DETAILED ACTION
The amendment filed on 03/03/2026 has been entered and fully considered. Claims 181-210 are pending, of which Claims 205-210 are newly added.
Response to Amendment
In response to amendment, the examiner 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) 181-182, 184-188, 190-195 and 201-210 is/are rejected under 35 U.S.C. 103 as being unpatentable over Vliet et al. (Journal of Virological Methods, 2009, IDS) (Vliet) in view of Bark et al. (J. Am. Chem. Soc. 2001, IDS) (Bark).
Regarding claim 181, Vliet teaches a method of detecting post-translational modifications of viral proteins (VPs) in a preparation of adeno-associated virus (AAV) particles (abstract), the method comprising
a) denaturing the AAV particles (boiled for 3 min) (page 169, par 4);
b) subjecting the denatured AAV particles to enzymatic digestion to form peptide fragments (page 169, par 5);
c) subjecting the peptide fragments to liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) peptide mapping analysis (page 169, par 6, page 170, par 2);
d) determining the masses of one or more of the peptide fragments (page 169, par 6); and
e) determining any deviation (a different mass than expected) of the determined masses of the one or more peptide fragments from the theoretical masses of corresponding peptide fragments that have not undergone post-translational modifications to detect a deviation in the compared masses (page 172, par 2), wherein the VPs comprise VP1, VP2 and VP3 capsid proteins (Table 2, page 171, par 1).
Vliet does not specifically teach wherein the method is performed in the absence of a gel separation step. However, Bark teaches directly subjecting the denatured and digested virus particle (HK97) to liquid chromatography/mass spectrometry (LC/MS) without a gel separation step (Fig. 1, page 1774, par 4).
Bark teaches that “The primary techniques for proteomic analysis consist of 2-D gel electrophoresis followed by proteolytic digestion, mass spectrometry, and computer-facilitated data analysis.1 Masses for proteolytic fragments of the target protein generated by enzymatic degradation can be measured directly with high accuracy ((0.005 Da for a 1000 Da peptide). Alternatively, liquid chromatography tandem mass spectrometry can be used to analyze the protein digest fragments. This information is then compared with the theoretical proteolytic fragments predicted for proteins within a database, and matches are statistically evaluated. The success of this strategy relies on the existence of the protein sequence within the database, but with the sequences of whole genomes being completed, the likelihood for matches is high.” (page 1774, par 1). Here, the “alternatively” is the opposite to the “primary technology”. Bark teaches that in primary technology, the mixture of proteins is separated by 2D gel, the separated protein is then enzyme digested and analyzed by mass spectrometry. The advantage of this method less complex peptide mixture per spot, so as simplifies MS analysis. The disadvantage of this method is labor-intensive and low throughput. Bark further teaches that alternatively, the mixture of proteins is directly digested by enzyme, the digested peptides are separated by liquid chromatography, and analyzed by mass spectrometry. The advantage of this method is high throughput, the disadvantage of this method is complex peptide mixture requires the existence of the protein sequence within the database, but with the sequences of whole genomes being completed, the likelihood for matches is high. Thus, it would have been obvious to one of ordinary skill in the art to separate peptide mixture by liquid chromatography in the absence of gel separation step, in order to high throughput the process. With the sequences of whole genomes being completed, the likelihood for matches is high.
The applicant is advised that the Supreme Court recently clarified that a claim can be proved obvious merely by showing that the combination of known elements was obvious to try. In this regard, the Supreme Court explained that, “[w]hen there is a design need or market pressure to solve a problem and there are a finite number of identified, predictable solutions, a person of ordinary skill in the art has a good reason to pursue the known options within his or her technical grasp.". See KSR Int’l v. Teleflex Inc., 127 Sup. Ct. 1727, 1742, 82 USPQ2d 1385, 1397 (2007) (see MPEP § 2143). In this case, the market pressure to solve a problem is low throughput of gel separation. A finite number of identified, predictable solutions is liquid chromatography separation.
Regarding claim 190, Vliet teaches a method of identifying deamidated viral proteins (VPs) in a preparation of adeno-associated virus (AA V) particles (abstract), the method comprising
a) denaturing the AAV particles to form viral proteins (boiled for 3 min) (page 169, par 4);
b) subjecting the denatured viral proteins to enzymatic digestion to form peptide fragments (page 169, par 5);
c) subjecting the peptide fragments in the digest to liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) peptide mapping analysis (page 169, par 6);
d) determining the masses of one or more peptide fragments containing amino acid residues with potential deamidation sites (page 169, par 8); and
e) determining any deviation (a different mass than expected) of the determined masses of the one or more peptide fragments corresponding to removal of one or more amide functional groups in step d) from the theoretical masses of corresponding peptide fragments that have not undergone deamidation to detect a deviation in the compared masses (page 172, par 2),
wherein the VPs comprise VP1, VP2 and VP3 capsid proteins (page 171, par 1).
