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 .
Priority
This is a National Stage Entry under 35 U.S.C. 371 of International Patent Application No.
PCT/EP2022/078725, filed October 14, 2022, which claims priority to US Application Nos. 63/368,803, filed July 19, 2022, and 63/256,111, filed October 15, 2021. This application claims priority to EP provisional application EP21205732.7, filed on October 29, 2021.
Claim Objections
Claims 1 and 2 are objected to because of the following informalities:
Claims 1 and 2 recite a series of acts but fail to properly identify the series as “steps.”
Claim 1 should be amended to recite:
“A method of preparing a digested virus protein, comprising the steps of:
a. precipitating a virus protein from a sample containing the virus protein;
b. dissolving the virus protein in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-Maltoside (DDM) to generate a solution; and
c. digesting the virus protein with a protease.”
Claim 2 should be amended to recite:
“A method of preparing a digested virus protein, comprising the steps of:
a. precipitating a virus protein from a sample containing the virus protein;
b. dissolving the virus protein in a mixture comprising sodium deoxycholate (SDC) and N-dodecyl-beta-D-Maltoside (DDM) to generate a solution;
c. digesting the virus protein with a protease; and
e. analyzing the digested virus protein via liquid chromatography-tandem mass spectrometry (LC-MS/MS).”
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 3, 10 – 12, 17 – 19, 21, 23, 24, and 26 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The term “about” in claims 10 – 12, 17 – 19, 21, 23, and 26 is a relative term which renders the claim indefinite. The term “about” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
Claim 10 recites “SDC at about 0.01% to 1.5% (w/w) and DDM at about 0.01% to 1.0% (w/w)”
Claim 11 recites “SDC at about 0.5% to 1.5% (w/w) and DDM at about 0.01% to 1.0% (w/w)”
Claim 12 recites “SDC at about 0.5% to 1.5% (w/w) and DDM at about 0.2% to 1.0% (w/w), 2) SDC at about 0.75% to 1.25% (w/w) and DDM at about 0.5% to 0.8% (w/w), 3) SDC at about 0.01% to 0.6% (w/w) and DDM at about 0.01% to 1% (w/w), 4) 3DC at about 0.01% to 0.6% (w/w) and DDM at about 0.01% to 0.6% (w/w) or 5) wherein the mixture comprises SDC at about 0.2% to 0.4% (w/w) and DDM at about 0.05% to 0.2% (w/w)”
Claim 17 recites “a ratio of about 1:0.5 w/w or about 3.5:1 w/w (SDC:DDM)”
Claim 18 recites “at about pH 6.0 to about pH 9.0”
Claim 19 recites “at about 30°C to 40°C, for a period of about 2 to 12 hours”
Claim 21 recites “a ratio of about 20:1 to about 100:1 w:w of virus protein: trypsin”
Claim 23 recites “virus protein is about 3 to 70 amino acids in length”
Claim 26 recites “virus protein of about 0.001 mg/mL to about 0.10 mg/mL”
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.
Claim(s) 1 – 3, 10 – 12, 17, 19 and 21 are rejected under 35 U.S.C. 103 as being unpatentable Sacramento et al (Molecular Systems Biology, 2020, 10.15252/msb.20209596, hereinafter, “Sacramento”), Lu et al (Frontiers in Cellular and Infection Microbiology, September 2021, 10.3389/fcimb.2021.734578, hereinafter, “Lu”), and Moser et al (WO2006045532A2).
Sacramento teaches the characterization and quantification of the proteome during brain aging in Nothobranchius furzeri by combining transcriptomics and proteomics (Abstract). Sacramento teaches the preparation and analysis of proteins using liquid chromatography and mass spectrometry (Materials and Methods). Sacramento teaches that these methods enable detailed information of the proteome allowing for peptide mapping and analysis of post-translational modifications (PTMs) (Abstract).
