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 § 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, 11, 12, 17, 18 and 19 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.
Claim 3 recites the limitation "the anion exchanger residues" in line 1. There is insufficient antecedent basis for this limitation in the claim. Claim 2 introduces the anion exchanger residues but claim 3 is dependent on claim 1.
Claim 11 recites the limitation " the anion exchanger residues " in line 3. There is insufficient antecedent basis for this limitation in the claim. Claim 2 introduces the anion exchanger residues but claim 11 is dependent on claim 10 and claim 10 is dependent upon claim 1. Claims 12 and 18 rejected by virtue of its dependency on claim 11.
Claim 17 recites the limitation " the anion exchanger residues " in line 3. There is insufficient antecedent basis for this limitation in the claim. Claim 2 introduces the anion exchanger residues but claim 17 is dependent on claim 16 and claim 16 is dependent upon claim 1. Claim 19 rejected by virtue of its dependency on claim 17.
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 for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. 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, 4, 6, 13 are rejected under 35 U.S.C. 103 as being unpatentable over Kinsey et al. (Determination of lipid content and stability in lipid nanoparticles using ultrahigh-performance liquid chromatography in combination with a Corona Charged Aerosol Detector, 2022, Electrophoresis, 43, 1091-1100) in view of Horlitz et al. (WO 2013045432 A1).
With respect to claim 1, Kinsey et al. teaches a method of preparing a sample for compositional analysis (Page 1092: paragraph starting with “For the aforementioned”), the method comprising: providing a sample comprising a lipid particle encapsulated nucleic acid (Page 1092: paragraph starting with, “ Then lipid nanoparticles” Kinsey specifically teaches the lipid nanoparticles were produced by simultaneous T-mixing of the lipid mixture with an aqueous solution mRNA being submitted for analysis. Kinsey et al. teaches a UHPLC method for separating lipids using a C18 reverse phase column (Page 1093: UHPLC-CAD conditions, paragraph starting with “Samples were”).
dissolving the sample comprising the lipid particle encapsulated nucleic acid in an organic solvent to provide a solution comprising dissolved lipids and solubilized nucleic acid; (Page 1092: paragraph starting with “Each lipid,” Kinsey teaches each LNP sample was treated with a diluent consisting of ethanol: formamide. Page 1093: paragraph starting with “Another dilution”, Kinsey teaches measuring an LNP sample containing 0.05 mg/ml mRNA was analyzed using dilution of 4 and 10 ethanol: formamide).
However, Kinsey et al. does not teach adsorbing the solubilized nucleic acid on a weak anion exchange (WAX) solid phase sorbent.
Horlitz et al. was used to remedy this, as Horlitz et al. teaches nucleic acid is bound to sorbent in a binding mixture, the sorbent is removed, washed and nucleic acids are eluted (Page 6: line 6-18). Horlitz et al. teaches extracellular nucleic acids such as DNA and/or RNA in many medical conditions, malignancies, and infectious processes is of interest inter alia for screening, diagnosis, prognosis, surveillance for disease progression, for identifying potential therapeutic targets, and for monitoring treatment response (Page 2: line 16). Horlitz et al. further teaches isolating extracellular nuclear acid using with a solid phase sorbent that has anion exchange groups (Page 6: line 7).
Horlitz et al. further teaches and adsorbing the solubilized nucleic acid on a weak anion exchange (WAX) solid phase sorbent (Page 30: line 40 to Page 31: line 2, Horlitz teaches anion exchange groups having a protonatable group with a positive charge. Page 30: “line 24,” Horlitz teaches the anion exchange group comprises a group selected from the group consisting of primary, secondary and tertiary amines. Page 20: “line 19,” Horlitz teaches a solid phase comprising anion exchange groups is used which comprise diethylamino groups, preferably diethylaminopropyl groups. “Page 30: line 4,” Horlitz teaches the pKa value of the (protonated) protonatable group is in the range of from about 8 to about 13).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Kinsey et al. UHPLC method to incorporate the teachings of anion exchange groups on a solid phase sorbent, having a protonatable group with a positive charge as taught by Horlitz et al. (On Page 30: line 40 to Page 31: line 2) to provide: and adsorbing the solubilized nucleic acid on a weak anion exchange (WAX) solid phase sorbent. It would have been obvious to combine Horlitz et al. adsorbing the solubilized nucleic acid on a weak anion exchange (WAX) solid phase sorbent with Kinsey et al. UHPLC method for separating lipids from lipid encapsulated nucleic acids because Kinsey et al. teaches an elimination of bonds between lipids and nucleic acids when mixed with a diluent, while Horlitz et al. separates isolating extracellular nucleic acids from biological samples, which achieves that same outcome. Additionally, Horlitz et al. teaches that the isolation method according to present invention can also be used as a pretreatment protocol for isolating extracellular nucleic acids from large sample volumes. In this combination, both Kinsey et al. method and Horlitz et al. analysis performing the same functions they would if they were separate. The person of ordinary skill in the art would have found it obvious to combine the elements because ordinarily skilled artisans would have recognized the reasons for applying Modified Kinsey et al UHPLC method for separating lipids from lipid encapsulated nucleic using Horlitz et al. adsorbing the solubilized nucleic acid on a weak anion exchange (WAX) solid phase sorbent and would have known how to do so. The person of ordinary skill in the art would further have predicted that the combination would allow for the identification of potential therapeutic targets because Horlitz et al. teaches that isolated extracellular nucleic acids can be directly analyzed using analytical methods for diagnostic and therapeutic testing (Page 33: line 20-line 41 to Page 34: line 2 ). MPEP 2143 (I)(A)
With respect to claim 2, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 1.
Modified Kinsey et al. also teaches wherein the WAX solid phase sorbent comprises a surface having anion exchanger residues disposed thereon (Page 30: line 40 to Page 31: line 2).
the anion exchanger residues comprising a primary amine, a secondary amine, a tertiary amine, or a combination thereof (On Page 30: line 24, Horlitz teaches the anion exchange group comprises a group selected from the group consisting of primary, secondary and tertiary amines).
Modified Kinsey teaches the anion exchanger residues having a pKa in a range from about 8 to about 13 (Horlitz et al., Page 30: line 4) and therefore fails to teach a pka in a range from about 5 to about 11 as claimed. However, 8 to about 13 overlaps with applicants claimed range of 5 to 11.
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to modify the pKa of Modified Kinsey et al. to select the pKa value of the (protonated) protonatable group in the range of from about 5 to about 11 because in the case where the claimed ranges overlap or lie inside ranges disclosed by the prior art a prima facie case of obviousness exists. MPEP § 2144.05.
With respect to claim 4, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 1. Modified Kinsey et al. further teaches wherein the organic solvent comprises a C1-C4 alcohol, acetonitrile, dimethyl formamide, dimethyl sulfoxide, or a combination thereof (Page 1092: paragraph starting with “Each lipid” Kinsey teaches each LNP sample was treated with a diluent consisting of ethanol: formamide).
With respect to claim 6, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 1. Modified Kinsey et al. teaches wherein the organic solvent further comprises an additive selected from the group consisting of acids, bases, detergents, buffers, salts, and combinations thereof (Page 1093: paragraph starting with “The first diluent,” Kinsey teaches when analyzing samples containing Cationic Lipid 1, 0.5% Formic Acid: Triethylamine Complex (5:2) was added to the assay diluent. When analyzing samples containing Cationic Lipid 2, 19.2mM glacial acetic acid and 16.2mM triethylamine were added to the assay diluent).
With respect to claim 13, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 1.
Kinsey et al. teaches further comprising analyzing one or more of the lipid sample by high performance liquid chromatography, mass spectrometry, or a combination thereof (On page 1092: paragraph starting with “For the aforementioned” Kinsey teaches lipid analysis of mRNA loaded lipid nanoparticles using UHPLC).
