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 Claims
Claims 1-3, 5-10, and 12-19 were pending. Claim 1 is amended.
Claims 1-3, 5-10, and 12-19 are examined herein.
Withdrawn Rejections
The rejection of claims 1-3, 5-10, and 12-19 under 35 U.S.C. § 112(b) is withdrawn in view of claim 1 amendments.
The rejection of claims 1-3, 5-10, and 12-19 under 35 U.S.C. 103 is withdrawn in view of claim 1 amendments. The amendments necessitated a new prior art search and new grounds of rejection have been found in view of newly found prior art reference of Lin.
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 1-2, 5-7, 9-10, 12-13 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Lundell et al. (Analytical biochemistry vol. 266,1 (1999): 31-47), in view of Thermo Scientific (Tech Tip #19, 2010; pp. 1-3), Jin et al. (Human gene therapy methods vol. 28,5 (2017): 255-267), and Lin et al. (J Am Soc Mass Spectrom 16, 581–588 (2005), and as evidenced by Ahmad et al. (Biochim Biophys Acta. 2000 Jul 14;1480(1-2):201-10).
Regarding claim 1, Lundell teaches “Sample Preparation for Peptide Mapping: A Pharmaceutical Quality-Control Perspective” (Title), protein characterization of therapeutic proteins by peptide mapping and steps prior to the separation of the generated peptides, including denaturation, reduction, alkylation, buffer exchange, solubilization, and digestion (Abstract). Lundell also teaches a method of characterizing proteins in a sample, the method comprising:
- denaturing a protein sample using GndHCl (pg. 32, Col. 2, last par.);
- subjecting the protein sample to denaturing size-exclusion chromatography to form a denatured sample – specifically, Lundell teaches protein solution separated on a gel filtration column equilibrated with elution buffer (10 mM TFA with 5 mM methionine) (pg. 32, Col. 2, last paragraph). The TFA component (trifluoroacetic acid) of the elution buffer provides the denaturing properties to this solution, as evidenced by Ahmad – “in the presence of an increasing concentration of TFA, cytochrome c was found to undergo extensive unfolding characterized by a significant breakdown of the secondary and tertiary structure of protein” (Abstract);
- eluting the denatured sample via liquid chromatography to collect fractions of the sample, wherein the fractions of the sample include a protein fraction – specifically, Lundell teaches that 400 µl of sample was applied to the column and the protein was eluted using 900 µl of elution buffer (pg. 33, Col. 1, 2nd paragraph) – not every fraction from the chromatography column was collected, only 900 µl fraction containing purified protein was collected for further experiments;
- digesting the denatured protein fraction with an enzyme - Lundell teaches trypsin solution added to the eluate (pg. 33, Col. 1, 2nd paragraph); and
- analyzing the digested protein fraction - Lundell teaches digests separated by reversed-phase chromatography (pg. 35, Col. 1, 2nd paragraph in section “Digestion Conditions for Trypsin”).
Additionally, Lundell teaches using non-ionic detergents (n-octylglucoside and n-dodecylmaltoside) an alternative to GndHCl for protein denaturation during trypsin digestion (pg. 34, section “Evaluation of Tryptic Activity”).
Lundell and Ahmad teach (a) using GndHCl for denaturing protein sample and removing GndHCl using SEC and (b) using non-ionic surfactant for denaturing protein sample, but do not teach removing non-ionic surfactant from the sample via denaturing size-exclusion chromatography.
However, size-exclusion chromatography is known in the art for its ability to remove detergents/surfactants from protein samples as taught by Thermo Scientific “Gel filtration removes detergents by size exclusion. Detergent monomers and micelles that are smaller than the filtration-resin pore size (i.e., molecular-weight cutoff, MWCO) can be separated from proteins and other macromolecules that are much larger” (pg. 1, 2nd paragraph of section “Overview of Removal Methods”). The terms detergent and surfactants are interchangeably used in the art. Table 2 indicates some non-ionic detergents that can be removed by desalting: Octyl-ß-Glucoside and OTG, because of their high critical micelle concentration and/or a low micelle molecular weight (pg. 2, 2nd paragraph). Detergents with a low CMC and a high molecular weight (e.g., Triton X-100) are very difficult to remove from solution (id.). Using denaturing size-exclusion chromatography with trifluoroacetic acid taught by Lundell (pg. 32, Col. 2, last paragraph) solves this problem, because TFA denatures surfactant micelles by disrupting hydrophobic interactions - the same interactions that stabilize proteins.