Regarding claims 1 and 2, Sacramento teaches a general workflow for preparing proteins for LC-MS (Section: Sample preparation for mass spectrometry analysis and data acquisition). Sacramento teaches the precipitation of proteins using ice-cold acetone followed by a digestion of the precipitated proteins (Section: Sample preparation for proteome analysis (Nothobranchius furzeri)). Sacramento teaches the digestion of the proteins can be performed by a physical process executed at a pH of 8.0 followed by enzymatic digestion with a protease at a ratio of 1:100 enzyme:protein (Section: Sample preparation for proteome analysis (Nothobranchius furzeri)). Sacramento teaches that the prepared proteins and peptides can then by analyzed using LC-MS (Section: Sample preparation for proteome analysis (Nothobranchius furzeri)).
Sacramento does not teach the use of sodium deoxycholate (SDC) and N-dodecyl-beta-D-Maltoside (DDM) in a solution.
However, Lu teaches methods for identifying membrane proteins using different digestion methods and extraction detergents for label-free quantitative and targeted proteome (Abstract). Lu teaches performing the proteome using LC-MS (Abstract). Furthermore, teaches the effects of lingering detergents on enzymatic and LC-MS quality (Discussion, ¶2).
Regarding claims 1 and 2, Lu teaches a SDC or DDM solution for protein dissolution and digestion (Section: Cell Culture and Sample Preparation). Lu teaches that in view of peptide loss that may be caused by detergent removal, SDC and DDM are preferred detergents (Discussion ¶2). Lu also teaches using a protease at a ratio of 1:50 or 1:25 ratio before using LC-MS to analyze the digest proteins and peptides (Section: Cell Culture and Sample Preparation, Nano LC−MS/MS and Data Acquired).
Sacramento do not teach the use of both SDC and DDM in a solution. While Lu teaches SDC and DDM are preferred detergents, Lu does not teach the use of both SDC and DDM in a single solution.
However, Moser teaches a virosome comprising at least one lipid and nucleocapsid proteins of an enveloped virus (Abstract, Page 1 Line 6). Moser teaches the proteomic analysis of virosome particles to understand the proteolysis resistant fragments (Page 34 Line 7 – 19). Moser teaches the digestion of viral proteins using 0.5% SDC and the non-ionic detergent 1% Triton X-100 followed by protease digestion (Page 34 Line 7 – 19).
Regarding claims 1 and 2, Moser teaches a solution to digest viral proteins using an ionic and non-ionic detergent (Page 34 Line 7 – 19). Moser also teaches other non-ionic detergents, such as Tween 20/80, that can be substituted for Triton X-100 in a solution (Page 31 Line 10 - 15).
Sacramento, Lu, and Moser are analogous because they teach methods of digesting proteins for proteomic analysis. Sacramento teaches a general workflow for preparing proteins for LC-MS (Section: Sample preparation for mass spectrometry analysis and data acquisition). Sacramento teaches the precipitation of proteins using ice-cold acetone followed by a digestion of the precipitated proteins followed by the digestion of the proteins using a followed by LC-MS (Section: Sample preparation for proteome analysis (Nothobranchius furzeri)). Lu teaches that DDM, a non-ionic detergent, is used to dissolve proteins in solution (Section: Cell Culture and Sample Preparation). Moser teaches that ionic detergents such as SDC and non-ionic detergents such as Triton X-100 can be used in a protein digestion solution together (Page 34 Line 7 – 19).
Together, one of ordinary skill in the art would have substituted the known element of a non-ionic detergent, such as Triton X-100, for another non-ionic detergent, such as DDM, in a protein dissolution or digestion solution as the results of the substitution would have been predictable. Therefore, it would have been prima facie obvious before the effective filing date of the claimed invention to have used a solution comprising SDC and DDM to dissolve or digest viral proteins before LC-MS because doing so would enable a thorough analysis of a viral proteome. One of ordinary skill in the art would have had a reasonable expectation of success in using a solution comprising SDC and DDM to dissolve or digest viral proteins before LC-MS given that the breakdown of proteins using ionic and nonionic detergents for thorough digestion is well known, has been successfully demonstrated, and commonly used in the prior art.