However, Kinsey et al. does not teach analyzing the nucleic acid sample by high performance liquid chromatography, mass spectrometry, or a combination thereof.
Horlitz teaches extracellular nucleic acids such as DNA and/or RNA in many medical conditions, malignancies, and infectious processes is of interest inter alia for screening, diagnosis, prognosis, surveillance for disease progression, for identifying potential therapeutic targets, and for monitoring treatment response (Page 2: line 16).
Horlitz et al. teaches analyzing the nucleic acid sample by high performance liquid chromatography, mass spectrometry, or a combination thereof (On page 34: line 1 to line 10, Horlitz teaches the isolated extracellular nucleic acids can be analyzed with mass spectrometry.)
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Modified Kinsey et al. analyzing lipids using a UHPLC to incorporate the teachings of analyzing isolated extracellular nucleic acid using mass spectrometry as taught by Horlitz et al to provide: analyzing the nucleic acid sample by high performance liquid chromatography, mass spectrometry, or a combination thereof. The skilled artisan would have expected success in combining Kinsey et al. the analysis of lipids using UHPLC with the analysis of nucleic acids using mass spectrometry in Horlitz et al because Kinsey et al.” teaches a detection method that supports various mRNA-LNP vaccine programs and Horlitz et al. teaches the isolation of mRNA (Page 12: line 4). MPEP 2143 (I)(B) Doing so would have a reasonable expectation of in separation of nucleic acids and lipids from a LNP sample. Additionally, Kinsey et al. teaches the reversed phase liquid chromatography coupled with a CAD method, could even be modified for mass spectrometry for investigational impurity identification (Page 1092, paragraph starting with “Charged aerosol”). Because Modified Kinsey et al. teaches further comprising analyzing one or more of the lipid and the nucleic acid sample by high performance liquid chromatography, mass spectrometry, or a combination thereof., a prima facie case of obviousness exists.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Kinsey et al. (Kinsey et al., Determination of lipid content and stability in lipid nanoparticles using ultrahigh-performance liquid chromatography in combination with a Corona Charged Aerosol Detector, 2022, Electrophoresis, 43, 1091-1100) in view of Horlitz et al. (WO 2013045432 A1) as applied to claim 1, in further view of Shao (WO0162976 A1).
With respect to claim 5, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 1.
Modified Kinsey et al. also teaches dissolving the sample comprising the lipid particle encapsulated nucleic acid in an organic solvent to provide a solution comprising dissolved lipids and solubilized nucleic acid (Kinsey et al., Page 1092: paragraph starting with “Each lipid”). Kinsey et al. specifically teaches each LNP sample was treated with a diluent consisting of ethanol: formamide (Page 1092: paragraph starting with “Each lipid”).
Modified Kinsey et al. did not teach wherein the organic solvent comprises n-propanol.
Shao teaches a method for purifying, isolating and separating nucleic acids (Page 3: paragraph starting with “The method”). Shao specifically teaches the inclusion of a solid support that is washed (Page 4: paragraph starting with “In yet another”). Shao teaches solid surfaces may be in the forms of beads, particles, membranes, or filters (Page 4: paragraph starting with “The solid”). Shao teaches the sample solution may contain a mixture of nucleic acids, nucleotides, enzymes and proteins in general, carbohydrates, lipids, and generally, any component of a prior purification or molecular manipulation of a biological sample (Page 4: paragraph starting with “The sample”).
Shao teaches wherein the organic solvent comprises n-propanol (Page 2: paragraph starting with “In accomplishing those,” Shao teaches isolation and purification of nucleic acids from an aqueous sample. Page 1: paragraph starting with “The present,” the mixing an aqueous sample solution comprising a nucleic acid, an organic solvent miscible in water, and a solid surface collecting the solid surface on which the nucleic acid is precipitated; and eluting the nucleic acid from the solid surface. Page 3: paragraph starting with “In accordance with yet another embodiment, the organic,” Shao further teaches the organic solvent is an alcohol. Shao specifically teaches preferred alcohols are ethanol, isopropanol, n-propanol, and butanol).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the methods of Modified Kinsey et al. to treat LNP samples with a diluent consisting of ethanol with n-propanol instead as taught by Shao (Page 2, paragraph starting with “In accomplishing those) to provide: the organic solvent comprises n-propanol. The skilled artisan would have expected success in substituting Kinsey et al. treat LNP samples with a diluent consisting of ethanol exemplified in Shao alcohol solvent because Kinsey et al. teaches an ethanol solvent and Horlitz et al. teaches the mixing of a nucleic acid sample with an organic solvent that is an alcohol such as ethanol or isopropanol. MPEP 2143 (I)(B) The person of ordinary skill in the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that wherein the organic solvent comprises n-propanol, results in nucleic acid separation from the solution. The claimed method requires wherein the organic solvent comprises n-propanol. Because Modified Kinsey et al. treat LNP samples with a diluent consisting of ethanol, a prima facie case of obviousness exists.
Claim 7, 10, 14 and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Kinsey et al. (Kinsey et al., Determination of lipid content and stability in lipid nanoparticles using ultrahigh-performance liquid chromatography in combination with a Corona Charged Aerosol Detector, 2022, Electrophoresis, 43, 1091-1100) in view of Horlitz et al. (WO 2013045432 A1) as applied to claims 1 and 6, in further view of Laugharn et al. (WO 9922868 A1).
With respect to claim 7, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 6.
Modified Kinsey et al. teaches wherein the organic solvent further comprises an additive selected from the group consisting of acids, bases, detergents, buffers, salts, and combinations thereof (Kinsey et al., Page 1093: paragraph starting with “The first diluent”) Modified Kinsey et al. teaches when analyzing samples containing Cationic Lipid 1, 0.5% Formic Acid: Triethylamine Complex was added to the assay diluent. When analyzing samples containing Cationic Lipid 2, 19.2mM glacial acetic acid and 16.2mM triethylamine were added to the assay diluent).
Modified Kinsey et al. does not teach wherein the additive comprises an ionizable cationic base.
Laugharn et al. teaches purification od nucleic acids from a sample (Section 3: line 28) such as RNA, mRNA, viral RNA or DNA (Section 6: line 15). Laugharn et al. further teaches isolating biological components such as lipids and nucleic acids (Section 11: line 3). Laugharn et al. teaches adding a sample to one or more reagents to form a reaction mixture (Section 11: line 23). Laugharn et al. teaches adding a sample to immobilized solid phase and dissociating the nucleic acid sample (Section 12: line 19).
Laugharn et al. further teaches wherein the additive comprises an ionizable cationic base (Section 29: line 1, Laugharn teaches lysis extraction solvents such as phenol/chloroform extraction and treatment with sodium hydroxide. In example 7, Laugharn specifically teaches isolation of RNA using potassium hydroxide).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Modified Kinsey et al. organic diluent to incorporate the teachings of using sodium hydroxide or potassium hydroxide as taught by Laugharn et al. (Section 29: line 1 and Example 7) to provide: wherein the additive comprises an ionizable cationic base. It would have been obvious to combine Modified Kinsey et al. ethanol: formamide diluent with Laugharn et al. method containing reagents wherein the additive comprises an ionizable cationic base because Kinsey et al. adds acid and buffer to diluent to increase lysis. In this combination, both Modified Kinsey et al. method and Laugharn et al. lysis performing the same functions they would if they were separate. The person of ordinary skill in the art would have found it obvious to combine the elements because ordinarily skilled artisans would have recognized the reasons for applying Kinsey et al. diluent using Laugharn et al. cationic base lysing method and would have known how to do so. The person of ordinary skill in the art would further have predicted that the combination would allow for the identification of potential therapeutic targets because Laugharn et al. sodium hydroxide being used as a lysing solvent is, a known in the art (Section 29, line 1). MPEP 2143 (I)(A) Laugharn et al. further teaches that purification of lipids and nucleic acids (Section 11, line 3).