Vliet does not specifically teach wherein the method is performed in the absence of a gel separation step. However, Bark teaches directly subjecting the denatured and digested virus particle (HK97) to liquid chromatography/mass spectrometry (LC/MS) without a gel separation step (Fig. 1, page 1774, par 4).
Bark teaches that “The primary techniques for proteomic analysis consist of 2-D gel electrophoresis followed by proteolytic digestion, mass spectrometry, and computer-facilitated data analysis.1 Masses for proteolytic fragments of the target protein generated by enzymatic degradation can be measured directly with high accuracy ((0.005 Da for a 1000 Da peptide). Alternatively, liquid chromatography tandem mass spectrometry can be used to analyze the protein digest fragments. This information is then compared with the theoretical proteolytic fragments predicted for proteins within a database, and matches are statistically evaluated. The success of this strategy relies on the existence of the protein sequence within the database, but with the sequences of whole genomes being completed, the likelihood for matches is high.” (page 1774, par 1). Here, the “alternatively” is the opposite to the “primary technology”. Bark teaches that in primary technology, the mixture of proteins is separated by 2D gel, the separated protein is then enzyme digested and analyzed by mass spectrometry. The advantage of this method less complex peptide mixture per spot, so as simplifies MS analysis. The disadvantage of this method is labor-intensive and low throughput. Bark further teaches that alternatively, the mixture of proteins is directly digested by enzyme, the digested peptides are separated by liquid chromatography, and analyzed by mass spectrometry. The advantage of this method is high throughput, the disadvantage of this method is complex peptide mixture requires the existence of the protein sequence within the database, but with the sequences of whole genomes being completed, the likelihood for matches is high. Thus, it would have been obvious to one of ordinary skill in the art to separate peptide mixture by liquid chromatography in the absence of gel separation step, in order to high throughput the process. With the sequences of whole genomes being completed, the likelihood for matches is high.
Regarding claim 201, Vliet teaches a method of preparing a pharmaceutical composition of adeno-associated virus (AAV) particles (abstract), the method comprising:
monitoring AAV particles for unwanted deamidation (page 169, par 8);
wherein the AAV particles comprise viral proteins (VPs) comprising VP1, VP2 and VP3 capsid proteins of an AA V particle capsid (Table 2, page 171, par 1);
wherein the AAV particles are monitored for unwanted deamidation by:
a) extracting an aliquot of an AAV particle preparation (page 168, par 4);
b) denaturing the AAV particles (page 169, par 4);
c) subjecting the denatured AAV particles to enzymatic digestion to form peptide fragments (page 169, par 5);
d) subjecting the peptide fragments to liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) peptide mapping analysis (page 169, par 6);
e) determining the masses of one or more of the peptide fragments (page 172, par 2); and
f) comparing the determined masses of the one or more peptide fragments to theoretical masses of corresponding peptide fragments, wherein the theoretical masses of corresponding peptide fragments are those of known peptide fragments from AA V serotypes that have not undergone unwanted deamidation (page 172, par 2);
g) determining if there is any deviation of the determined masses of the one or more peptide fragments from the theoretical masses of the corresponding peptide fragments (page 172, par 2);
wherein the determination of any deviation of the determined masses of the one or more peptide fragments from the theoretical masses of corresponding peptide fragments monitors the AAV particles for unwanted deamidation (page 173, par 1); and
wherein if less than an undesirable amount of unwanted deamidation of peptide fragments is detected, the AA V particles are combined with one or more pharmaceutically acceptable excipients to form the pharmaceutical composition (page 168, par 3).
Vliet does not specifically teach that wherein the monitoring for unwanted deamidation is performed in the absence of a gel separation step. However, Bark teaches directly subjecting the denatured and digested virus particle (HK97) to liquid chromatography/mass spectrometry (LC/MS) without a gel separation step (Fig. 1, page 1774, par 4). Bark teaches that “The primary techniques for proteomic analysis consist of 2-D gel electrophoresis followed by proteolytic digestion, mass spectrometry, and computer-facilitated data analysis. Masses for proteolytic fragments of the target protein generated by enzymatic degradation can be measured directly with high accuracy (±0.005 Da for a 1000 Da peptide). Alternatively, liquid chromatography tandem mass spectrometry can be used to analyze the protein digest fragments” (page 1774, par 1). Here Bark teaches that alternatively, one can directly subjects the denatured and digested virus particle (HK97) to liquid chromatography/mass spectrometry (LC/MS) without a gel separation step. At time before the filing, it would have been obvious to one of ordinary skill in the art to directly subjecting the denatured and digested AAV particle to liquid chromatography/mass spectrometry (LC/MS) without a gel separation step, in order to save time that is required for gel separation.