Regarding claim 10, 11, 12, and 17 Lu teaches using a solution comprising 2% SDC or a solution comprising 1% DDM (Section: Cell Culture and Sample Preparation). Moser teaches a digestion buffer comprising 0.5% SDC and 1% Triton X-100. It would have been a matter of routine experimentation using standard laboratory techniques available at the time of filing to determine the optimal concentration of SDC and DDM for use in the method taught by Sacramento, Lu, and Moser to maximize the digestion of proteins with a reasonable expectation of success. Generally, differences in concentration will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such differences are critical. “[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). Here, the instant claims encompass routing concentration ranges for protein digestion which evidences that a particular concentration is not critical and readily obtained by routine optimization.
Regarding claim 3, Lu teaches the removal of SDC from the solution using 1% formic acid or acetone (Discussion ¶2).
Regarding claim 19, Sacramento teaches applying protease to digest protein at 37°C for 4 hours (Section: Sample preparation for proteome analysis (Nothobranchius furzeri)).
Regarding claim 21, Lu teaches digesting with trypsin (Section: Cell Culture and Sample Preparation).
Therefore, it would have been prima facie obvious before the effective filing date of the claimed invention to have used a solution comprising SDC and DDM to dissolve or digest viral proteins and remove SDC from the solution before subsequent trypsin digest at 37°C for 4 hours before LC-MS because doing so would enable a thorough analysis of a viral proteome. One of ordinary skill in the art would have had a reasonable expectation of success in using a solution comprising SDC and DDM to dissolve or digest viral proteins before LC-MS given that the removal of detergents before protease digestion is well known, has been successfully demonstrated, and commonly used in the prior art.
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill in the art at the time of filing especially in the absence of evidence to the contrary.
Claims 4, 5, 23, and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Sacramento, Lu, and Moser as applied to claims 1 – 3, 10 – 12, 17, 19 and 21 above, and further in view of Toole et al (Analytical Chemistry, July 2021, 10.1021/acs.analchem.1c02117, hereinafter, “Toole”).
As discussed above, claims 1 – 3, 10 – 12, 17, 19 and 21 were rendered prima facie obvious by Sacramento, Lu, and Moser. While the references teach a general workflow for preparing proteins for LC-MS including the precipitation of proteins using ice-cold acetone, the use of a dissolution solution comprising SDC and DDM, and a protease digestion, the references fail to teach the digestion of a AAV viral protein or wherein the sample of virus protein is at a concentration of 0.001 mg/ml to 0.10 mg/ml.
However, Toole teaches the motivation of virus peptide mapping and digestion (Abstract). Toole teaches the peptide mapping of AAVs to confirm sequence, product purity, PTMs, and stability (Abstract). Toole teaches digesting AAVs for viral capsid denaturation and improved cleave site accessibility (Abstract). Toole teaches using trypsin or pepsin to digest AAV2 viral capsid protein followed by LC-MS to analyze the protein and peptide (Abstract, Section: Trypsin High-Temperature Proteolysis, Pepsin High-Temperature Proteolysis, LC-MS of AAV Digests).
Regarding claims 4 and 5, Toole teaches the digestion of and subsequent LC-MS analysis of AAV2 viral capsid proteins (Abstract, Section: Trypsin High-Temperature Proteolysis, Pepsin High-Temperature Proteolysis, LC-MS of AAV Digests).
Regarding claim 23, Toole teaches digest viral proteins that are 8 amino acids in length (“Even the short peptide FMVPQYGY resulting from digestion with pepsin is retained strongly and elutes late in the gradient at a retention time of 103 min”, Figure 2).
Regarding claim 26, Toole teaches a sample containing 1ug/25ul or 0.04mg/ml (0.001mg/0.025ml= 0.04mg/ml) of the virus protein.
