives.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because 5% acetic acid is compatible with immune-purification of antibodies, as demonstrated by Mills.
Response to Arguments
Applicant’s arguments filed October 27, 2025 have been fully considered.
Claims 1, 53, 56, 57, 60-69, and 75-77 were rejected under 35 U.S.C § 112(b) as being indefinite. Applicant argues that “claim 1, as amended, is clear and unambiguous” (pg. 6, par. 1). The argument is persuasive; therefore, the rejection is withdrawn.
Claims 1, 53, 60-69 and 75-77 were rejected under 35 U.S.C. § 103(a) as being unpatentable over the cited prior art references. On pg. 6, par. 3-5 and pg. 7, par. 1-4 Applicant summarizes the essence of the claimed method, the teachings of the cited prior art, and argues that “these references, either alone or in combination, fail to teach or suggest that the IgG subclass could be identified by a mass spectrometry technique either by mass or by sequence. As such, the combinations of cited references simply cannot be considered as teaching or suggesting that the subclass of an IgG could be identified by a mass spectrometry technique based on the sequence of the heavy chain acid hydrolysis product where the IgG is determined to be: a) IgG1 when the sequence at the N-terminus of the heavy chain acid hydrolysis products is PEVKFNWYVD (SEQ ID NO:5), b) IgG2 or IgG4 when the sequence at the N-terminus of the heavy chain acid hydrolysis products is PEVQFNWYVD (SEQ ID NO:6), or c) IgG3 when the sequence at the N-terminus of the heavy chain acid hydrolysis products is PEVQFKWYVD (SEQ ID NO:7)” (pg. 7, last par. – pg. 8, par. 1).
The argument is not persuasive because the combined prior art references do teach identification of IgG1 (see §103 rejection above for details). Briefly, the acid fragmentation method taught by Vlasak cleaves the D270-P271 peptide bond in IgG1 antibodies and makes the N-terminal sequence PEVKFNWYVD (SEQ ID NO:5) (Wang, pg. 3060, Col. 2, last par.) available for mass spectrometry identification; therefore, meeting limitation (a) of claim 1 (underlined above). The mass spectrometry identification taught by Barnidge is capable of analyzing even unfragmented IgG light and heavy chains (pg. 4, lines 30-31 and pg. 5, lines 1-3). The acid fragmentation taught by Vlasak reduces the sizes of the IgG fragments to be analyzed. Analysis of shorter protein fragments is preferred over the analysis of longer fragments because of better resolution of smaller molecular weight fragments. One having ordinary skill in the art would have been motivated to make such combination of mass spectrometry analysis and acid fragmentation because acid fragmentation of the heavy chains makes them shorter and simplifies mass spectrometry analysis of IgG heavy chains.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, 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 nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
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