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 .
DETAILED ACTION
Claims 1-19 are pending and under examination herein.
Priority
This application, filed on 7/30/2024, is a 371 of PCT/IB2023/053829 filed 4/14/2023, which claims benefit of INDIA 202211023269 filed on 4/20/2022. The effective filing date of the current application is April 20, 2022.
Information Disclosure Statement
The information disclosure statement filed on 7/30/2024 complies with 37 CFR 1.98(a)(2), which requires a legible copy of each cited foreign patent document; each non-patent literature publication or that portion which caused it to be listed; and all other information or that portion which caused it to be listed. All references were considered.
Claim Objections
Claim 18 is objected to because of the following informalities:
Claim 18 recites at “1210C for 15 min” in step iii) line 1, which uses a zero instead of a degree symbol, and should be amended to recite “121°C for 15 min”.
Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-2, 4-11, and 13-19 are rejected under 35 U.S.C. 101 because the claimed invention is directed to
Claim 1 recites a buffer composition comprising a nuclease quenching agent, a stabilizer, a salt and a cryoprotectant. Claim 2 recites the nuclease quenching agent is selected from citric acid and glycine, which are naturally occurring. Claim 5 recites the stabilizer is selected from sodium salt, citric acid, potassium citrate, sodium citrate, and potassium phosphate, which are naturally occurring. Claim 8 recites the salt is selected from sodium sulfate, potassium sulfate, and ammonium sulfate, which are naturally occurring. Claim 11 recites the cryoprotectant is selected from sugar, polyol, glycerol and amino acid, which are naturally occurring.
Angumeenal et al. (“An overview of citric acid production”, LWT – Food Science and Technology, 2013, Vol. 50, pp.367-370) identifies citric acid as a naturally occurring substance present in juice of citrus fruits and pineapple (p.367, 2. Chemistry of citric acid).
Li et al. (“Roles of dietary glycine, proline, and hydroxyproline in collagen synthesis and animal growth”, Amino Acids, 2018, Vol. 50, pp.29-38) identifies glycine as the simplest amino acid in nature (p.29, 2nd column – 1st paragraph). Li discloses that collagen is formed from amino acids (p.33, 1st column collagen synthesis and processing). Li further identifies all of the amino acids and the type of collagen they are found in (p.35, Table 4), thus identifying that amino acids including glycine are naturally occurring substances.
Hocking (Chapter 4: Natural and Derived Sodium and Potassium Salts, Modern Chemical Technology and Emission Control, 1985, pp.105-121) discloses outdoor recovery of sodium chloride by evaporation of sea water or natural brines in areas with high evaporation rate and low rainfall (p.106, 1st column – 4.1.1. Solar Salt). Hocking teaches that sodium sulfate is recovered from natural sources, generally from captive lake basins in areas with high evaporation rates or from aquifers with a high dissolved sodium sulfate content (p.118, 2nd column – 4.3.1. Production and Use pattern for Sodium Sulfate). Hocking identifies that Potassium is recovered from the Great Salt Lake, mainly as the sulfate (p.113, 1st column top paragraph), thus identifying that sodium salt, sodium sulfate, and potassium sulfate are naturally occurring substances.
Singh et al. (Chapter 4: Sugar and Sugar Substitutes: Recent Developments and Future Prospects; Sugar and Sugar Derivatives – Changing Consumer Preferences Book; 2020, pp.39-75) identifies that sugar is found inherently in fruits, vegetables, dairy goods and grains (abstract). Singh discloses that sugar alcohols (polyols) are present naturally in various foods including fruit, vegetables, grains and dairy (p.49, 4.5.3 Sugar Alcohol (Polyols)). Singh further identifies glycerol, also known as glycerine, as a natural sugar alcohol generally produced by animal or vegetable fat (p.51, 4.5.3.8 Glycerol).