Lundell, Ahmad and Thermo Scientific do not teach analysis of adeno-associated virus capsid proteins.
Regarding claim 1, Jin teaches “Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins” (Title). Jin also teaches adeno-associated virus capsid proteins. Specifically, Jin teaches similar sample preparation steps as in Lundell (Abstract) and “AAV capsid proteins were directly analyzed by reverse-phase liquid chromatography/mass spectrometry (LC/MS) and characterized in detail by LC/MS/MS after in-solution enzymatic digestion” (pg. 257, Col. 1, 2nd paragraph). AAV is adeno-associated virus.
Lundell does not teach lyophilization and reconstitution of the lyophilized protein in the same experiment with size-exclusion chromatography and protein digestion. However, Lundell does teach protein lyophilization and reconstitution after a reverse-phase chromatography (pg. 35, Col. 1, 2nd paragraph), for increasing protein concentration in the sample. Fig. 1 demonstrates reverse-phase chromatography separation of reduced IFN protein sample discussed in the 2nd paragraph. As can be seen on the separation profile, each of the four protein peaks is eluted in volumes approaching 1 ml and reconstituted in smaller volume “collected fractions were lyophilized and dissolved in 400 µl of denaturation solution” (id.). As such, Lundell teaches lyophilization to increase protein concentration.
Lundell also teaches the molar ratio of the reducing agent to the cysteine residues in protein of interest is > 25:1.
Specifically, Lundell teaches reducing a sample of human interferon dissolved at a concentration of 4.8 µM (pg. 32, Col. 2, paragraph 1) using a reducing agent DTT (id., last paragraph). The reaction components were mixed in the following proportions: 150 µl IFN solution, 270 µl of denaturation solution and 10 µl of reduction solution. The final concentration of IFN in the mixture was 4.8 µM x 150 µl / 430 µl (total volume) = 1.7 µM. There were three cysteine residues in the IFN molecule, therefore, concentration of the cysteine residues was 3 x 1.7 µM = 5.1 µM. The final concentration of DTT in the mixture was 800 mM x 10 µl / 430 µl (total volume) = 18.6 mM or 18600 µM. The ratio of the reduction agent to the cysteine residues was 18,600 µM / 5.1 µM = 3,647-fold excess, meeting the limitation of claim 1 reciting the ratio of > 25:1.
In one of the examples ([0037]), the specification discloses using AAV concentration of 1x1013 vg/mL. Viral capsid proteins VP1, VP2 and VP3 are present in the capsid in 1:1:10 ratio. Molar concentration (M) of each capsid protein = 1x1013 vg/mL x ratio x 1,000 (mL/L) / 6x1023 (Avogadro number): VP1 – 1/6 x 10-7, VP2 – 1/6 x 10-7, and VP3 – 10/6 x 10-6. Total concentration of all VP proteins is 1/6 x 10-7 + 1/6 x 10-7 + 10/6 x 10-7 = 2x10-7 M. Each VP protein has 5 cysteine residues. Therefore, total concentration of cysteine residues is 1 x 10-6 M or 1 µM. At DTT concentration of 18,600 µM (see above IFN example) the molar ratio of the reducing agent to the cysteine residues is 18,600-fold, meeting the limitation of claim 1 reciting the ratio of > 25:1.
Lundell does not specifically teach reconstituting the lyophilized protein fraction with a buffer comprising a reducing agent, instead, Lundell teaches reducing protein fraction prior to the size-exclusion chromatography (pg. 33, Col. 1, par. 2). As such, Lundell teaches a different sequence of adding the reducing agent while achieving the same result of preventing reassociation of the sulfhydryl groups during trypsin digestion.
Additionally, some reducing agents, such as glutathione, are known for not evaporating during lyophilization process. Therefore, glutathione added to the protein fraction prior lyophilization stays there and does not require replenishment. As such, Lundell performs reconstituting the lyophilized protein fraction with a buffer comprising both a surfactant and a reducing agent.
