DETAILED ACTION
The amendment filed on 03/02/2026 has been entered and fully considered. Claims 1-7, 9, 11-12, 14-15, 19,24, 29-31, 34-35 and 39 are pending. Claims 6, 7, 11 and 35 have been withdrawn from consideration. Claims 1-5, 9, 11-12, 14-15, 19, 24, 29-31, 34 and 39 are considered on merits, of which Claim 39 is newly added.
Response to Amendment
In response to amendment, the maintains rejection over the prior art established in the previous Office 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 .
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim(s) 1-5, 9, 14-15, 19, 24, 29-31, 34 and 39 is/are rejected under 35 U.S.C. 103 as being unpatentable over DeGraan-Weber et al. (J. Am. Soc. Mass Spectrom., 2016, IDS) (Weber).
Regarding claim 1, Weber teaches a method of processing a protein, the method comprising:
(a) digesting a protein into peptides (many peptides obtained from standard trypsin digestion) (page 2050, par 0),
(b) subjecting the peptides to mass spectrometry and measuring fragmentation, by mass spectrometry, of a peptide bond N-terminal to an isoAsp (abstract, Fig. 1, page 2043, par 4);
(c) comparing the fragmentation to a threshold, wherein the fragmentation exceeding the threshold is indicative of the presence of the isoAsp in the protein (Fig. 1, page 2043, par 5).
Weber does not specifically teach (d) rejecting the protein comprising the isoAsp, or engineering the protein to remove the isoAsp. However, Weber teaches that “Protein modifications, such as the conversion of aspartic acid or asparagine residues to isoaspartic acid, can lead to conformational changes, reduced function, and aggregation [1–9]. Their occurrence in the complementarity determining regions (CDRs) of antibodies can decrease the affinity of antigen binding. For example, rhuMAb HER2 is a therapeutic monoclonal antibody for metastatic breast cancer that contains an aspartic acid in a heavy chain CDR. Isomerization of this residue reduces the activity by 79%–91% of the unmodified antibody [10]” (page 2041, par 1). Thus, it would have been obvious to one of ordinary skill in the art to reject the antibodies comprising the isoAsp, for quality control purpose.
Regarding claim 2, Weber teaches that the method further comprising charge reduction prior to (b) (Fig. 1, Scheme 1, page 2043, par 4).
Regarding claim 3, Weber teaches that wherein the peptides subject to mass spectrometry in (b) are singly-charged (Fig. 1, Scheme 1, page 2043, par 4).
Regarding claim 4, Weber teaches that wherein the fragmentation is measured as a b-series peak, a y-series peak, or both (Fig. 1).
Regarding claim 5, Weber teaches that wherein the mass spectrometry is matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDITOF/TOF) (page 2043, par 4).
Regarding claim 9, Weber teaches that wherein (a) comprises enzymatic digestion with a proteolytic enzyme (trypsin) (page 2050, par 0).
Regarding claim 14, Weber teaches that wherein the fragmentation exceeds the threshold when a b-peak and/or a y-peak of the fragmented peptide bond N-terminal to isoAsp is present (Fig. 1, page 2043, par 4).
Regarding claim 15, Weber teaches that wherein the threshold is a fragmentation of a peptide bond N-terminal to an L-Asp in a control peptide (Fig. 1, page 2043, par 5).
Regarding claim 19, Weber teaches that wherein the protein is a large peptide, antibody, antibody fragment, antibody fusion peptide or antigen-binding fragment thereof (page 2040, par 1).
Regarding claim 24, as has been discussed regarding claim 1 above, Weber teaches that “Protein modifications, such as the conversion of aspartic acid or asparagine residues to isoaspartic acid, can lead to conformational changes, reduced function, and aggregation [1–9]. Their occurrence in the complementarity determining regions (CDRs) of antibodies can decrease the affinity of antigen binding. For example, rhuMAb HER2 is a therapeutic monoclonal antibody for metastatic breast cancer that contains an aspartic acid in a heavy chain CDR. Isomerization of this residue reduces the activity by 79%–91% of the unmodified antibody [10]” (page 2041, par 1). Thus, it would have been obvious to one of ordinary skill in the art to reject the antibodies comprising the isoAsp comprises rejecting a quantity of product comprising the antibody, or rejecting a clone that produces the antibody, for quality control purpose.
Regarding claim 29, Weber teaches that wherein at least one of the peptides comprises a positive charge at the N terminus of the peptide (TMPP tagging) (page 2042, par 6).
Regarding claim 30, Weber teaches that the method further comprising introducing a positive charge to the N terminus of the peptides (TMPP tagging) (page 2042, par 6).