With respect to claim 10, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 1.
Kinsey et al. teaches separating lipids from a lipid encapsulated nucleic acid particles using a C18 reverse phase column (Page 1093: UHPLC-CAD conditions, paragraph starting with “Samples were”). Kinsey et al. teaches extracting LNPs in a diluent and the ethanol: formamide diluent disrupting all non-covalent interactions between the mRNA and lipids and maintaining solubility for both mRNA and lipids during analysis (Page 1093: paragraph starting with “Another dilution”).
However, Kinsey et al. does not teach further comprising eluting the adsorbed nucleic acid from the WAX solid phase sorbent, forming a nucleic acid sample which is substantially free of lipids.
Horlitz et al. was used to remedy this, as Horlitz et al. teaches nucleic acid is bound to sorbent in a binding mixture, the sorbent is removed, washed and nucleic acids are eluted (Page 6: line 6-18). Horlitz et al. teaches extracellular nucleic acids such as DNA and/or RNA in many medical conditions, malignancies, and infectious processes is of interest inter alia for screening, diagnosis, prognosis, surveillance for disease progression, for identifying potential therapeutic targets, and for monitoring treatment response (Page 2: line 16). Horlitz et al. further teaches isolating extracellular nuclear acid using with a solid phase sorbent that has anion exchange groups (Page 6: line 7).
Horlitz et al. teach further comprising eluting the adsorbed nucleic acid from the WAX solid phase sorbent, forming a nucleic acid sample (On page 30: line 40 to Page 31: line 2, Horlitz teaches anion exchange groups having a protonatable group with a positive charge. On Page 36: line 24, Horlitz teaches wherein for isolation, extracellular nucleic acids are bound to solid phase that contains anion exchange residues. On page 18: line 4, Horlitz teaches using an anion exchange matrix for binding nucleic acids, the bound nucleic acids can be eluted from the matrix. On page 15: line 25-30, Horlitz teaches washing the sorbent and a recommendation of at least two washes to remove potential impurities).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Kinsey et al. to disrupt all non-covalent interactions between mRNA and lipids using a diluent to incorporate the teachings of eluting nucleic acids that are bound to a WAX sorbent as taught by Horlitz et al. (Page 18: line 4) to provide: further comprising eluting the adsorbed nucleic acid from the WAX solid phase sorbent, forming a nucleic acid sample. Doing so would have a reasonable expectation of successfully isolating nucleic acids because Kinsey et al. analyzes lipids separated from lipid encapsulated nanoparticles containing nucleic acids for the development of therapeutic formulations, while Horlitz et al. isolating nucleic acids by washing away impurities and eluting from a WAX sorbent achieves that same outcome. In this combination, both Kinsey et al. method and Horlitz et al. purification method performing the same functions they would if they were separate. The person of ordinary skill in the art would further have predicted that the combination would allow for the identification of potential therapeutic targets such as LNPs because Horlitz et al. teaches that extracellular nucleic acids can be rapidly isolated from various samples with high yield (Page 9: line 35). MPEP 2143 (I)(A) Additionally, Horlitz et al. teaches the monitoring of anti-viral therapy can be improved and when aiming at isolating viral nucleic acids, it is preferred to use the present method as pre-treatment protocol and addition to another nucleic acid isolation protocol (Page 12: line 22-31.)
However, Modified Kinsey et al. does not explicitly teach forming a nucleic acid sample which is substantially free of lipids.
Laugharn et al. teaches a method for separating compounds in a mixture. Laugharn et al. teaches purification od nucleic acids from a sample (Section 3: line 28) such as RNA, mRNA, viral RNA or DNA (Section 6: line 15). Laugharn et al. further teaches isolating biological components such as lipids and nucleic acids (Section 11: line 3). Laugharn et al. teaches adding a sample to one or more reagents to form a reaction mixture (Section 11: line 23). Laugharn et al. teaches adding a sample to immobilized solid phase and dissociating the nucleic acid sample (Section 1: line 19). Laugharn et al. teaches the solid phase can be made from any substance that selectively binds the desired biomolecule such as anion-exchange column (Section 20: line 2-6).
Laugharn et al. further teaches forming a nucleic acid sample which is substantially free of lipids. (Section 12: line 34-36, Laugharn teaches a biomolecule to be purified is introduced onto a solid phase. Section 19: line 14-20, the solid phase, to which the biomolecule present in the solution should is bound, washed with a buffered second solution, in which the desired biomolecule will remain bound to the solid phase at elevated pressure, whereas the undesirable contaminants (e.g., proteins and lipids) will be removed from the solid phase. Section 19: line 11-26, Laugharn teaches after washing, the desired biomolecule to be freed from the solid phase. Section 20: line 22, Laugharn teaches washing the liberated biomolecule away from the solid phase and into a collection vessel and eluting the impurities away from solid phase).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Modified Kinsey et al. of removing impurities to incorporate the teachings of removing lipids as taught by Laugharn et al (Section 19: line 14-20) to provide: forming a nucleic acid sample which is substantially free of lipids. The skilled artisan would have expected success in substituting Modified Kinsey et al. forming a nucleic acid sample after elution in Laugharn et al. elution of biomolecules where impurities are washed away because Modified Kinsey et al.” teaches at least two washes to remove potential impurities and Horlitz et al. teaches washing impurities such as lipids away from the solid phase attached biomolecule. MPEP 2143 (I)(B) Doing so would have a reasonable expectation of success in eluting biomolecules from a solid phase after washing results in separation of nucleic acids and lipids (Laugharn et al., Section 19: line 14-20). The claimed method requires further comprising eluting the adsorbed nucleic acid from the WAX solid phase sorbent, forming a nucleic acid sample which is substantially free of lipids. Because Modified Kinsey et al. further comprising eluting the adsorbed nucleic acid from the WAX solid phase sorbent, forming a nucleic acid sample which is substantially free of lipids a prima facie case of obviousness exists.
With respect to claim 14, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 10.
Kinsey et al. teaches when analyzing samples containing Cationic Lipid 1, 0.5% Formic Acid: Triethylamine Complex was added to the assay diluent. When analyzing samples containing Cationic Lipid 2, 19.2mM glacial acetic acid and 16.2mM triethylamine were added to the assay diluent (Kinsey et al., Page 1093: paragraph starting with “The first diluent”). Kinsey et al. teaches the ethanol: formamide diluent was able to disrupt all non-covalent interactions between the mRNA and lipids and maintain solubility for both mRNA and lipids during analysis (Page 1093: paragraph starting with “Another dilution”).
Kinsey et al. did not teach further comprising digesting at least a portion of the nucleic acid present in the nucleic acid sample with a nuclease.
Horlitz et al. teaches nucleic acid is bound to sorbent in a binding mixture, the sorbent is removed, washed and nucleic acids are eluted (Page 6: line 6-18). Horlitz et al. teaches extracellular nucleic acids such as DNA and/or RNA in many medical conditions, malignancies, and infectious processes is of interest inter alia for screening, diagnosis, prognosis, surveillance for disease progression, for identifying potential therapeutic targets, and for monitoring treatment response (Page 2: line 16). Horlitz et al. further teaches isolating extracellular nuclear acid using with a solid phase sorbent that has anion exchange groups (Page 6: line 7).