Regarding claim 182, Vliet teaches that wherein the post-translational modifications are selected from the group consisting of acetylation, deacetylation, deamidation, glycosylation, truncation and ubiquitination (page 173, par 1).
Regarding claim 184, 192 and 203, Vliet teaches that wherein the liquid chromatography is reverse phase chromatography (page 169, par 6).
Regarding claim 185, 193 and 204, while Vliet teaches that wherein the reverse phase chromatography is a C18 reverse chromatography (page 169, par 6), Vliet does not teach that wherein the reverse phase chromatography is a C4 or C8 reverse chromatography. However, like C18, C4 and C8 are known reverse phase columns. C18 has 18 carbon atoms while C8 has only 8 carbon atoms. C18 has a longer carbon chain, but C8 has a shorter one. C18 has higher retention while C8 has shorter retention. C18 has higher hydrophobicity, but C8 has a lower hydrophobicity. At time before the filing, it would have been obvious to one of ordinary skill in the art to optimize the hydrophobicity of the reverse phase column, by routine experimentation.
Regarding claim 186, 191 and 202, Vliet teaches that wherein the AAV particles are denatured using detergent, heat, high salt, or buffer with low or high pHs (page 169, par 4).
Regarding claim 187, Vliet teaches that the method further comprising determining the sequence of one or more VPs that has undergone post-translational modifications (page 172, par 2).
Regarding claim 188, Vliet teaches that wherein the post-translational modifications are selected from the group consisting of acetylation, deacetylation, deamidation, glycosylation, truncation and ubiquitination (page 173, par 1).
Regarding claim 194, Vliet teaches that the method further comprising determining the sequence of one or more VPs that has been deamidated (page 172, par 2; page 173, par 1).
Regarding claim 195, Vliet teaches that wherein the sequences of VP 1, VP2 and VP3 are determined (page 172, par 2; page 173, par 1).
Regarding claim 205, Vliet teaches detecting post-translational modifications of one or more VP1 or VP2 proteins in the preparation of AAV particles (Fig. 2, page 170).
Regarding claim 206, Vliet determining the sequence of one or more VP1 or VP2 VPs that has undergone post-translational modifications (Fig. 2, page 170).
Regarding claim 207, Vliet teaches detecting the post-translational modifications of all of the VP1, VP2, and VP3 VPs in the preparation of AAV particles (Fig. 2, page 170).
Regarding claim 208, Vliet teaches detecting deamidation of one or more VP1 or VP2 proteins in the preparation of AAV particles (Fig. 5, page 173)
Regarding claim 209, Vliet teaches determining the sequence of one or more VP1 or VP2 VPs that has been deamidated (Fig. 5, page 173).
Regarding claim 210, Vliet teaches detecting deamidation of all of the VP1, VP2, and VP3 VPs in the preparation of AAV particles (Fig. 5, page 173).
Claim(s) 183 and 189 is/are rejected under 35 U.S.C. 103 as being unpatentable over Vliet in view of Bark as applied to claims 181-182, 184-188, 190-195 and 201-204 above, and further in view of Senesac (US 2004/0106184).
Regarding claim 183 and 189, Vliet does not specifically teach that wherein the post-translational modification is N-terminal acetylation. However, Senesac teaches that the post-translational modification is N-terminal acetylation (par [0272]). It would have been obvious to one of ordinary skill in the art to include N-terminal acetylation in the post- translational modifications, in order to find all variations.
Claim 196-200 is/are rejected under 35 U.S.C. 103 as being unpatentable over Vliet in view of Bark as applied to claims 181-182, 184-188, 190-195 and 201-204 above, and further in view of Taylor et al. (US 2013/0217789)(Taylor).
Regarding claim 196, Vliet teaches that the method further comprising determining the sequence of one or more VPs that has been deamidated (page 172, par 2; page 173, par 1).
Vliet does not specifically teach that the method further comprising quantifying levels of deamidation at deamidation sites of the VPs. However, teaches quantifying mass spectrometry with assisted calibration and sodium iodide is used as a calibrant (par [0125]). At time before the filing, it would have been obvious to one of ordinary skill in the art to incorporate assisted calibration in mass spectrometry and use sodium iodide as a calibrant, in order to quantifying levels of deamidation at deamidation sites of the VPs.