Regarding claim 31, Weber teaches that wherein the positive charge is introduced by incubating the peptide with an N-terminal charge-derivatizing reagent (page 2042, par 6).
Regarding claim 34, Weber teaches that wherein the isoAsp is within 5 amino acid residues of a C-terminus of at least one of the peptides (page 2043, par 4).
Regarding claim 39, Weber teaches a method of processing a protein, the method comprising:
(a) digesting a protein into peptides (many peptides obtained from standard trypsin digestion) (page 2050, par 0),
(b) subjecting the peptides to mass spectrometry, wherein the peptides subject to mass spectrometry are singly-charged (Fig. 1, Scheme 1, page 2043, par 4), and measuring fragmentation, by mass spectrometry, of a peptide bond N-terminal to an isoAsp (Fig. 1, Scheme 1, page 2043, par 4);
(c) comparing the fragmentation to a threshold, wherein the fragmentation exceeding the threshold is indicative of the presence of the isoAsp in the protein (abstract, Fig. 1, page 2043, par 5).
Weber does not specifically teach (d) rejecting the protein comprising the isoAsp, or engineering the protein to remove the isoAsp. However, Weber teaches that “Protein modifications, such as the conversion of aspartic acid or asparagine residues to isoaspartic acid, can lead to conformational changes, reduced function, and aggregation [1–9]. Their occurrence in the complementarity determining regions (CDRs) of antibodies can decrease the affinity of antigen binding. For example, rhuMAb HER2 is a therapeutic monoclonal antibody for metastatic breast cancer that contains an aspartic acid in a heavy chain CDR. Isomerization of this residue reduces the activity by 79%–91% of the unmodified antibody [10]” (page 2041, par 1). Thus, it would have been obvious to one of ordinary skill in the art to reject the antibodies comprising the isoAsp, for quality control purpose.
Claim(s) 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over DeGraan-Weber et al. (J. Am. Soc. Mass Spectrom., 2016, IDS) (Weber) in view of Yi et al. (Journal of Pharmaceutical Sciences, 2013) (Yi).
Regarding claim 12, Weber does not specifically teach that wherein the isoAsp is adjacent to an adjacent aspartate residue in the amino acid sequence of the protein. However, Yi teaches a monoclonal antibody wherein the isoAsp is adjacent to an adjacent aspartate residue in the amino acid sequence of the antobody (abstract). Thus, it would have been obvious to one of ordinary skill in the art to apply Weber’s method to the protein wherein the isoAsp is adjacent to an adjacent aspartate residue in the amino acid sequence of the protein, in order to monitor the quality of the monoclonal antibody wherein the isoAsp is adjacent to an adjacent aspartate residue in the amino acid sequence of the antibody.
Response to Arguments
Applicant's arguments filed 03/02/2026 have been fully considered but they are not persuasive.
Applicant argues that Weber allegedly provides no teaching or reason to digest a protein in connection with fragmentation by mass spectrometry, no reason to fragment singly charged ions in a manner that distinguishes isoAsp from Asp, no teaching of comparison to a threshold, and no teaching or reason for the rejection/engineering step of part (d). Applicant further argues that Weber is directed to synthetic peptides rather than proteins or antibodies in a quality-control setting.
These arguments are not persuasive. First, Weber expressly concerns the problem addressed by the claim, namely distinguishing aspartic acid from isoaspartic acid in peptides by mass spectrometric fragmentation methods. Weber states in the abstract that six fragmentation techniques were compared for distinguishing Asp from isoAsp, including PSD, photodissociation, charge tagging, CID, ETD, and FRIPS, and that diagnostic ions and significant fragment-intensity differences can be used for such distinction. Weber further explains that protein modifications converting Asp or Asn to isoAsp can lead to conformational changes, reduced function, and aggregation, and specifically notes that in antibodies, isomerization at CDR Asp residues can markedly reduce binding activity.
Applicant’s argument regarding digestion of a protein into peptides is not persuasive because Weber is plainly directed to peptide analysis by mass spectrometry and expressly teaches LC separation and MS fragmentation of peptide samples by CID, including separation on a reversed-phase LC column prior to introduction into the ESI source. Weber also teaches that ETD is limited because many peptides obtained from standard trypsin digestions are not large enough to accommodate three charges. Thus, Weber does not ignore peptide digests; rather, it expressly recognizes peptide digests as a conventional source of analytes and discusses the practical consequences of digest-derived peptide charge state for different fragmentation methods. A disclosure that a particular method has limitations for some tryptic peptides does not amount to a teaching away from analyzing digested proteins as peptides.