Horlitz et al. teaches further comprising digesting at least a portion of the nucleic acid present in the nucleic acid sample with a nuclease (Page 4: line 20, Horlitz teaches a lysis step for nucleic acid isolation. Page 4: line 20, Horlitz teaches the lysis step to denature and/or digest protein contaminations or other contaminating substances that could interfere with the binding of the nucleic acid to the solid phase and/or could lead to an improper purification. Page 32: line 39, Horlitz further teaches further comprising digesting at least a portion of the nucleic acid present in the nucleic acid sample with a nuclease. Page 33: line 5 to 7, Horlitz more specifically teaches it is also within the scope of the present invention to digest the non-target nucleic acid using nucleases after isolation).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Kinsey et al. to disrupt all non-covalent interactions between mRNA and lipids to incorporate the teachings of digesting nucleic acid with nuclease as taught by Horlitz et al (Page 33: line 5 to 7) to provide: further comprising digesting at least a portion of the nucleic acid present in the nucleic acid sample with a nuclease. It would have been obvious to combine Horlitz et al. digestion of nucleic acids with Kinsey et al. method of extraction method of LNPs because Kinsey et al. disrupts the interactions of lipids and mRNA from lipid encapsulated nanoparticles containing nucleic acids using diluents for the development of therapeutic formulations, while Horlitz et al. mixes the nucleic acid sample with an organic solvent and adds a lysing and digesting step to release nucleic acids which achieves that same outcome. In this combination, both Kinsey et al. method and Horlitz et al. analysis performing the same functions they would if they were separate. The person of ordinary skill in the art would further have predicted that the modification would allow for the identification of potential therapeutic targets because Horlitz et al. teaches that extracellular nucleic acids can be rapidly isolated from various samples with high yield (Page 9: line 35). MPEP 2143 (I)(A) Additionally, Horlitz et al. teaches the monitoring of anti-viral therapy can be improved and when aiming at isolating viral nucleic acids, it is preferred to use the present method as pre-treatment protocol and addition to another nucleic acid isolation protocol (Page 12: line 22-31.)
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Kinsey et al. (Kinsey et al., Determination of lipid content and stability in lipid nanoparticles using ultrahigh-performance liquid chromatography in combination with a Corona Charged Aerosol Detector, 2022, Electrophoresis, 43, 1091-1100) in view of Horlitz et al. (WO 2013045432 A1) as applied to claim 1, in further view of Davis et al. (US 5660984 A).
With respect to claim 8, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 1. Modified Kinsey et al. teaches separating lipids from a lipid encapsulated nucleic acid particles using a C18 reverse phase column (Kinsey et al., Page 1093: UHPLC-CAD conditions, paragraph starting with “Samples were”). Modified Kinsey et al. teaches the ethanol: formamide diluent was able to disrupt all non-covalent interactions between the mRNA and lipids and maintain solubility for both mRNA and lipids during analysis (Kinsey et al., Page 1093: paragraph starting with “Another dilution”). Modified Kinsey et al. further teaches adsorbing the solubilized nucleic acid on a weak anion exchange (WAX) solid phase sorbent. (Horlitz et al. Page 14: line 4), (Horlitz et al., Page 5: line 6)). Modified Kinsey et al. teaches wherein for isolation extracellular nucleic acids are bound to solid phase (Horlitz et al., Page 36: line 24).
Modified Kinsey et al. further teaches the extracellular nucleic acids that are bound to the solid phase are separated from the remaining sample (Page 30: line 40 to Page 31: line 2). Modified Kinsey et al. teaches further comprising eluting the extracellular nucleic acid from the solid phase (Horlitz et al., Page 3: line 34). More specifically, Modified Kinsey et al. teaches wherein for isolation, extracellular nucleic acids are bound to solid phase (Horlitz et al., Page 36: line 24). More specifically, Modified Kinsey et al. teaches extracellular nucleic acids such as disease-associated, viral associated nucleic acids and biological sample extracellular nucleic acids (Horlitz et al., Page 12: line 1-31). Modified Kinsey et al. teaches washing and a recommendation of at least two washes to remove potential impurities (Horlitz et al., page 15: line 25-30).
However, Modified Kinsey et al. does not teach further comprising removing the dissolved lipids from the WAX solid phase sorbent, forming a lipid sample which is substantially free of nucleic acid, wherein removing the dissolved lipids comprises allowing the dissolved lipids to pass through the WAX solid phase sorbent.
Davis et al. is used to remedy this, as Davis et al. teaches isolating a DNA sample from a heterogeneous mixture (Section 4: line 12) with a column using an anion-exchange resin (Section 4: line 16). Davis et al. teach the DNA sample is further purified from such heterologous components by washing the column (Section 4: line 20).
Davis et al. also teaches further comprising removing the dissolved lipids from the WAX solid phase sorbent, forming a lipid sample which is substantially free of nucleic acid, wherein removing the dissolved lipids comprises allowing the dissolved lipids to pass through the WAX solid phase sorbent (Section 7: line 48, Davis teaches contaminating species that do not efficiently bind to the resin are washed from the column by passing the unbound heterogenous mixture through the column. Section 7: line 54, Davis teaches the DNA sample remains bound to the column during this passage and is thus purified from such contaminating species. Section 7: line 56, Davis further teaches the column can be washed any appropriate number of times using a buffer wherein the DNA sample remains bound to the anion exchange resin in the column).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Modified Kinsey et al. washing impurities from the sorbent to incorporate the teachings of the DNA staying bound to the column and separated from lipid sample as taught by Davis et al. (Section 7: line 54) to provide: further comprising removing the dissolved lipids from the WAX solid phase sorbent, forming a lipid sample which is substantially free of nucleic acid, wherein removing the dissolved lipids comprises allowing the dissolved lipids to pass through the WAX solid phase sorbent. Doing so would have a reasonable expectation of successful because Modified Kinsey et al. teaches washing impurities from WAX sorbent, while Davis et al. teaches eluting contaminating species such as lipids from the anion resin, which achieves that same outcome. In this combination, both Kinsey et al. method and Davis et al. analysis performing the same functions they would if they were separate. The person of ordinary skill in the art would have found it obvious to combine the elements because ordinarily skilled artisans would have recognized the reasons for applying Modified Kinsey et al. removal of dissolved lipids from WAX sorbent using Davis et al. forming a lipid sample which is substantially free of nucleic acid, wherein removing the dissolved lipids comprises allowing the dissolved lipids to pass through the WAX solid phase sorbent and would have known how to do so. The person of ordinary skill in the art would further have predicted that the combination would allow for separation of lipids and nucleic acids because Davis et al. teaches that the method allows for contaminating species such as lipids to be eluted from the resin (Section 4: line 61). MPEP 2143 (I)(A)
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Kinsey et al. (Kinsey et al., Determination of lipid content and stability in lipid nanoparticles using ultrahigh-performance liquid chromatography in combination with a Corona Charged Aerosol Detector, 2022, Electrophoresis, 43, 1091-1100) in view of Horlitz et al. (WO 2013045432 A1) in further view of Davis et al (US 5660984 A) in further view of Van de Bittner et al. (US20200393342A1).
With respect to claim 9, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 8.
Modified Kinsey et al. teaches adsorbing the solubilized nucleic acid on a weak anion exchange (WAX) solid phase sorbent (Page 30: line 40 to Page 31: line 2). Modified Kinsey et al. teaches wherein for isolation extracellular nucleic acids are bound to solid phase (Horlitz et al., Page 36: line 24). Modified Kinsey et al. teach the DNA sample is further purified from such heterologous components by washing the column (Davis et al., Section 4: line 20). Modified Kinsey et al. teaches contaminating species that do not efficiently bind to the resin are washed from the column by passing the unbound heterogenous mixture through the column (Davis et al., Section 7: line 48). The DNA sample remains bound to the column during this passage and is thus purified from such contaminating species (Davis et al., Section 7: line 54). Modified Kinsey et al. further teaches the column can be washed any appropriate number of times using a buffer wherein the DNA sample remains bound to the cholestyramine anion exchange resin in the column (Davis et al., Section 7: line 56).
However, Modified Kinsey et al. does not teach wherein removing the dissolved lipids further comprises: flowing the organic solvent through the WAX solid phase sorbent and collecting the organic solvent flowed therethrough to provide one or more washings; and combining the one or more washings with the dissolved lipids which have been allowed to pass through the WAX solid phase sorbent.