Regarding claim 197 and 200, while Vliet teaches that wherein the reverse phase chromatography is a C18 reverse chromatography (page 169, par 6), Vliet does not teach that wherein the reverse phase chromatography is a C4 or C8 reverse chromatography. However, like C18, C4 and C8 are known reverse phase columns. C18 has 18 carbon atoms while C8 has only 8 carbon atoms. C18 has a longer carbon chain, but C8 has a shorter one. C18 has higher retention while C8 has shorter retention. C18 has higher hydrophobicity, but C8 has a lower hydrophobicity. At time before the filing, it would have been obvious to one of ordinary skill in the art to optimize the hydrophobicity of the reverse phase column, by routine experimentation.
Regarding claim 198, Vliet teaches that the method further comprising determining the sequence of one or more VPs that has been deamidated (page 172, par 2; page 173, par 1).
Regarding claim 199, Vliet teaches that wherein the sequences of VP1, VP2 and VP3 are determined (page 172, par 2; page 173, par 1).
Response to Arguments
Applicant's arguments filed 03/03/2026 have been fully considered but they are not persuasive.
Applicant argues that Bark does not provide motivation to eliminate gel separation and that Bark allegedly teaches away. This argument is not persuasive.
Bark expressly teaches that, as an alternative to gel-based proteomic workflows, protein digests may be analyzed directly using LC/MS/MS:
“Alternatively, liquid chromatography tandem mass spectrometry can be used to analyze the protein digest fragments.” (Bark, page 1774)
This statement is not limited to any particular protein or system and reflects a recognized alternative analytical workflow in proteomics. The Examiner relies on this explicit disclosure to show that gel separation is optional, not required.
Vliet already teaches:
• digestion of AAV capsid proteins (VP1, VP2, VP3)
• LC/MS/MS peptide mapping
• PTM detection via mass deviation
Thus, Bark does not change the analytical principle of Vliet, but merely substitutes one known sample preparation approach (gel-based separation) with another known alternative (direct digestion followed by LC separation), which is a routine optimization.
Under KSR, substituting one known technique for another to improve throughput constitutes an obvious modification.
Applicant asserts that Bark teaches away because it shows incomplete peptide recovery in mixtures.
This argument is not persuasive for two reasons:
First, Bark does not criticize, discourage, or discredit LC/MS/MS of protein mixtures. Rather, Bark explicitly uses LC/MS/MS on mixtures and identifies expected analytical limitations. Recognition of limitations does not constitute teaching away.
Second, Bark’s disclosure demonstrates that:
• LC/MS/MS of complex mixtures was known and practiced
• Analytical trade-offs (complexity vs throughput) were understood
A reference that merely discusses limitations of a technique does not teach away unless it discourages its use, which Bark does not do.
Applicant argues that a skilled artisan would not expect success due to:
• low abundance of VP1/VP2
• complex mixtures
• presence of excipients
• structural similarity of capsid proteins
These arguments are not commensurate with the scope of the claims.
As shown in amended claim 181, the claim requires only:
• detecting any deviation in peptide mass
• to identify a post-translational modification
The claim does not require:
• full sequence coverage
• detection of all VPs
• quantitative accuracy
• detection at low abundance thresholds
• analysis free of excipients
• complete peptide recovery
Therefore, even partial peptide detection, as demonstrated in Bark, would satisfy the claim.
A reasonable expectation of success exists where the technique is known to work for its intended purpose, even if results are not optimal.
Applicant attempts to distinguish AAV as uniquely complex.
However:
• Vliet already successfully applies LC/MS/MS to AAV capsid proteins
• Bark teaches the general applicability of LC/MS/MS to protein mixtures
• The modification (removal of gel step) does not depend on virus type
The combination does not require adapting Bark to AAV-specific biology, but merely applying a known analytical workflow variation to Vliet’s already functional system.
Applicant argues that achieving high sequence coverage and detection of VP1/VP2 PTMs is unexpected.
This argument is not persuasive because:
The claims do not recite:
• full sequence coverage
• detection of all PTMs
• improved sensitivity
• specific performance thresholds
Alleged advantages in the specification (e.g., 100% coverage, low sample amounts) are not claimed limitations
Unexpected results must be commensurate in scope with the claims, which is not the case here
Applicant argues that detection of VP1/VP2 is particularly challenging.
However:
• Vliet explicitly analyzes VP1, VP2, and VP3 capsid proteins
• The modification from Bark (removal of gel step) does not eliminate detectability of these proteins
• The claims still do not require detection of all peptides or complete coverage
Thus, these limitations do not distinguish over the combination.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to XIAOYUN R XU, Ph. D. whose telephone number is (571)270-5560. The examiner can normally be reached M-F 8am-5pm.
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/XIAOYUN R XU, Ph.D./ Primary Examiner, Art Unit 1797