Regarding amended claim limitation of “buffer composition improves solubility of denatured proteins with minimal impact on enzymatic activities” in step (d), Lundell teaches trypsin digestion in the presence of non-ionic detergents (n-octylglucoside and n-dodecylmaltoside) (pg. 34, section “Evaluation of Tryptic Activity”). It is the primary function of the non-ionic detergents to improve protein solubility. Additionally, Lundell performed trypsin digestion in the presence of non-ionic detergents, therefore the buffer composition of Lundell had minimal impact on enzymatic activities of trypsin. Finally, Lundell teaches step (e) - mixing the denatured protein fraction (denatured with one of the non-ionic detergents) with an enzyme for proteolytic digestion (trypsin).
Regarding claim limitation of “steps (d) and (e) constitute a one-pot denaturation and digestion method”, Lundell teaches that protein samples denatured with the non-ionic detergent are digested with trypsin (pg. 34, section “Evaluation of Tryptic Activity”). Lundell does not teach sample transfer from one test tube to another, or purification of the denatured protein prior to trypsin addition, therefore, Lundell does teach one-pot denaturation and digestion method.
Lundell, Ahmad, Jin, and Thermo Scientific do not specifically teach denaturation and digestion which occurs within 2.5 hours.
Regarding claim 1, Lin teaches accelerated enzymatic digestion of proteins for mass spectrometry analysis (Abstract). Lin also teaches digestion method occurs within 2.5 hours.
Specifically, Lin teaches sample digestion in the presence of organic solvents heated under microwave irradiation for 10 min using a commercial microwave applicator (Abstract), meeting the limitation of claim 1 reciting digestion which occurs within 2.5 hours. Lin does not specifically teach that denaturation also occurs within 2.5 hours, but when proteins are digested with a protease into small fragments in the presence of organic solvents, they lose their native structure and denature. Therefore, Lin inherently teaches denaturation also occurs within 2.5 hours.
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 Lundell and Ahmad by employing denaturation of the protein fraction using non-ionic surfactant as taught by Lundell (pg. 34, section “Evaluation of Tryptic Activity”), as an obvious matter of simple substitution of one known element (denaturation using non-ionic surfactant) for another (denaturation using GndHCl) to obtain predictable results, namely denaturing the protein fraction.
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 Lundell and Ahmad by employing size-exclusion chromatography as taught by Thermo Scientific, in order to provide a method for protein analysis with protein sample containing non-ionic surfactant which is difficult to remove from solution using non-denaturing size-exclusion chromatography. One having ordinary skill in the art would have been motivated to combine these teachings because some of the most common surfactants used in the art, such as, Triton X-100 are difficult to remove from solution using non-denaturing size-exclusion chromatography. The combination would have been desirable to those of ordinary skill in the art for the ability to analyze protein samples containing different surfactants.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because Lundell teaches denaturing size-exclusion chromatography for proteins, and proteins and surfactants are stabilized by the same hydrophobic interactions and are equally sensitive to denaturing conditions.
Although, Lundell does not teach removing non-ionic surfactant from the sample, the prior art method of Thermo Scientific teaches removing non-ionic surfactant, therefore it meets the claim.
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to apply the method of Lundell, Ahmad and Thermo Scientific for analysis of adeno-associated virus capsid proteins as taught by Jin, as an obvious matter of simple substitution of one known element (adeno-associated virus capsid proteins of Jin) for another (therapeutic proteins of Lundell) to obtain predictable results.
Both adeno-associated virus capsid proteins taught by Jin and therapeutic proteins of Lundell are proteins, therefore one of ordinary skill would have a reasonable expectation of success.
Lundell teaches a different sequence of adding a reducing agent while achieving the same result of preventing reassociation of the sulfhydryl groups during trypsin digestion, therefore Lundell meets the claim. Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.). See MPEP 2144.04.