Van de Bittner et al. teaches a method for separating metabolites which metabolites, including polar metabolites and/or lipid metabolites, may be extracted from a biological sample [0101]. Van de Bittner et al. also teaches the mixture may further comprise nucleic acids [0102]. Van de Bittner et al. teaches any biomolecules such as lipids, proteins, and/or nucleic acids may be also isolated from the same biological sample and analyzed in parallel to polar metabolites or instead of polar metabolites [0103]. Van de Bittner et al. teaches metabolites are separated from lipids at least partially by capturing at least some of the lipids on a solid-phase extraction (SPE) matrix [0170].
Van de Bittner et al. further teaches wherein removing the dissolved lipids further comprises: flowing the organic solvent through the WAX solid phase sorbent and collecting the organic solvent flowed therethrough to provide one or more washings; and combining the one or more washings with the dissolved lipids which have been allowed to pass through the WAX solid phase sorbent (In [0052], Van de Bittner teaches collecting the metabolites in a flow-through solution; [0053] optionally, washing the solid-phase extraction matrix with a wash buffer and optionally, combining the wash buffer with the flow-through solution and thereby obtaining a solution comprising metabolites. In [0268], Van de Bittner et al. teaches an example where the CAPTIVA™ EMR-lipid plate was washed with a 50% water solution containing organic solvents, and the wash was combined with the eluate containing the polar metabolites).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to combine the methods of Modified Kinsey et al. washing the impurities from the WAX sorbent to incorporate the teachings of collecting the metabolites in a flow-through solution as taught by Van de Bittner et al. [0052] to provide: wherein removing the dissolved lipids further comprises: flowing the organic solvent through the WAX solid phase sorbent and collecting the organic solvent flowed therethrough to provide one or more washings; and combining the one or more washings with the dissolved lipids which have been allowed to pass through the WAX solid phase sorbent. It would have been obvious to combine Van de Bittner et al. flowing the organic solvent through the WAX solid phase sorbent and collecting the organic solvent flowed therethrough with Modified Kinsey et al. method of removing contaminates from the WAX solid phase sorbent because Modified Kinsey et al. separates a DNA sample by binding the nucleic acid to a solid phase WAX sorbent and washing the sorbent to remove contaminants while Van di Bittner teaches wherein removing the dissolved lipids. In this combination, Modified Kinsey et al. method and Van de Bittner purification of samples containing lipids and nucleic acids performs the same functions they would if they were separate. The person of ordinary skill in the art would have found it obvious to combine the elements because ordinarily skilled artisans would have recognized the reasons for applying Modified Kinsey et al. removal of dissolved lipids from WAX sorbent using Van de Bittner method of wherein removal of dissolved lipids and would have known how to do so. The person of ordinary skill in the art would further have predicted that the combination would allow for the isolation of nucleic acids in lipid containing samples Modified Kinsey et al. teaches that the method allows for contaminating species such as lipids to be eluted from the resin (Davis et al., Section 4, line 61). MPEP 2143 (I)(A)
Claims 11, 12, 16, 19, 20 are rejected under 35 U.S.C. 103 as being unpatentable over Kinsey et al. (Kinsey et al., Determination of lipid content and stability in lipid nanoparticles using ultrahigh-performance liquid chromatography in combination with a Corona Charged Aerosol Detector, 2022, Electrophoresis, 43, 1091-1100) in view of Horlitz et al. (WO 2013045432 A1) as applied to claims 10, 11, 1, 17 and 16 in further view of Hollander et al. (WO 201110432 A1)
With respect to claim 11, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 10.
Kinsey et al. teaches wherein eluting comprises flowing an aqueous buffer (On page 1092, paragraph starting with “When measuring” Kinsey teaches LNP samples with lipid 1 having a mobile phase composition mobile phase A consisted of water: methanol with 0.5% Formic Acid: Triethylamine Complex at a pH of 3.5 and mobile phase B was composed of methanol with 0.5% Formic Acid: Triethylamine Complex).
However, Kinsey et al. does not teach wherein eluting comprises flowing an aqueous buffer through the WAX solid phase sorbent, through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent, said aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent.
Horlitz et al. teaches wherein eluting comprises flowing an aqueous buffer through the WAX solid phase sorbent, through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent (Page 16: line 5, Horlitz teaches eluting the extracellular nucleic acid from the WAX sorbent where elution occurs at the second pH and the choice of the second pH is suitable for eluting the extracellular nucleic acids from the anion exchange groups that are present on the solid phase. Page 16: line 11, Horlitz et al. further teaches the second pH may be below, at or above the pKa of a protonatable group of the anion exchange group. Page 16: line 36, Horlitz further teaches elution is achieved by contacting the extracellular nucleic acids that are bound to the solid phase with an elution solution. Page 17: line 27, Horlitz et al. teaches buffers such as Tris (Page 16, line 39), HEPES, Tris-Borate and MOPS).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Kinsey et al. eluting lipids using a buffer mobile phase to incorporate the teachings of eluting the extracellular nucleic acid at second pH that is below, at or above the pKa of a protonatable groups on WAX sorbent as taught by Horlitz et al. (Page 16: line 11) to provide: wherein eluting comprises flowing an aqueous buffer through the WAX solid phase sorbent, through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent. It would have been obvious to combine Kinsey et al. wherein eluting comprises flowing an aqueous buffer through the WAX solid phase sorbent with Horlitz et al. wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent because Kinsey et al. teaches measuring LNP samples with a mobile phase containing aqueous buffer while Horlitz et al. eluting using an aqueous buffer with a pKa greater than the anion exchange residues which achieves that same outcome. In this combination, Kinsey et al. method, and Horlitz et al. eluting with an aqueous buffer performs the same functions they would if they were separate. The person of ordinary skill in the art would have found it obvious to combine the elements because ordinarily skilled artisans would have recognized the reasons for applying Kinsey et al. wherein eluting comprises flowing an aqueous buffer with Horlitz et al. method through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent and would have known how to do so. MPEP 2143 (I)(A)
However, Modified Kinsey et al. does not teach said aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent.
Hollander et al. teaches a method of isolating RNA from contaminants (Page 4: line 3). Hollander et al. teaches degrading a sample (Page 5: line 21) and binding RNA to a solid phase (Page 4: line 9). Hollander et al. teaches a membrane that can bind nucleic acids is ion exchange (Page 14: line 29). Hollander et al. further teaches organic solvents such as alcohols that can be used to degrade the sample (Page 9: line 5). Hollander et al. further comprising: digesting at least a portion of the solubilized nucleic acid adsorbed on the sorbent, forming an adsorbed mixture of nucleic acid digestion products, wherein digesting comprises contacting the sorbent with a nuclease; and eluting the mixture of nucleic acid digestion products from the WAX solid phase sorbent, forming a nucleic acid digestion sample (Hollander et al. teaches a method for isolating nucleic acids including DNA and RNA (Page 3: line 19).