Sacramento, Lu, Moser, and Toole are analogous because they teach methods of digesting proteins for proteomic analysis. Sacramento teaches a general workflow for preparing proteins for LC-MS (Section: Sample preparation for mass spectrometry analysis and data acquisition). Lu teaches that DDM, a non-ionic detergent, is used to dissolve proteins in solution (Section: Cell Culture and Sample Preparation). Moser teaches that ionic detergents such as SDC and non-ionic detergents such as Triton X-100 can be used in a protein digestion solution together (Page 34 Line 7 – 19). Toole teaches LC-MS of AAV2 capsid proteins after digestion (Abstract, Section: Trypsin High-Temperature Proteolysis, Pepsin High-Temperature Proteolysis, LC-MS of AAV Digests).
Therefore, it would have been prima facie obvious before the effective filing date of the claimed invention to have used a solution comprising SDC and DDM to digest AAV2 viral capsid proteins because doing so would enable a thorough analysis of a capsid proteome. One of ordinary skill in the art would have had a reasonable expectation of success in using a solution comprising SDC and DDM to digest AAV viral capsid proteins given that the digestion and proteomic probing of AAV viral capsid proteins is well known, has been successfully demonstrated, and commonly used in the prior art.
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill in the art at the time of filing especially in the absence of evidence to the contrary.
Claims 6 – 8 are rejected under 35 U.S.C. 103 as being unpatentable over Sacramento, Lu, and Moser as applied to claims 1 – 3, 10 – 12, 17, 19 and 21 above, and further in view of Benevento et al (JBC, 2014, 10.1074/jbc.M113.537498, hereinafter, “Benevento”).
As discussed above, claims 1 – 3, 10 – 12, 17, 19 and 21 were rendered prima facie obvious by Sacramento, Lu, and Moser. While the references teach a general workflow for preparing proteins for LC-MS including the precipitation of proteins using ice-cold acetone, the use of a dissolution of digestion solution comprising SDC and DDM, and a protease digestion, the references fail to teach the digestion of adenovirus 5 protein.
However, Benevento teaches a high-resolution MS-based proteomic method that achieves high sequence coverage for adenovirus proteins (Abstract). Benevento teaches that digesting proteins allowed the identification of additional proteins as well as define the stoichiometry of each adenovirus protein (Discussion). Benevento also teaches that digestion of adenovirus particles with SDC can be used for other viruses (Discussion).
Regarding claims 6 – 8, Benevento teaches the digestion of adenovirus 5 using an initial solution comprising SDC (Section: Experimental procedures- Protein Extraction and Digestion).
Sacramento, Lu, Moser, and Benevento are analogous because they teach methods of digesting proteins for proteomic analysis. Sacramento teaches a general workflow for preparing proteins for LC-MS (Section: Sample preparation for mass spectrometry analysis and data acquisition). Lu teaches that DDM, a non-ionic detergent, is used to dissolve proteins in solution (Section: Cell Culture and Sample Preparation). Moser teaches that ionic detergents such as SDC and non-ionic detergents such as Triton X-100 can be used in a protein digestion solution together (Page 34 Line 7 – 19). Benevento teaches the digestion of adenovirus using an initial solution comprising SDC (Section: Experimental procedures- Protein Extraction and Digestion).
Therefore, it would have been prima facie obvious before the effective filing date of the claimed invention to have used a solution comprising SDC and DDM to digest adenovirus 5 viral proteins because doing so would enable a thorough analysis of a virus proteome. One of ordinary skill in the art would have had a reasonable expectation of success in using a solution comprising SDC and DDM to digest adenovirus 5 viral capsid proteins given that the digestion and proteomic probing of adenovirus 5 viral proteins is well known, has been successfully demonstrated, and commonly used in the prior art.
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill in the art at the time of filing especially in the absence of evidence to the contrary.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Sacramento, Lu, and Moser as applied to claims 1 – 3, 10 – 12, 17, 19 and 21 above, and further in view of Schlatzer et al (Analytical Chemistry, 2017, 10.1021/acs.analchem.6b05070, hereinafter, “Schlatzer”).