Applicant’s argument focused on singly charged ions is likewise unpersuasive as to claim 1, because claim 1 does not require that the peptides subjected to mass spectrometry be singly charged. That limitation appears in separate dependent claims, not in claim 1 itself. Claim 1 broadly recites subjecting peptides to mass spectrometry and measuring fragmentation of a peptide bond N-terminal to an isoAsp. Accordingly, Weber’s teachings regarding multiply charged ETD ions, charge-tagged peptides, PSD, photodissociation, and CID remain pertinent to claim 1.
Applicant’s argument that Weber does not teach fragmentation of a peptide bond N-terminal to an isoAsp is also unpersuasive. Weber expressly teaches that MALDI PSD and CID generate b- and y-type ions and explains that, for isoaspartic acid, the side chain can interact with the amide oxygen on its N-terminal side, leading to formation of diagnostic b+H2O and y-46 ions (Fig. 1, Scheme 1, page 2043, par 4). Weber further states in Table 1 that MALDI PSD and photodissociation provide “b+H2O/y-46, enhanced b-/y-type ion N-terminal to iso-D,” and Figure 1 / Scheme 1 expressly identify an “enhanced b- or y-type ion on the N-terminal side of isoaspartic acid.” Weber also teaches that, in spectra of RAFVDiSLT versus RAFVDSLT, the b4+H2O ion is a diagnostic ion for isoAsp and that the intensities of diagnostic ions may be compared with neighboring fragments such as b4, c4, and b5, which relative measurements may improve the ability to quantitate the presence of Asp and isoAsp.
Applicant’s argument that Weber lacks a threshold is not persuasive. Weber expressly teaches that fragment-ion intensity differences can distinguish Asp from isoAsp, that “for some molecules, aspartic and isoaspartic acid yield ion fragments with significantly different intensities,” (abstract) and that “quantitation of isoaspartic acid can be performed through many of these methods.” (page 2651, par 3). Weber additionally teaches comparing diagnostic ion intensities with neighboring fragment intensities to improve quantitation (page 2043, par 5). In view of these teachings, using a threshold for the compared fragmentation would have been no more than a routine optimization of Weber’s expressly taught quantitative comparison of fragment intensities in order to classify a peptide as containing isoAsp. The claimed threshold does not require any particular mathematical form beyond using compared fragmentation as an indicator of isoAsp presence.
Applicant’s argument regarding part (d) is also not persuasive. Weber expressly teaches that Asp/isoAsp conversion in proteins, including antibodies, leads to deleterious consequences such as conformational changes, reduced function, aggregation, and substantially reduced antibody binding activity (page 2041, par 1). Thus, Weber provides a clear reason why a skilled artisan would identify isoAsp-containing proteins: because such modified proteins are undesirable and functionally compromised. Once Weber’s analytical method identifies a protein or peptide as containing isoAsp, it would have been an obvious and routine quality or design decision either to reject the isoAsp-containing material as unsuitable or to engineer the protein sequence to remove the isoAsp liability site, especially in view of Weber’s express discussion that isoAsp formation in antibody CDRs significantly decreases activity. The claimed action in part (d) is therefore an obvious consequence of Weber’s teaching that isoAsp is a harmful protein modification.
Applicant’s emphasis that Weber used synthetic peptides also does not overcome the rejection. Weber selected peptide models precisely to study fragmentation behavior that distinguishes Asp from isoAsp, and those model peptides were chosen in view of antibody-relevant sequence contexts, with Weber noting that hydrophobic amino acids are commonly found in monoclonal antibody CDRs and that the residue on the C-terminal side of Asp/isoAsp was selected as Ser or His because such residues are known to promote isomerization and deamidation. Weber further discusses actual antibody examples, including rhuMAb HER2 and E25, to explain the practical importance of detecting isoAsp-related degradation. Thus, Weber is not limited to an abstract synthetic-peptide exercise divorced from proteins; rather, it teaches a peptide-based analytical approach directed to a known protein degradation problem.
Accordingly, Weber teaches or at least strongly suggests each feature of claim 1, including digest-derived peptide analysis by mass spectrometry, measurement of fragmentation associated with a peptide bond N-terminal to isoAsp, comparison of fragment behavior indicative of isoAsp presence, and acting on the identified isoAsp-containing protein because isoAsp formation is known to impair protein quality and function. Therefore, the rejection of claim 1 under 35 U.S.C. § 103 is maintained.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any 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 mailing date of this final action.
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/XIAOYUN R XU, Ph.D./ Primary Examiner, Art Unit 1797