Sabboh et al. (“Effects of plant food potassium salts (citrate, galacturonate or tartrate) on acid–base status and digestive fermentations in rats”, British Journal of Nutrition, 2007, Vol. 98, Issue 1, pp. 72-77) identifies that fruits and vegetables are practically the unique source of K organic salts, predominantly potassium malate and potassium citrate (p.72, 1st column).
This judicial exception is not integrated into a practical application, because the buffer can comprise naturally occurring substances, and do not possess any markedly different characteristics from the naturally occurring
Thus, claims 1-2, 4-11 and 13-19 are rejected as being directed towards a naturally occurring product without significantly more.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 18-19 are rejected under 35 U.S.C. 112(a) because the specification, while being enabling for autoclaving solutions containing cryoprotectants selected from polymer and glycerol, does not reasonably provide enablement for autoclaving other cryoprotectants including sugar, polyol, amino acids or dimethyl sulfoxide (DMSO). The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
In making a determination that a disclosure does not satisfy the enablement requirement, the factors that may be considered include: (A) the breadth of the claims, (B) the nature of the invention, (C) the state of the prior art, (D) the level of one of ordinary skill, (E) the level of predictability in the art, (F) the amount of direction provided by the inventor, (G) the existence of working examples, and (H) the quantity of experimentation needed to make or use the invention based on the content of the disclosure. While it is not essential that every factor be examined in detail, those factors deemed most relevant should be considered.
Nature of the invention. Claim 18 is drawn to a method of preparing the buffer composition comprising the steps of dissolving salt, nuclease quenching agent and stabilizer in water to obtain a solution; adding cryoprotectant to the solution to obtain a uniform solution; and autoclaving said solution at 121°C for 15 minutes to obtain the buffer solution. Claim 19 is drawn to the method of claim 18, wherein the dissolving step occurs at 45°C. Enablement of the claimed invention is based on whether one of ordinary skill in the art can reliably autoclave a solution comprising any cryoprotectant at 121°C for 15min.
Breadth of the claims. The breadth of the claims is exceedingly large and fails to receive adequate support in the specification. Claims 18-19 encompass adding any cryoprotectant, including glycerol, any sugar, any polyol, any polymer, any amino acid, skim milk, and dimethyl sulfoxide (DMSO), into the solution prior to autoclaving at 121°C for 15 minutes.
Guidance in the specification and working examples. The specification describes cryoprotectants selected from sugar, polyol, polymer, glycerol and amino acid (p.4, lines 10-12). Example 1 describes a composition comprising glycerol (5-10%) as the cryoprotectant (p.6, Example 1). Example 2 describes a composition comprising glycerol that is autoclaved before use (p.8, Example 2).
State of the prior art and unpredictability. No prior art references demonstrate the addition of any other cryoprotectant and then autoclaving the solution.
Prior art surrounding buffer chemistry and cryopreservation is well developed and establishes that, when autoclaving cryoprotectant solutions (such as glycerol, sucrose, or skim milk), follow a standard liquid cycle: 121°C for 15 to 20 minutes using the slow exhaust (or liquid) setting. This prevents the superheated liquid from boiling over. However, DMSO must never be autoclaved, as extreme heat causes it to break down and become highly toxic to cells.
Moore (US3852155, published Dec. 3, 1974) describes cryopreservation of equine cell cultures (title). Moore tests different cryopreservation agents including glycerol, glucose, sucrose, dimethyl sulfoxide, and polyvinylpyrrolidone (PVP) in cryopreservation of equine cell cultures (Col. 3, lines 31-32). Moore identifies that glycerol and PVP were sterilized by autoclaving at 18 psi. pressure and 121°C for 15 minutes (Col. 3, lines 33-35), while other cryoprotective agents were filtered through a 450 mµ millipore filter (Col. 3 line 35 – Col. 4, line 1), suggesting that cryoprotectants glucose, sucrose and dimethyl sulfoxide cannot be autoclaved.