As discussed above, claims 1 – 3, 10 – 12, 17, 19 and 21 were rendered prima facie obvious by Sacramento, Lu, and Moser. The references fail to teach the digestion of lentivirus protein.
However, Schlatzer teaches the detection of HIV from infected patients using the GSG structural proteins as a marker (Abstract). Schaltzer teaches the identification of HIV viral proteins by first digesting viral proteins followed by LC-MS (Section: Data-Dependent (DDA) LC-MS/MS Detection of Gag in Purified Viruses, Infected Cells, and Immunoprecipation Experiments). Schaltzer teaches a sequence coverage of 84% for HIV viral proteins (HIV-1 Gag Structural Proteins Are the Most Abundant Viral Protein in Purified Particles and Infected CD4+ T Cells).
Regarding claim 9, Shaltzer teaches digestion of HIV protein using trypsin at a ratio of 1:20 at 37°C overnight (Materials and Methods Sample Preparation for Purified Virus or Cells. Digestion protocol).
Sacramento, Lu, Moser, and Shaltzer are analogous because they teach methods of digesting proteins for proteomic analysis. Sacramento teaches a general workflow for preparing proteins for LC-MS (Section: Sample preparation for mass spectrometry analysis and data acquisition). Lu teaches that DDM, a non-ionic detergent, is used to dissolve proteins in solution (Section: Cell Culture and Sample Preparation). Moser teaches that ionic detergents such as SDC and non-ionic detergents such as Triton X-100 can be used in a protein digestion solution together (Page 34 Line 7 – 19). Shaltzer teaches digestion of HIV protein using trypsin at a ratio of 1:20 at 37°C overnight (Section: Sample Preparation for Purified Virus or Cells).
Therefore, it would have been prima facie obvious before the effective filing date of the claimed invention to have used a solution comprising SDC and DDM to dissolve or digest lentiviral proteins because doing so would enable a thorough analysis of a viral proteome. One of ordinary skill in the art would have had a reasonable expectation of success in using a solution comprising SDC and DDM to dissolve or digest lentivirus proteins given that the digestion and proteomic probing of lentivirus proteins is well known, has been successfully demonstrated, and commonly used in the prior art.
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill in the art at the time of filing especially in the absence of evidence to the contrary.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Sacramento, Lu, and Moser as applied to claims 1 – 3, 10 – 12, 17, 19 and 21 above, and further in view of Levin et al (bioRxiv, February 2021, 10.1101/2021.02.08.430232, hereinafter, “Levin”) and Liu et al (Analytical Chemistry 2015, 10.1021/ac504700t, hereinafter, “Liu”).
As discussed above, claims 1 – 3, 10 – 12, 17, 19 and 21 were rendered prima facie obvious by Sacramento, Lu, and Moser. While the references teach a general workflow for preparing proteins for LC-MS including the precipitation of proteins using ice-cold acetone, the use of a dissolving solution comprising SDC and DDM, and a protease digestion, the references fail to teach a dissolving solution between pH 6.0 and 9.0.
However, Liu teaches testing different concentrations of DDM to solubilize proteins without changing their structure (Abstract). Liu teaches that DDM can be used in solutions and is compatible with trypsin digest (Supplemental information Section: Compatibility with trypsin activity and MS analysis). Liu teaches that the use of DDM allows for trypsin digestion without detergent removal steps therefore, increases “sensitivity, reliability, and accuracy of proteomic analysis especially for the post translational modifications (PTMs) analysis of a minute amount of protein samples” (¶1,2).
Levin teaches the photosynthetic properties of a desert algae that thrives at extreme high light in-tensities (Abstract). Levin teaches a proteomic analysis of proteins using LC-MS/MS of digested proteins (Section: Sample preparation for mass spectroscopy analysis, LC-MS/MS). Levin teaches to digesting proteins using a solution of SDC at pH 8.0 for LC-MS/MS (Section: Sample preparation for mass spectroscopy analysis, LC-MS/MS).