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 Lundell for characterizing proteins comprising denaturing size-exclusion chromatography and trypsin digestion by employing lyophilization and reconstitution steps as taught by Lundell for a related chromatography experiment, to provide a method of characterizing proteins comprising denaturing size-exclusion chromatography with lyophilization and reconstitution steps and trypsin digestion, as an obvious matter of use of known technique (lyophilization plus reconstitution) to improve similar method (size exclusion chromatography) in the same way (increasing protein concentration the same way it was increased after reverse phase chromatography experiment (pg. 35, Col. 1, 2nd paragraph)). One having ordinary skill in the art would have been motivated to combine these teachings because lyophilization increases concentration of proteins and therefore increases detection sensitivity of subsequent methods of analysis. The combination would have been desirable to those of ordinary skill in the art for the ability to analyze diluted protein samples.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because Lundell teaches that lyophilization and reconstitution steps performed after chromatography separation do not negatively affect protein digestion.
It would have been a necessary property of the method taught by Lundell, Ahmad, Thermo Scientific, and Jin that using DTT concentration of 18,600 µM and an AAV sample with 1x1013 vg/mL would achieve the molar ratio of the reducing agent to the cysteine
residues in the adeno-associated virus capsid proteins of 18,600-fold, meeting the limitation of claim 1 reciting the ratio of > 25:1.
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 Lundell, Ahmad, Thermo Scientific, and Jin for analysis of adeno-associated virus capsid proteins by employing accelerated enzymatic digestion as taught by Lin, in order to accelerate sample preparation for mass spectrometry. One having ordinary skill in the art would have been motivated to combine these teachings because denaturation helps enzymatic digestion to run faster and shorter sample preparation time leads to increased mass spectrometry productivity. The combination would have been desirable to those of ordinary skill in the art for the reasons mentioned above.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because both Lundell, Ahmad, Thermo Scientific, and Jin and Lin are similarly drawn to preparation of protein samples for mass spectrometry. Lundell, Ahmad, Thermo Scientific, and Jin are generic on samples treatment using microwaves.
Regarding claim 2, Lundell teaches adding methionine to the protein fraction, prior to lyophilizing the protein fraction. Specifically, Lundell teaches elution of the protein sample from the gel filtration column with the elution buffer containing 5 mM methionine (pg. 32, Col. 2, last paragraph), therefore methionine is present in the protein fraction prior to lyophilization.
Regarding claims 5-6, Lundell teaches analyzing the digested protein fraction via
liquid chromatography-mass spectrometry comprises analyzing intact mass of the digested protein fraction “the molecular weight of a protein or peptide was determined by first lyophilizing the sample and then dissolving … prior to introduction to the electrospray” (pg. 32, col. 1, paragraph 1).
Regarding claim 12, Lundell teaches the buffer further comprises a reducing agent and a metal chelator. Specifically, Lundell teaches mixing protein sample with the denaturation and the reduction solutions (pg. 33, Col. 1, 2nd paragraph), wherein the denaturation buffer contained a chelator EDTA and the reduction solution contained a reducing agent DTT and the chelator EDTA (pg. 32, Col. 2, last paragraph). Therefore, the reconstitution buffer used to solubilize the lyophilized protein fraction contained the reducing agent and the chelator, because DTT and EDTA are not volatile compounds and stay in the protein fraction during lyophilization.
Regarding claim 13, Lundell teaches the enzyme is trypsin “trypsin digestion buffer and 10 µl of trypsin solution were now added to the eluate” (pg. 33, Col. 1, 2nd paragraph).
Regarding claim 17, Lundell teaches digesting the denatured protein fraction with an enzyme is carried out at a temperature ranging from about 30°C to about 50°C. Specifically, Lundell teaches “digestion was performed overnight at approximately 14°C (refrigerator), 23°C (room temperature), or 37°C (water bath)” (pg. 35, 2nd paragraph in section “Digestion conditions for trypsin”). Temperature of 37°C is falling within and therefore meeting the limitation of claim 17, which recites a range of 30-50°C.
Regarding claim 7, Lundell does not specifically teach a benchtop Time-of-Flight (ToF) mass spectrometer.
Regarding claim 7, Jin teaches a benchtop Time-of-Flight (ToF) mass spectrometer for analysis of AAV capsid proteins.
Specifically, Jin teaches “AAV capsid proteins were directly analyzed by reverse-phase liquid chromatography/ mass spectrometry (LC/MS) and characterized in detail by LC/MS/MS after in-solution enzymatic digestion” (pg. 257, Col. 1, 2nd paragraph) and “protein digests were also analyzed by UPLC/MS in an Acquity UPLC-Xevo G2XS QTOF MS system (Waters)” (pg. 258, Col. 2, section “UPLC/MS peptide mapping”), a Time-of-Flight (ToF) mass spectrometer.