Vedamuthu (US3975545, published August 17, 1976) describes freezing stabilization of bacterial concentrates of lactic acid producing bacteria in compositions comprising skim milk and a freezing stabilizing agent including an alkali metal salt of glycerophosphoric acid in an amount sufficient to reduce damage to the cells as a result of freezing (Col. 1, lines 35-40). Vedamuthu discusses that alkali metal salts of glycerophosphoric acid can be used alone or preferably in admixture with glycerol or other known freezing stabilizing agents which are usually of the class of hygroscopic, di- and polyhydric alkyl alcohols, such as mannitol, containing between 3 to 6 carbon atoms and which can be used in foods. Vedamuthu further identifies that usually between about 0.1 to 5 percent by weight salt is used based upon the volume of the bacterial concentrate (Col. 2, lines 10-18). When glycerol or other stabilizing agents are used in addition, they are used in an amount between about 1 to 20 percent by volume of the concentrate (Col. 2, lines 18-20). Vedamuthu describes a stock solution containing 25% by weight NaGP was prepared and stored in refrigerator; the glycerol level used in all cases was 10% by volume; and both of these compounds were sterilized by autoclaving at 121°C for 15 minutes (Col. 2, lines 64-68).
Coté (Unit 1.4 “Sterilization and Filtration”, Current Protocols in Cell Biology, 1999, Supplement 1, pages 1-21) discusses sterilization and filtration methods (title). Coté identifies that the autoclave cycle is based on the time it takes the material being sterilized to be in contact with saturated steam at 121°C for 15 min (p.1, 3rd paragraph). Coté identifies that problematic filtration needs often appear in cell culture applications, such as the need to filter sterilize the chemically aggressive reagent dimethyl sulfoxide (DMSO) (p.12, 2nd paragraph). Coté states that DMSO is used as the cryoprotectant for liquid-nitrogen preservation of cell cultures, and further that the reagent is not stable to autoclaving conditions, and must be filter sterilized (p.14, 1st paragraph). Coté further identifies that DMSO is an aggressive solvent that dissolves general-use filter units such as cellulose acetate or cellulose nitrate as well as the polystyrene filter units themselves (p.14, 1st paragraph), suggesting that DMSO as a cryoprotectant cannot be sterilized easily, nor autoclaved as required by the inventive method.
Amount of experimentation necessary. Based on the evidence regarding each of the above factors, the specification, at the time the application was filed, would not have taught one skilled in the art how to practice the full scope of the claimed invention without undue experimentation. The claimed method is broad and encompasses autoclaving a solution comprising cryoprotectants that cannot be autoclaved, including glucose, sucrose, and DMSO, as some cryoprotectants can break down and become toxic to cells. Therefore, the disclosure in view of the current state of art does not enable the full scope of the claimed method.
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 1-13 and 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (“The maintenance of microbial community in human fecal samples by a cost effective preservation buffer”, Nature Scientific Reports, June 2021, Volume 11, Article 13453, 10 pages) in view of Whaley et al. (“Cryopreservation: An Overview of Principles and Cell-Specific Considerations”, Cell Transplantation, 2021, Vol. 30, article 0963689721999617, 12 pages).
Regarding claim 1, Wu teaches the maintenance of microbial community in human fecal samples by a cost effective preservation buffer (title). Wu teaches the utilization of self-made preservation buffer (PB) could facilitate stabilizing microbiome in human fecal samples (p.5, last sentence). Wu teaches the recipe for the self-made preservation buffer (PB) consisted of 20 mM ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate, 25 mM sodium citrate trisodium salt dihydrate, and 5.3M ammonium sulfate (p.8 – Materials and methods, sample collection and storage).
Wu does not disclose a cryoprotectant.