Hollander et al. further teaches said aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent (Page 16: line 4, Hollander teaches an aqueous buffer to wash RNA bound to a solid phase. Page 16: line 9, Hollander teaches the wash solvent containing a buffering component. Example 3: Lysis method D, Hollander teaches an alcohol such as ethanol-containing wash buffer. Page 16: line 20, Hollander teaches the alcohol being a concentration of 30% v/v, preferably at least 50% v/v. Page 16: line 22, Hollander et al. also teaches a buffer such as Tris.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Modified Kinsey et al. buffer elution solution to incorporate the teachings of 30% alcohol wash solution as taught by Hollander G et al (Example 3: Lysis method) to provide: aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent. It would have been obvious to combine Modified Kinsey et al. wherein eluting comprises flowing an aqueous buffer through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent with Hollander et al. said aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent. Because Modified Kinsey et al. dissolves the lipid nucleic acid sample using a diluent with a buffer and acid while Hollander et al. teaches the wash solvent containing a buffering component and an alcohol, which achieves that same outcome. In this combination, Modified Kinsey et al. method, and Hollander et al. purification of samples containing lipids and nucleic acids performs the same functions they would if they were separate. The person of ordinary skill in the art would have found it obvious to combine the elements because ordinarily skilled artisans would have recognized the reasons for applying Modified Kinsey et al. wherein eluting comprises flowing an aqueous buffer through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent with Hollander et al. method, said aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent. and would have known how to do so. MPEP 2143 (I)(A)
With respect to claim 12, Modified Kinsey et al. and Hollander et al. teaches all of the elements of the current invention as stated above with respect to claim 11.
Kinsey et al. teaches LNP samples with lipid 1 having a mobile phase composition mobile phase A consisted of water: methanol with 0.5% Formic Acid: Triethylamine Complex at a pH of 3.5 and mobile phase B was composed of methanol with 0.5% Formic Acid: Triethylamine Complex (Page 1092: paragraph starting with “When measuring”). Kinsey et al further teaches when measuring LNP samples containing Cationic Lipid 2, the following mobile phases were used: mobile phase A was composed of water: methanol with 19.2mM glacial acetic acid and 16.2 mM triethylamine, and mobile phase B was composed of methanol with 19.2 mM glacial acetic acid and 16.2 mM Triethylamine (Page 1092: paragraph starting with “When measuring”).
However Kinsey et al. does not teach wherein the aqueous buffer has a pH from about 8 to about 12.5, or from about 8.5 to about 12.
Horlitz et al. further teaches elution is achieved by contacting the extracellular nucleic acids that are bound to the solid phase with an elution solution (Page 16: line 36). Horlitz et al. teaches biological buffers can be used such as Tris (Page 16: line 39), HEPES, Tris-Borate and MOPS (Page 17: line 27).
Horlitz et al. teaches elution occurs at the second pH and the choice of the second pH is suitable for eluting the extracellular nucleic acids from the anion exchange groups that are present on the solid phase (Page 16: line 5). Horlitz et al. further teaches an embodiment, the second pH is in the range of from about 7.5 to 10.5, preferably from about 8 to 10, 8.2 to 9.5, preferably about 8.5 to 9 (Page 18: line 27) and therefore fails to teach a pH from about 8 to about 12.5, or from about 8.5 to about 12. However, 7.5 to about 10.5 overlaps with applicants claimed range of from about 8 to about 12.5, or from about 8.5 to about 12.
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to modify the pH of Kinsey et al. to select the pH value of aqueous buffer in range of about 8 to about 12.5, or from about 8.5 to about 12. because in the case where the claimed ranges overlap or lie inside ranges disclosed by the prior art a prima facie case of obviousness exists. MPEP § 2144.05.
With respect to claim 15, Modified Kinsey et al. and teaches all of the elements of the current invention as stated above with respect to claim 10.
Modified Kinsey et al. teaches a lysis step for nucleic acid isolation (Horlitz et al., Page 4: line 20). Modified Kinsey et al. teaches the lysis step to denature and/or digest protein contaminations or other contaminating substances that could interfere e.g. with the binding of the nucleic acid to the solid phase and/or could lead to an improper purification (Horlitz et al., Page 4, line 20).
Modified Kinsey et al., more specifically teaches it is also within the scope of the present invention to digest the non-target nucleic acid using nucleases after isolation (Horlitz et al., Section 32: line 3- 6).
However, Modified Kinsey et al. does not specifically teach further comprising hydrolyzing at least a portion of the nucleic acid present in the nucleic acid sample to form a mixture of constituent residues.
Hollander et al. teaches a method of isolating RNA from contaminants (Page 4: line 3). Hollander et al. teaches degrading a sample (Page 5, line 21) and binding RNA to a solid phase (Page 4: line 9). Hollander et al. further teaches organic solvents such as alcohols that can be used to degrade the sample (Page 9: line 5).
Hollander et al. further teaches further comprising hydrolyzing at least a portion of the nucleic acid present in the nucleic acid sample to form a mixture of constituent residues (Page 10: line 33, Hollander teaches a DNase refers to any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA. Page 10: line 39, Hollander teaches performing DNase digest before the RNA is isolated from the degraded sample. Page 4: line 29, Hollander teaches further teaches separating undissolved constituents from a degraded sample. Page 5: line 34, Hollander teaches degradation assists with the release of nucleic acids from complexing sample constituents).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Modified Kinsey et al. to digest the non-target nucleic acid using nucleases after isolation to incorporate the teachings of performing DNase digest before the RNA is isolated as taught by Hollander et al. (Page 10: line 39) to provide: further comprising hydrolyzing at least a portion of the nucleic acid present in the nucleic acid sample to form a mixture of constituent residues. It would have been obvious to combine Hollander et al. method to hydrolyzing at least a portion of the nucleic acid present in the nucleic acid sample to form a mixture of constituent residues with Modified Kinsey et al. to digest the non-target nucleic acid using nucleases after isolation because Modified Kinsey et al. teaches the lysis step to denature protein contaminations and digestion of nucleic acids, while Hollander et al. teaches hydrolytic cleavage of phosphodiester linkages in the DNA, which achieves that same outcome. In this combination, both Kinsey et al. method and Horlitz et al. analysis performing the same functions they would if they were separate. The person of ordinary skill in the art would have found it obvious to combine the elements because ordinarily skilled artisans would have recognized the reasons for applying Modified Kinsey et al. digest the non-target nucleic acid using nucleases after isolation using Hollander et al. hydrolytic cleavage of phosphodiester linkages in the DNA and would have known how to do so. The person of ordinary skill in the art would further have predicted that the combination would allow for the purification of nucleic acids because Modified Kinsey et al. teaches that isolated extracellular nucleic acids can be directly analyzed using analytical methods for diagnostic and therapeutic testing (Horlitz et al., Page 33: line 20-line 41 to Page 34: line 2 ). MPEP 2143 (I)(A)
With respect to claim 16, Modified Kinsey et al. teaches all of the elements of the current invention as stated above with respect to claim 1.
Modified Kinsey et al. teaches adsorbing the solubilized nucleic acid on a weak anion exchange (WAX) solid phase sorbent (Page 30: line 40 to Page 31: line 2). Modified Kinsey et al. further teaches eluting the extracellular nucleic acid from the solid phase (Page 36: line 34). Modified Kinsey et al. teaches a lysis step for nucleic acid isolation (Horlitz et al., Page 4: line 20). Modified Kinsey et al. teaches the lysis step to denature and/or digest protein contaminations or other contaminating substances that could interfere e.g. with the binding of the nucleic acid to the solid phase and/or could lead to an improper purification (Horlitz et al., Page 4: line 20).
Modified Kinsey et al. teaches further comprising digesting at least a portion of the nucleic acid present in the nucleic acid sample with a nuclease (Horlitz et al., Page 32: line 39). However, Modified Kinsey et al. does not teach further comprising: digesting at least a portion of the solubilized nucleic acid adsorbed on the sorbent, forming an adsorbed mixture of nucleic acid digestion products, wherein digesting comprises contacting the sorbent with a nuclease; and eluting the mixture of nucleic acid digestion products from the WAX solid phase sorbent, forming a nucleic acid digestion sample.
Hollander et al. teaches a method of isolating RNA from contaminants (Page 4: line 3). Hollander et al. teaches degrading a sample (Page 5: line 21) and binding RNA to a solid phase (Page 4: line 9). Hollander et al. teaches a membrane that can bind nucleic acids is ion exchange (Page 14: line 29). Hollander et al. further teaches organic solvents such as alcohols that can be used to degrade the sample (Page 9: line 5).