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 Lundell, Ahmad, and Thermo Scientific by employing a Time-of-Flight MS instrument as taught by Jin, to provide a method of characterizing AAV proteins, as an obvious matter of simple substitution of one known element (a ToF MS instrument of Jin) for another (an electrospray MS instrument of Lundell) to obtain predictable results.
Regarding claims 9-10, Lundell does not teach a specific protein sequence coverage.
Regarding claims 9-10, Jin also teaches analyzing the digested protein fraction comprises providing greater than 95% and greater than 97% protein sequence coverage.
Specifically, Jin teaches “AAV capsid proteins were directly analyzed by reverse-phase liquid chromatography/ mass spectrometry (LC/MS) and characterized in detail by LC/MS/MS after in-solution enzymatic digestion. For AAV2, complete sequence coverage of VP1, VP2, and VP3 was achieved” (pg. 257, Col. 1, 2nd paragraph). The complete coverage is falling within and therefore meeting the limitations of claims 9 and 10, which recite greater than 95% and greater than 97% protein sequence coverage.
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to apply the method of Lundell, Ahmad, and Thermo Scientific modified with a Time-of-Flight MS instrument as taught by Jin, and expect the complete sequence coverage of the AAV proteins as an inherent matter of MS analysis of the AAV proteins analyzed by the Time-of-Flight MS instrument as taught by Jin.
Claims 3 and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Lundell in view of Thermo Scientific, Jin, and Lin, and as evidenced by Ahmad, as applied to claim 1 above, and further in view of Russell et al. (Analytical chemistry vol. 83,6 (2011): 2187-93), for reasons of record which are reiterated herein below.
The teachings of Lundell, Ahmad, Thermo Scientific, Jin, and Lin have been set forth above.
Regarding claims 3 and 8, Lundell, Ahmad, Thermo Scientific, Jin, and Lin fail to teach the protein fraction is less than 10 µg, and analyzing the digested protein fraction comprises measuring viral protein expression with fluorescence detection.
Regarding claim 3, Russell teaches “Parallel Detection of Intrinsic Fluorescence from Peptides and Proteins for Quantification during Mass Spectrometric Analysis” (Title). Russel also teaches the protein fraction is less than 10 µg.
Specifically, Russel teaches “MS SICs provided lower limits of detection (~5 fmol) by a factor of 3-5. However, when peptides contained multiple nonadjacent tryptophan residues, UV-IF produced limits of detection approaching subfemtomole levels” (pg. 2190, Col. 2, last paragraph, and pg. 2191, Col. 1, 1st paragraph). Let’s convert detection levels from femtomoles as reported by Russel into picograms as recited in claim 3 and compare them. Let’s use 10 femtomole as a conservative detection limit (in comparison to 5 fmol and subfemtomole levels). For a protein with a molecular weight of 65 kDa (such as VP2 protein) the detection limit of 10 femtomole corresponds to 65 x 103 x 10 x 10-15 = 6.5 x 10-10g or 0.65 µg - the value falling within and therefore meeting the limitation of claim 3, which recites the protein fraction is less than 10 µg. As such, even a conservative lower limit of detection reported by Russel (10 fmol) corresponds to more than 10-fold better sensitivity than recited in claim 3.
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 teachings of Lundell, Ahmad, Thermo Scientific, Jin, and Lin by employing the method as taught by Russel, in order to provide a method capable of analyzing less than 10 µg of protein fraction, as an inherent matter of the analytic sensitivity of the applied methods.
Regarding claim 8, Russell also teaches analyzing the digested protein fraction comprises measuring viral protein expression with fluorescence detection. Specifically, Russell teaches “the implementation of a fluorescence detection system for measurement of the UV-excited intrinsic fluorescence (UV-IF) from peptides and proteins” and “fluorescence signal is linear over 3 orders of magnitude with on-column limits of detection in the low femtomole range” (Abstract).