However, Whaley teaches that successful low-temperature cell preservation consists of the addition of a cryoprotective agent (CPA) alone or in combination with additional permeating or non-permeating agents, cooling rates of approximately 1°C/min, and storage in either liquid or vapor phase nitrogen (abstract). Whaley further teaches that the use of glycerol as a medium increased the survivability of spermatozoa in subfreezing (-70°C) temperatures, and using glycerol as a medium effectively served to protect cells from rapid formation of ice crystals during the preservation process (p.2, 1st column – history of cryopreservation). Whaley teaches that permeating agents such as glycerol, dimethyl sulfoxide, ethylene glycol and propanediol exist, and must be highly water soluble at low temperatures, able to easily cross biological membranes and ideally, be minimally toxic (p2, 2nd column – permeating agents).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add a cryoprotective agent, glycerol, as taught by Whaley, to the buffer composition of Wu, because Whaley teaches that glycerol effectively served to protect cells from rapid formation of ice crystals during the preservation process. One of ordinary skill in the art would have been motivated to add a cryoprotective agent to the buffer composition because Whaley teaches that glycerol effectively protected cells from rapid formation of ice crystals during the preservation process and increased survivability of spermatozoa in subfreezing temperatures.
Regarding claims 2-4, Wu teaches the preservation buffer contains 20 mM ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate (p.8 – Materials and methods, sample collection and storage).
Regarding claims 5-7, Wu teaches the preservation buffer contains 25 mM sodium citrate trisodium salt dihydrate (p.8 – Materials and methods, sample collection and storage).
Regarding claims 8-10, Wu teaches the preservation buffer contains 5.3M ammonium sulfate (p.8 – Materials and methods, sample collection and storage).
Regarding claims 11-12, Wu does not teach a cryoprotectant of polyol or glycerol.
However, Whaley teaches permeating cryoprotective agents including glycerol, dimethyl sulfoxide, ethylene glycol and propanediol (propylene glycol) (p.2, 2nd column – Permeating agents). Whaley teaches non-permeating agents include polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), raffinose, sucrose and trehalose (p.2, 1st column – Non-permeating agents). Glycerol is a polyol. Whaley teaches non-permeating agents induce vitrification by the same mechanism as permeating agents but extracellularly and to a lesser extent (p.3, 1st column – Non-permeating agents). Whaley further teaches that the use of glycerol as a medium increased the survivability of spermatozoa in subfreezing (-70°C) temperatures, and using glycerol as a medium effectively served to protect cells from rapid formation of ice crystals during the preservation process (p.2, 1st column – history of cryopreservation).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add glycerol taught by Whaley to the composition of Wu, because Whaley teaches that glycerol effectively served to protect cells from rapid formation of ice crystals during the preservation process. One of ordinary skill in the art would have been motivated to add the cryoprotective agent glycerol to the buffer composition of Wu because Whaley teaches that glycerol effectively protected cells from rapid formation of ice crystals during the preservation process and increased survivability of spermatozoa in subfreezing temperatures.
Regarding claim 13, Wu does not teach the cryoprotectant is present in a range of 5% to 10% of the composition.
However, Whaley teaches adding DMSO at 5%, evidence suggests DMSO decreases membrane thickness and increases membrane permeability. Whaley teaches that at commonly used concentrations of 10%, water pore formation in biological membranes is induced, which can be advantageous as intracellular water can be more readily replaced by cryoprotectants that promote vitrification (p.2, 2nd column last paragraph – p.3, 1st column top paragraph). Whaley further teaches that at higher toxic concentrations (40%), lipid bilayers can begin to disintegrate.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add the cryoprotectant at 5% to 10%, as taught by Whaley, to the composition of Wu, because Whaley teaches that at 10% concentration, water pore formation is induced in the cell membrane, which could be advantageous as intracellular water can be replaced by the cryoprotectant that promotes vitrification. One of ordinary skill in the art would have found it beneficial to use a concentration that protects cell structural integrity during freezing and thus improve cell viability.
Regarding claim 15, Wu teaches the maintenance of microbial community in human fecal samples by a cost effective preservation buffer (title). Wu further teaches that microbial consortia in human fecal samples were substantially preserved within a temporary storage of 4h, independent of the storage temperature (abstract).