Hollander et al. teaches further comprising: digesting at least a portion of the solubilized nucleic acid adsorbed on the sorbent, forming an adsorbed mixture of nucleic acid digestion products, wherein digesting comprises contacting the sorbent with a nuclease; and eluting the mixture of nucleic acid digestion products from the WAX solid phase sorbent, forming a nucleic acid digestion sample (Page 10: line 33, Hollander teaches a DNase refers to any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA. Page 10: line 20, Hollander teaches performing DNase digest before the RNA is isolated from the degraded sample. Page 4: line 29, Hollander further teaches separating undissolved constituents from a degraded sample. Page 5: line 34, Hollander teaches degradation assists with the release of nucleic acids from complexing sample constituents. Page 3: line 25, Hollander teaches DNase digestion on eluted RNA or if using a nucleic acid binding solid phase comprised in a column. In Example 1, Hollander further teaches eluting the RNA from a RNeasy MinElute column after digestion).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Modified Kinsey et al. to digest the non-target nucleic acid using nucleases to incorporate the teachings of DNase digestion on eluted RNA or if using a nucleic acid binding solid phase comprised in a column as taught by Hollander et al. (Page 3: line 25) to provide: further comprising: digesting at least a portion of the solubilized nucleic acid adsorbed on the sorbent, forming an adsorbed mixture of nucleic acid digestion products, wherein digesting comprises contacting the sorbent with a nuclease; and eluting the mixture of nucleic acid digestion products from the WAX solid phase sorbent, forming a nucleic acid digestion sample. The person of ordinary skill of the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that , further comprising: digesting at least a portion of the solubilized nucleic acid adsorbed on the sorbent would enhance miRNA purification efficiency from tissue samples (Page 35, line 20). The skilled artisan would have had a reasonable expectation of success in a further comprising: digesting at least a portion of the solubilized nucleic acid adsorbed on the sorbent, forming an adsorbed mixture of nucleic acid digestion products, wherein digesting comprises contacting the sorbent with a nuclease; and eluting the mixture of nucleic acid digestion products from the WAX solid phase sorbent, forming a nucleic acid digestion sample because Modified Kinsey et al. teaches digesting nucleic acid after isolation for nucleic acid purification and Hollander et al. purification of nucleic acids on the column (Page 26, line 14). MPEP 2143 (I)(B)
With respect to claim 17, Modified Kinsey et al. and Hollander et al. teaches all of the elements of the current invention as stated above with respect to claim 16.
Kinsey et al. teaches LNP samples with lipid 1 having a mobile phase composition mobile phase A consisted of water: methanol with 0.5% Formic Acid: Triethylamine Complex at a pH of 3.5 and mobile phase B was composed of methanol with 0.5% Formic Acid: Triethylamine Complex (Page 1092: paragraph starting with “When measuring”). Kinsey et al further teaches when measuring LNP samples containing Cationic Lipid 2, the following mobile phases were used: mobile phase A was composed of water: methanol with 19.2mM glacial acetic acid and 16.2 mM triethylamine, and mobile phase B was composed of methanol with 19.2 mM glacial acetic acid and 16.2 mM Triethylamine (Page 1092: paragraph starting with “When measuring”).
However Kinsey et al. does not teach through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent, said aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent.
Horlitz et al. teaches wherein eluting comprises flowing an aqueous buffer through the WAX solid phase sorbent, through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent (Page 16: line 5, Horlitz teaches eluting the extracellular nucleic acid from the WAX sorbent where elution occurs at the second pH and the choice of the second pH is suitable for eluting the extracellular nucleic acids from the anion exchange groups that are present on the solid phase. Page 16: line 11, Horlitz et al. further teaches the second pH may be below, at or above the pKa of a protonatable group of the anion exchange group. Page 16: line 36, Horlitz further teaches elution is achieved by contacting the extracellular nucleic acids that are bound to the solid phase with an elution solution. Page 17: line 27, Horlitz et al. teaches buffers such as Tris (Page 16: line 39), HEPES, Tris-Borate and MOPS).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Kinsey et al. eluting lipids using a buffer mobile phase to incorporate the teachings of eluting the extracellular nucleic acid at second pH that is below, at or above the pKa of a protonatable groups on WAX sorbent as taught by Horlitz et al (Page 16: line 11) to provide: wherein eluting comprises flowing an aqueous buffer through the WAX solid phase sorbent, through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent. It would have been obvious to combine Kinsey et al. wherein eluting comprises flowing an aqueous buffer through the WAX solid phase sorbent with Horlitz et al. wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent. Because Kinsey et al. teaches measuring LNP samples with a mobile phase containing aqueous buffer while Horlitz et al. eluting using an aqueous buffer with a pKa greater than the anion exchange residues which achieves that same outcome. In this combination, Kinsey et al. method, and Horlitz et al. eluting with an aqueous buffer performs the same functions they would if they were separate. The person of ordinary skill in the art would have found it obvious to combine the elements because ordinarily skilled artisans would have recognized the reasons for applying Kinsey et al. wherein eluting comprises flowing an aqueous buffer with Horlitz et al. method through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent and would have known how to do so. MPEP 2143 (I)(A)
However, Modified Kinsey et al. does not teach said aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent.
Hollander et al. teaches a method of isolating RNA from contaminants (Page 4: line 3). Hollander et al. teaches degrading a sample (Page 5: line 21) and binding RNA to a solid phase (Page 4: line 9). Hollander et al. teaches a membrane that can bind nucleic acids is ion exchange (Page 14: line 29). Hollander et al. further teaches organic solvents such as alcohols that can be used to degrade the sample (Page 9: line 5). Hollander et al. further comprising: digesting at least a portion of the solubilized nucleic acid adsorbed on the sorbent, forming an adsorbed mixture of nucleic acid digestion products, wherein digesting comprises contacting the sorbent with a nuclease; and eluting the mixture of nucleic acid digestion products from the WAX solid phase sorbent, forming a nucleic acid digestion sample (Hollander et al. teaches a method for isolating nucleic acids including DNA and RNA (Page 3: line 19).
Hollander et al. further teaches said aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent (Page 16: line 4, Hollander teaches an aqueous buffer to wash RNA bound to a solid phase. Page 16: line 9, Hollander teaches the wash solvent containing a buffering component. Example 3: Lysis method D, Hollander teaches an alcohol such as ethanol-containing wash buffer. Page 16: line 20, Hollander teaches the alcohol being a concentration of 30% v/v, preferably at least 50% v/v. Page 16: line 22, Hollander et al. also teaches a buffer such as Tris.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to combine the methods of Modified Kinsey et al. buffer elution solution with 30% alcohol wash solution as taught by Hollander G et al (Example 3: Lysis method) to provide: aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent. It would have been obvious to combine Modified Kinsey et al. wherein eluting comprises flowing an aqueous buffer through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent with Hollander et al. said aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent. Because Modified Kinsey et al. dissolves the lipid nucleic acid sample using a diluent with a buffer and acid while Hollander et al. teaches the wash solvent containing a buffering component and an alcohol, which achieves that same outcome. In this combination, Modified Kinsey et al. method, and Hollander et al. purification of samples containing lipids and nucleic acids performs the same functions they would if they were separate. The person of ordinary skill in the art would have found it obvious to combine the elements because ordinarily skilled artisans would have recognized the reasons for applying Modified Kinsey et al. wherein eluting comprises flowing an aqueous buffer through the WAX solid phase sorbent, wherein the aqueous buffer has a pH greater than or equal to the pKa of the anion exchanger residues of the WAX solid phase sorbent with Hollander et al. method, said aqueous buffer comprising up to about 50% by volume of a non-aqueous solvent. and would have known how to do so. MPEP 2143 (I)(A)
With respect to claim 18, Modified Kinsey et al. and Hollander et al. teaches all of the elements of the current invention as stated above with respect to claim 11.