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 Lundell, Ahmad, Thermo Scientific, Jin, and Lin by employing the fluorescence detection method as taught by Russel, as an obvious matter of simple substitution of one known element (fluorescence detection of Russel) for another (MS detector of Lundell or Jin) to obtain predictable results.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Lundell, in view of Thermo Scientific, Jin, and Lin, and as evidenced by Ahmad, as applied to claim 1 above, and further in view of Park et al. (Analytical chemistry vol. 72,11 (2000): 2667-70).
The teachings of Lundell, Ahmad, Thermo Scientific, Jin, and Lin have been set forth above.
Regarding claim 14, Lundell, Ahmad, Thermo Scientific, Jin, and Lin fail to teach specific denaturation temperature.
Regarding claim 14, Park teaches “Thermal Denaturation: A Useful Technique in Peptide Mass Mapping” (Title) and “use of thermal denaturation of proteins prior to in-solution digestion and mass spectral peptide mass mapping” (Abstract). Park also teaches reconstituting the lyophilized protein fraction with a buffer comprising a surfactant to denature the protein is carried out at a temperature of greater than 65°C.
Specifically, Park teaches “aliquots from protein solutions were thermally denatured by incubating at 90°C” (pg. 2668, 2nd paragraph, “Experimental section”). Temperature of 90°C is falling within and therefore meeting the limitation of claim 14, which recites a temperature of greater than 65°C.
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 Lundell, Ahmad, Thermo Scientific, Jin, and Lin by employing the denaturation conditions as taught by Park, to provide a method of characterizing AAV proteins, wherein the trypsin digestion is as complete as possible. One having ordinary skill in the art would have been motivated to combine these teachings because the complete trypsin digestion provides good quality mass spectrometry results and avoids using other enzymes to fill the gap. The combination would have been desirable to those of ordinary skill in the art for the reason mentioned above.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because protein denaturation and enzymatic digestion are very well-studied reactions.
Claims 15-16, are rejected under 35 U.S.C. 103 as being unpatentable over Lundell, in view of Thermo Scientific, Jin, and Lin, and as evidenced by Ahmad, as applied to claim 1 above, and further in view of Proc et al. (Journal of proteome research, vol. 9,10 (2010): 5422-37), for reasons of record which are reiterated herein below.
The teachings of Lundell, Ahmad, Thermo Scientific, Jin, and Lin have been set forth above.
Regarding claims 15-16, Lundell, Ahmad, Thermo Scientific, Jin, and Lin fail to teach specific incubation time for protein denaturation prior to enzymatic digestion.
Regarding claims 15-16, Proc teaches “A Quantitative Study of the Effects of Chaotropic Agents, Surfactants, and Solvents on the Digestion Efficiency of Human Plasma Proteins by Trypsin” (Title). Proc also teaches reconstituting the lyophilized protein fraction with a buffer comprising a surfactant to denature the protein is carried out for less than about 5 minutes, or for about 3 minutes.
Specifically, Proc teaches “heat denaturation of plasma samples prior to chemical denaturation with urea improved the efficiencies of the urea digestion protocol for 32 out of the 45 proteins examined (Figure 2D and Supporting Information Figure 1)” (pg. 5433, section “Heat denaturation”). Proc teaches that not all proteins are equally sensitive to denaturation. For example, Fig. 4, shows relative digestion efficiencies for three groups of proteins: rapidly digested, moderately digested and proteins resistant to digestion (panels A, B and C correspondingly). For the last group incubation for 9 and 16 hours has not resulted in digestion as complete as for the first group. Individual protein sample require optimization of the denaturation conditions.
Generally, differences in incubation time will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. Applicant has not disclosed that the specific limitations recited in claims 15-16 for denaturation time, are for any particular purpose or solve any stated problem. Instant specification is silent on any details related to denaturation time. Furthermore, the limitation of 3-5 min of denaturation time is not specific enough without reciting incubation temperature and concentrations of surfactants. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
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 Lundell, Ahmad, Thermo Scientific, Jin, and Lin by employing the teachings of Proc for significant variability in protein digestion rates to discover the optimum workable ranges of the denaturation time by normal optimization procedures known in the art.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because protein denaturation is a very well-known process that can be optimized by varying buffer composition and denaturation temperature.