Regarding claim 18, Levin teaches to digesting proteins using a solution of SDC at pH 8.0 for LC-MS/MS (Section: Sample preparation for mass spectroscopy analysis, LC-MS/MS). Liu teaches that DDM in buffers and solutions at a pH of between 7.5 and 8.0 upstream of LC-MS/MS (Supplemental information Section: Compatibility with trypsin activity and MS analysis).
Sacramento, Lu, Moser, Levin and Liu are analogous because they teach methods of digesting proteins for proteomic analysis. Sacramento teaches a general workflow for preparing proteins for LC-MS (Section: Sample preparation for mass spectrometry analysis and data acquisition)Lu teaches that DDM, a non-ionic detergent, is used to dissolve proteins in solution (Section: Cell Culture and Sample Preparation). Moser teaches that ionic detergents such as SDC and non-ionic detergents such as Triton X-100 can be used in a protein digestion solution together (Page 34 Line 7 – 19). Levin teaches to digesting proteins using a solution of SDC at pH 8.0 for LC-MS/MS (Section: Sample preparation for mass spectroscopy analysis, LC-MS/MS). Liu teaches that DDM in buffers and solutions at a pH of between 7.5 and 8.0 upstream of LC-MS/MS (Supplemental information Section: Compatibility with trypsin activity and MS analysis).
Therefore, it would have been prima facie obvious before the effective filing date of the claimed invention to have used a solution comprising SDC and DDM at a pH between 6.0 and 9.0 to dissolve or digest proteins because doing so would enable a thorough analysis of a viral proteome. One of ordinary skill in the art would have had a reasonable expectation of success in using a solution comprising SDC and DDM at a pH between 6.0 and 9.0 to dissolve or digest lentivirus proteins given that the digestion and proteomic probing of proteins is well known, has been successfully demonstrated, and commonly used in the prior art.
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill in the art at the time of filing especially in the absence of evidence to the contrary.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Sacramento, Lu, and Moser as applied to claims 1 – 3, 10 – 12, 17, 19 and 21 above, and further in view of Saito et al (Methods Protoc, 2019, 10.3390/mps2020046, hereinafter, “Saito”) and Gin et al (protocols.io, 2020, 10.17504/protocols.io.bfx6jpre, hereinafter, “Gin”).
As discussed above, claims 1 – 3, 10 – 12, 17, 19 and 21 were rendered prima facie obvious by Sacramento, Lu, and Moser. While the references teach a general workflow for preparing proteins for LC-MS including the precipitation of proteins using ice-cold acetone, the use of a dissolving solution comprising SDC and DDM, and a protease digestion, the references fail to teach a chloroform/water/methanol-based protein precipitation.
However, regarding claim 20, Saito teaches the effected of protein precipitation on the stability and accuracy of urine proteomics (Abstract). Saito teaches the use of several methods for protein precipitation for preparing samples for LC-MS/MS (Abstract, Introduction ¶3). Saito teaches the comparison of ethanol, acetone, acetonitrile, and methanol/chloroform (Introduction ¶3).
Similarly, Gin teaches a protocol on a method for precipitating proteins using chloroform/water/methanol (Section: Protein extraction). Gin teaches first adding methanol to cells followed by chloroform and water before centrifuging and removing the solvent (Steps 1 – 10).
Sacramento, Lu, Moser, Saito and Gin are analogous because they teach methods of digesting proteins for proteomic analysis. Sacramento teaches a general workflow for preparing proteins for LC-MS (Section: Sample preparation for mass spectrometry analysis and data acquisition). Lu teaches that DDM, a non-ionic detergent, is used to dissolve proteins in solution (Section: Cell Culture and Sample Preparation). Moser teaches that ionic detergents such as SDC and non-ionic detergents such as Triton X-100 can be used in a protein digestion solution together (Page 34 Line 7 – 19). Saito teaches the comparison of ethanol, acetone, acetonitrile, and methanol/chloroform (Introduction ¶3). Gin teaches a protocol on a method for precipitating proteins using chloroform/water/methanol (Section: Protein extraction).