Modified Kinsey et al. teaches eluting the extracellular nucleic acid from the WAX sorbent (Page 36: line 34). Modified Kinsey et al. teaches elution occurs at the second pH and the choice of the second pH is suitable for eluting the extracellular nucleic acids from the anion exchange groups that are present on the solid phase (Horlitz et al., Page 16: line 5). Modified Kinsey et al. further teaches the second pH may be below, at or above the pKa of a protonatable group of the anion exchange group (Horlitz et al., Page 16: line 11).
Modified Kinsey et al. further teaches elution is achieved by contacting the extracellular nucleic acids that are bound to the solid phase with an elution solution (Horlitz et al., Page 16: line 36). Modified Kinsey et al. teaches buffers such as Tris (Horlitz et al., Page 16: line 39), HEPES, Tris-Borate and MOPS (Horlitz et al., Page 17: line 27).
However, Modified Kinsey et al. does not teach wherein the non-aqueous solvent comprises a C1-C4 alcohol, acetonitrile, dimethyl formamide, dimethyl sulfoxide, or a combination thereof.
Hollander et al. teaches a method of isolating RNA from contaminants (Page 4: line 3). Hollander et al. teaches degrading a sample (Page 5: line 21) and binding RNA to a solid phase (Page 4: line 9). Hollander et al. teaches a membrane that can bind nucleic acids is ion exchange (Page 14: line 29). Hollander et al. further teaches organic solvents such as alcohols that can be used to degrade the sample (Page 9: line 5).
Hollander et al. further teaches wherein the non-aqueous solvent comprises a C1-C4 alcohol, acetonitrile, dimethyl formamide, dimethyl sulfoxide, or a combination thereof (Page 16: line 4, Hollander teaches an aqueous buffer to wash RNA bound to a solid phase. Page 16: line 9, Hollander teaches the wash solvent containing a buffering component. Example 3: Lysis method D, Hollander teaches an alcohol such as ethanol-containing wash buffer. Page 16: line 20, Hollander et al. teaches the alcohol being a concentration of 30% v/v, preferably at least 50% v/v. Page 16: line 22, Hollander teaches also teaches a buffer such as Tris. And a biological buffer is preferably Tris at a pH of approx. 7 to 8.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the methods of Modified Kinsey et al. buffer solution to incorporate the teachings of a non-aqueous solvent as taught by Hollander et al. (Example 3) to provide: wherein the non-aqueous solvent comprises a C1-C4 alcohol, acetonitrile, dimethyl formamide, dimethyl sulfoxide, or a combination thereof. The skilled artisan would have expected success in substituting Modified Kinsey et al. Tris buffer exemplified in Hollander et al. buffer alcohol elution solution because Kinsey et al.” teaches an ethanol solvent and a buffer agent to adjust the mobile phase pH and Horlitz et al. teaches ethanol-containing wash buffer to wash RNA bound to a solid phase. MPEP 2143 (I)(B) The person of ordinary skill in the art would have found it obvious to make the substitution because ordinarily skilled artisans would have predicted that wherein the non-aqueous solvent comprises a C1-C4 alcohol, acetonitrile, dimethyl formamide, dimethyl sulfoxide, or a combination thereof results in nucleic acid separation from the solution. The claimed method requires wherein the non-aqueous solvent comprises a C1-C4 alcohol, acetonitrile, dimethyl formamide, dimethyl sulfoxide, or a combination thereof. Because Modified Kinsey et al. teaches buffers like Tris as an elution solvent, a prima facie case of obviousness exists.
With respect to claim 19, Modified Kinsey et al. and Hollander et al. teaches all of the elements of the current invention as stated above with respect to claim 17.
Kinsey et al. teaches LNP samples with lipid 1 having a mobile phase composition mobile phase A consisted of water: methanol with 0.5% Formic Acid: Triethylamine Complex at a pH of 3.5 and mobile phase B was composed of methanol with 0.5% Formic Acid: Triethylamine Complex (Page 1092, paragraph starting with “When measuring”). Kinsey et al further teaches When measuring LNP samples containing Cationic Lipid 2, the following mobile phases were used: mobile phase A was composed of water: methanol with 19.2mM glacial acetic acid and 16.2 mM triethylamine, and mobile phase B was composed of methanol with 19.2 mM glacial acetic acid and 16.2 mM
Triethylamine (Page 1092, paragraph starting with “When measuring”).
However, Kinsey et al. does not teach wherein the aqueous buffer has a pH of about 7 to about 12.5, or from about 8 to about 12.
Horlitz et al. teaches wherein the aqueous buffer has a pH of about 7 to about 12.5, or from about 8 to about 12 (Page 16: line 5, Horlitz teaches elution occurs at the second pH and the choice of the second pH is suitable for eluting the extracellular nucleic acids from the anion exchange groups that are present on the solid phase. Page 16: line 36, Horlitz teaches elution is achieved by contacting the extracellular nucleic acids that are bound to the solid phase with an elution solution. Page 16,: line 39, Horlitz teaches biological buffers can be used such as Tris, HEPES and Tris-Borate.
Horlitz et al. teaches the second pH is in the range of from about 7.5 to 10.5 (Page 18: line 27) and therefore fails to teach a pH of about 7 to about 12.5, or from about 8 to about 12 as claimed. However, about 7.5 to 10.5 overlaps with applicants claimed range of 7 to about 12.5, or from about 8 to about 12.
Therefore, it would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention to modify the pH of Modified Kinsey et al. to select a pH of about 7 to about 12.5, or from about 8 to about 12 because in the case where the claimed ranges overlap or lie inside ranges disclosed by the prior art a prima facie case of obviousness exists. MPEP § 2144.05.
With respect to claim 20, Modified Kinsey et al. and Hollander et al. teaches all of the elements of the current invention as stated above with respect to claim 16.
Modified Kinsey et al. fails to explicitly teach an embodiment where further comprising analyzing the nucleic acid digestion sample by high performance liquid chromatography, mass spectrometry, or a combination thereof. Modified Kinsey et al. teaches a lysis step for nucleic acid isolation (Horlitz et al., Page 4: line 20). Modified Kinsey et al. teaches the lysis step to denature and/or digest protein contaminations or other contaminating substances that could interfere e.g. with the binding of the nucleic acid to the solid phase and/or could lead to an improper purification (Horlitz et al., Page 4: line 20). Modified Kinsey et al. more specifically teaches it is also within the scope of the present invention to e.g. digest the non-target nucleic acid using nucleases after isolation (Horlitz et al., Page 32: line 39). Modified Kinsey et al. teaches analyzing nucleic acid samples by mass spectrometry (Horlitz et al., page 34, line 1 and line 10).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective
filing date of the claimed invention to modify the lipids analysis using UHPLC by Modified Kinsey et al. to incorporate the teachings of analyzing the nucleic acid digestion sample by mass spectrometry as taught by Horlitz (page 32: line 39 and page 32: line 39) to provide: further comprising analyzing the nucleic acid digestion sample by high performance liquid chromatography, mass spectrometry, or a combination thereof. Because Modified Kinsey et al. analyzes the extracellular isolated nucleic acid samples by mass spectrometry and teaches digestion of nucleic acid samples post separation, a prima facie case of obviousness exists. The person of ordinary skill in the art would have found it obvious to try because ordinarily skilled artisans would have predicted that further comprising analyzing the nucleic acid digestion sample by high performance liquid chromatography, mass spectrometry, or a combination thereof ,would result in a reasonable expectation of success. MPEP2143 (I)(E).
Conclusion
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/S.J.B./ Examiner, Art Unit 1758
/MARIS R KESSEL/ Supervisory Patent Examiner, Art Unit 1758
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