Regarding claim 16, Wu teaches that samples stored in liquid nitrogen retained similar viable microbial profiles to samples stored at room temperature and high temperature (p.4, Figure 2 A and B).
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (“The maintenance of microbial community in human fecal samples by a cost effective preservation buffer”, Nature Scientific Reports, June 2021, Volume 11, Article 13453, 10 pages) in view of Whaley et al. (“Cryopreservation: An Overview of Principles and Cell-Specific Considerations”, Cell Transplantation, 2021, Vol. 30, article 0963689721999617, 12 pages) as applied to claim 1 above, and further in view of Evans et al. (“Handbook on Isolation, Characterization and Cryopreservation of Leishmania”, 1989, World Health Organization, Geneva Switzerland).
The teachings of Wu et al. and Whaley et al. are discussed above.
Regarding claim 14, Wu and Whaley do not disclose pH of the buffer composition.
However, Evans teaches preparing a 200mM solution of EDTA by dissolving 7.4445 g of disodium EDTA dihydrate in 50mL of distilled water and adjusting the pH to 7.0 using 1.0M NaOH (p.16, 4.2.1.1 step b). Evans further teaches adding the stabilizer solution to the organisms, mixing and freezing (p.16, 4.2.1.1. Lysis steps b-c). Evans teaches adjusting the pH of the PBSS solution used as biphasic culture media for Leishmania to pH 7.2 (p.28, PBSS).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to adjust the pH of the buffer composition of Wu as modified by Whaley to 7.0–7.2, because Evans teaches pH 7.2 is a useful pH for biphasic culture media for Leishmania. One of ordinary skill in the art would reasonably expect that adjusting the pH of the buffer to pH 7.0-7.2 would predictably result in a neutral pH buffer that would preserve microorganisms at physiological pH, and adjusting the pH of buffers to 7.0-7.2 for bacteria was known in the art at the time of invention.
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (“The maintenance of microbial community in human fecal samples by a cost effective preservation buffer”, Nature Scientific Reports, June 2021, Volume 11, Article 13453, 10 pages) in view of Whaley et al. (“Cryopreservation: An Overview of Principles and Cell-Specific Considerations”, Cell Transplantation, 2021, Vol. 30, article 0963689721999617, 12 pages) as applied to claim 1 above, and further in view of Shamkhalichenar et al. (“An Impedimetric Sensing Probe Based on Printed Circuit Board Technology for Monitoring in Cryobiology Applications”, Journal of the Electrochemical Society, 2021, Vol. 168, article 067505, 9 pages).
The teachings of Wu et al. and Whaley et al. are discussed above.
Regarding claim 17, Wu and Whaley do not teach said composition has a resistivity in the range of 10 to 25 MΩ.
However, Shamkhalichenar teaches the resistivity of water is 18.15 MΩ-cm and glycerol is 15.62 MΩ-cm (p.5, 1st column last full paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have a resistivity between 10 and 25 MΩ-cm because Shamkhalichenar teaches that resistivity of water is 18.15 MΩ-cm and glycerol is 15.62 MΩ-cm. One of ordinary skill in the art would reasonably expect that a composition comprising water and glycerol would predictably result in a resistivity measurement between 10 to 25 MΩ-cm, because it would amount to a simple measurement of combined ingredients in a predictable way, and the resistivities of water and glycerol were known in the art at the time of invention.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (“The maintenance of microbial community in human fecal samples by a cost effective preservation buffer”, Nature Scientific Reports, June 2021, Volume 11, Article 13453, 10 pages) in view of Whaley et al. (“Cryopreservation: An Overview of Principles and Cell-Specific Considerations”, Cell Transplantation, 2021, Vol. 30, article 0963689721999617, 12 pages) as applied to claim 1 above, and further in view of Moore (US 3,852,155 issued on December 3, 1974).
The teachings of Wu et al. and Whaley et al. are discussed above.
Regarding claim 18, Wu teaches preparing self-made preservation buffer using 20 mM ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate, 25 mM sodium citrate trisodium salt dihydrate, 5.3 M ammonium sulfate (p.8, Materials and Methods – Sample collection and storage).