Therefore, it would have been prima facie obvious before the effective filing date of the claimed invention to have used a chloroform/water/methanol-based method for protein precipitation because doing so would retain a large amount of protein for downstream processing. One of ordinary skill in the art would have had a reasonable expectation of success in using a chloroform/water/methanol-based method for protein precipitation given that the method is well known, has been successfully demonstrated, and commonly used in the prior art.
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill in the art at the time of filing especially in the absence of evidence to the contrary.
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over Sacramento, Lu, and Moser as applied to claims 1 – 3, 10 – 12, 17, 19 and 21 above, and further in view of Chelius et al (J Proteome Research, 2002, 10.1021/pr025528, hereinafter, “Chelius”) and Liu.
As discussed above, claims 1 – 3, 10 – 12, 17, 19 and 21 were rendered prima facie obvious by Sacramento, Lu, and Moser. While the references teach a general workflow for preparing proteins for LC-MS including the precipitation of proteins using ice-cold acetone, the use of a dissolving solution comprising SDC and DDM, and a protease digestion, the references fail to teach injecting the digested virus protein into a LC-MS without first performing a buffer exchange or a desalting step.
However, Chelius teaches the analysis of Adenovirus 5 proteome by LC-MS/MS (Abstract). Chelius teaches whole virus was digested with trypsin and separated by either reversed-phase LB or by both cation exchange and reversed-phase chromatography (Abstract). Chelius also teaches that this method allowed for analysis of low abundance proteins and can be used to identity PTMs of the structural proteins of adenovirus (Abstract).
Regarding claim 24, Chelius teaches digesting whole virus before directing adding the sample to the LC-MS/MS system (Section: Experimental Section - Reduction, Alkylation, and Digestion, One-Dimensional LC/MS/MS, Two-Dimensional LC/MS/MS).
Similarly, Liu teaches DDM dissolution of proteins, followed by trypsin and direct addition of the digested protein into a LC-MS/MS system without desalting or buffer exchange (Supplemental information- Compatibility with trypsin activity and MS analysis).
Sacramento, Lu, Moser, and Chelius are analogous because they teach methods of digesting proteins for proteomic analysis. Sacramento teaches a general workflow for preparing proteins for LC-MS (Section: Sample preparation for mass spectrometry analysis and data acquisition). Lu teaches that DDM, a non-ionic detergent, is used to dissolve proteins in solution (Section: Cell Culture and Sample Preparation). Moser teaches that ionic detergents such as SDC and non-ionic detergents such as Triton X-100 can be used in a protein digestion solution together (Page 34 Line 7 – 19). Chelius teaches digesting whole virus before directing adding the sample to the LC-MS/MS system (Section: Experimental Section - Reduction, Alkylation, and Digestion, One-Dimensional LC/MS/MS, Two-Dimensional LC/MS/MS). Liu teaches DDM dissolution of proteins, followed by trypsin and direct addition of the digested protein into a LC-MS/MS system without desalting or buffer exchange (Supplemental information- Compatibility with trypsin activity and MS analysis).
Therefore, it would have been prima facie obvious before the effective filing date of the claimed invention to have directly analyzed digested proteins without a buffer exchange because doing so would retain more protein and save time. One of ordinary skill in the art would have had a reasonable expectation of success in directly analyzed digested proteins without a buffer exchange given that the method is well known, has been successfully demonstrated, and commonly used in the prior art.
Accordingly, the claimed invention was prima facie obvious to one of ordinary skill in the art at the time of filing especially in the absence of evidence to the contrary.
Conclusion
NO CLAIMS ARE ALLOWED
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/DANYAL HASSAN ALAM/Examiner, Art Unit 1672
/THOMAS J. VISONE/Supervisory Patent Examiner, Art Unit 1672
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