Claims 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Lundell, in view of Thermo Scientific, Jin, and Lin, and as evidenced by Ahmad, as applied to claim 1 above, and further in view of Promega ("Rapid Digestion - Trypsin and Rapid Digestion - Trypsin/Lys-C Kits. Instructions for use of products VA1060 and VA1061", 2017, pp. 1-11), for reasons of record which are reiterated herein below.
The teachings of Lundell, Ahmad, Thermo Scientific, Jin, and Lin have been set forth above.
Regarding claims 18-19, Lundell, Ahmad, Thermo Scientific, Jin, and Lin fail to teach incubation time for protein enzymatic digestion is 50-70 minutes.
Regarding claims 18-19, Promega teaches a product for rapid trypsin digestion of proteins (pg. 1, Description). Promega also teaches digesting the denatured protein fraction with an enzyme is carried out for about 50 minutes to about 70 minutes, or for about 60 minutes.
Specifically, Promega teaches “rapid digestion-Trypsin and rapid digestion Trypsin/Lys-C Kits are designed to shorten protein digestion times to 60 minutes versus the typical 4–18 hours” (pg. 1, section “Description”). Digestion for 60 minutes is falling within and therefore meeting the limitations of claims 18-19, which recite digestion times of 50-70 minutes and about 60 minutes.
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 Lundell, Ahmad, Thermo Scientific, Jin, and Lin by employing the denaturation conditions as taught by Promega, to provide a method of characterizing AAV proteins, wherein the trypsin digestion is short in duration, to provide the method of characterizing proteins with minimal deamidation (Lundell teaches that longer digestion leads to undesired, increased deamidation (Fig. 5)). One having ordinary skill in the art would have been motivated to combine these teachings because lower deamidation of proteins improves the quality of mass spectrometry results. The combination would have been desirable to those of ordinary skill in the art for the reason mentioned above.
One having ordinary skill in the art would have had a reasonable expectation of success in combining the prior art references because enzymatic digestion is a very well-studied reaction as applied to mass spectrometry.
Response to Arguments
Applicant's arguments filed August 29, 2025 have been fully considered.
Claims 1-3, 5-10, and 12-19 were rejected under 35 U.S.C. §112(b). Applicant argues that claim 1 amendment meets the requirements of 35 U.S.C. §112(b) (pg. 6, section II). The argument is persuasive and §112(b) rejection of claims 1-3, 5-10, and 12-19 is withdrawn.
Claims 1-3, 5-10, and 12-19 were rejected under 35 U.S.C. §103 as being unpatentable over the referenced prior art. Applicant argues that claim 1 amendment recites “denaturation and digestion method which occurs within 2.5 hours” (pg. 8, par. 1) and the teachings of the referenced prior art fail to teach, suggest, or make obvious the method of claim 1, wherein steps (d) and (e) constitute a one-pot denaturation and digestion method which occurs within 2.5 hours.
Regarding the new limitation of “denaturation and digestion method which occurs within 2.5 hours”, Lin teaches a sample preparation method with digestion time less than 2.5 hours (Abstract). Therefore, claim 1 is rejected under 103 in view of Lundell, Ahmad, Thermo Scientific, Jin, and Lin. This argument applies to all dependent claims mentioned by Applicant (pg. 7, section II – pg. 12). Please, see §103 rejection above for details.
Applicant argues that “contrary to the Examiner's interpretation of the experiment in question in Lundell, the method only describes a protein digestion method in the presence of a detergent or urea, not a "one pot denaturation and digestion method," as claimed” (pg. 8, par. 2).
The “one-pot denaturation and digestion” limitation is addressed in the non-final OA mailed June 2, 2025 - “Regarding amended claim limitation of “wherein steps (d) and (e) constitute a one-pot denaturation and digestion method”, Lundell teaches that protein samples denatured with the non-ionic detergent are digested with trypsin (pg. 34, section “Evaluation of Tryptic Activity”). Lundell does not teach sample transfer from one test tube to another, or purification of the denatured protein prior to trypsin addition, therefore, Lundell does teach one-pot denaturation and digestion method” (pg. 7, par. 2). Applicant recites a passage from the specification (pg. 8, last par.), but fails to address the Office argument specifically.
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.
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
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