Whaley teaches that the use of glycerol as a medium increased the survivability of spermatozoa in subfreezing (-70°C) temperatures, and using glycerol as a medium effectively served to protect cells from rapid formation of ice crystals during the preservation process (p.2, 1st column – history of cryopreservation).
Wu and Whaley do not teach autoclaving said solution at 121°C for 15 minutes.
However, Moore teaches cryopreservation of equine cell cultures (title). Moore tests different cryopreservation agents including glycerol, glucose, sucrose, dimethyl sulfoxide, and polyvinylpyrrolidone (PVP) in cryopreservation of equine cell cultures (Col. 3, lines 31-32). Moore teaches that glycerol and PVP were sterilized by autoclaving at 18 psi. pressure and 121°C for 15 minutes (Col. 3, lines 33-35), while other cryoprotective agents were filtered through a 450 mµ millipore filter (Col. 3 line 35 – Col. 4, line 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to autoclave the solution taught by Wu and Whaley at 121°C for 15 min as taught by Moore, because Moore teaches a cryopreservation solution containing glycerol can be sterilized by autoclaving. One of ordinary skill in the art would have been motivated to use glycerol as the cryoprotectant because Whaley teaches that glycerol effectively served to protect cells from rapid formation of ice crystals during the preservation process. One of ordinary skill in the art would have found it beneficial to use sterilized cryopreservation buffers to prevent contamination of the preserved samples.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (“The maintenance of microbial community in human fecal samples by a cost effective preservation buffer”, Nature Scientific Reports, June 2021, Volume 11, Article 13453, 10 pages) in view of Whaley et al. (“Cryopreservation: An Overview of Principles and Cell-Specific Considerations”, Cell Transplantation, 2021, Vol. 30, article 0963689721999617, 12 pages) as applied to claim 1 above, and further in view of Camacho-Sanchez et al. (“Preservation of RNA and DNA from mammal samples under field conditions”, Molecular Ecology Resources, 2013, Vol. 13, Issue 4, pp.663-673) and Hosseini et al. (“Preparation and Characterization of chitosan nanoparticles-loaded fish gelatin-based edible films”, Journal of Food Process Engineering, 2016, Vol. 39, Issue 5, pp.521-530).
Regarding claim 19, Wu teaches self-made preservation buffer (PB buffer) could enable fecal microbiota samples to endure a high temperature condition that mimics temperature variations in summer (abstract). Wu further teaches the PB (2w)-high temperature group included a pre-storage with PB buffer for 2 weeks followed by an extra 50°C preservation for 3/4/5 days (p.8, Sample collection and storage).
Wu and Whaley do not teach wherein the dissolving step is performed at 45°C.
Camacho-Sanchez teaches preparation of a preservation buffer for mammal samples (title). Camacho-Sanchez teaches a method of preparing the buffer by combining EDTA, sodium citrate sodium salt dihydrate and ammonium sulfate in water and stirring on low to moderate heat until the ammonium sulfate dissolves completely, which usually takes hours (p.11, Appendix 1 step 1).
Camacho-Sanchez does not teach the dissolving step is performed at 45°C.
Hosseini teaches preparing gelatin films by heating distilled water with gelatin to 45°C for 30min under continuous stirring; adding glycerol and warming and stirring at 45°C for 15 minutes (p.522, 2nd column – Film preparation).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to dissolve EDTA, sodium citrate and ammonium sulfate at 45°C taught by Hosseini, because Hosseini teaches adding glycerol to a water-based solution at 45°C. One of ordinary skill in the art would have been motivated to dissolve the ingredients at 45°C because Camacho-Sanchez teaches dissolving the components requires moderate heat and can take hours. One of ordinary skill in the art would have found it beneficial to dissolve the ingredients at 45°C to dissolve the components into solution.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/DEEPA MISHRA/Examiner, Art Unit 1657
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