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 .
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1-6, in the reply filed on 09/17/26 is acknowledged.
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.
Claim(s) 1-6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Eilam (Ribosomes and Polysomes in Cucumber Leaves during Growth and Senescence, 1970) in view of Abraham (Cucumber-Derived Exosomes-like Vesicles and PlantCrystals for Improved Dermal Drug Delivery, 2022).
Regarding claim 1, Eilam discloses an extraction method of a cucumber ribosome (ribosomal fraction of the first leaf of cucumber (Cucumis sativus), Abstract), comprising the following steps:
S1: mixing cucumber plants with a buffer solution (The leaves (10 to 40 g) were rapidly washed…and with one to two times their volume of tris-magnesium buffer…Preparation of Ribosomes and Polysomes, para. 1, page 317), and squeezing to obtain a mixture (The homogenate was squeezed through four layers of muslin, and the material in the muslin was extracted… Preparation of Ribosomes and Polysomes, para. 1, page 317); wherein a volume ratio of the cucumber leaves to the first buffer solution is 1-2 (…with one to two times their [cucumber leaves] their volume of tris-magnesium buffer… Preparation of Ribosomes and Polysomes, para. 1, page 317);
S2: performing a first centrifugation on the mixture obtained in S1, and taking a first supernatant; wherein the first centrifugation is performed at a centrifugal force of 2000-3000 g for 10 min (Both halves were centrifuged successively for 10 min at 2,000 g, Differential centrifugation, page 317);
S3: performing a second centrifugation on the first supernatant obtained in S2, and taking a second supernatant, wherein the second centrifugation is performed at a centrifugal force of 20,000 g for 20 min (Differential centrifugation, page 317);
S4: performing a third centrifugation on the second supernatant obtained in S3, and taking a third supernatant; wherein the third centrifugation is performed at a centrifugal force of 30,000 g for 30 min (Differential centrifugation, page 317);
S6: performing an ultracentrifugation on the filtrate (pellets, Differential centrifugation, page 317) obtained in S4, and discarding a fourth supernatant to obtain a precipitate; wherein the ultracentrifugation is performed at a centrifugal force of 100000-140000 g for 60-90 min (The pellets were discarded, and the final supernatants were centrifuged at 105,000g for 90 min. Differential centrifugation, page 317); and
S7: mixing the precipitate obtained in S6 with a second buffer solution (The pellet were washed once…at which time they were resuspended in tris-magnesium buffer and clarified for 10 min at 20,000g. Differential Centrifugation, page 317).
The differences between Eilam’s method and claimed invention are:
For step 1, the clamed invention uses cucumber cubes and PBS solution, whereas Eilam uses cucumber leaves and tris-magnesium buffer, and Eilam does not disclose the claimed weight ratio;
Step 2: the claim requires 20-30 min of centrifuge time, whereas Eilam centrifuges for about 10 minutes;
Step 3: the claim requires 5000-6000g for 60-90 min, whereas Eilam centrifuges for 20,000 g for 20 min;
Step 4: the claim requires 9000-10000g for 60-90 min, whereas Eilam centrifuges for 30,000 g for 30 min;
Step 5: Eilam does not disclose Step 5 regarding step of filtering the third supernatant obtained in S4 by using a filter membrane to obtain a filtrate; and
Step 7: the claim requires mixing the precipitate with a second PBS and performs 14,000-16,000 g, whereas Eilam mixes the precipitate with a second tris-magnesium buffer and performs centrifuge at 20,000 g.
With respect to the missing limitation of cubes, PBS, and filtering the third supernatant obtained in S5, analogous art Abraham discloses an extraction method of a cucumber exosome-like vesicle, comprising the following steps:
S1: mixing and squeezing cucumber cubes to obtain a mixture and mix the mixture with a PBS (The peeled cucumbers were cut into smaller pieces and juiced using a blender (Philips HR3655/00 Blender, Philips Electronics NV, Amsterdam, Netherlands). After adding the protease inhibitor cocktail, filtered (0.22 µm) PBS was added to reach a final volume of 1000 mL. 2.2.1 Preparation of Classic PEVs, page 3),
S2: performing a first centrifugation on the mixture obtained in S1, and taking a first supernatant; wherein the first centrifugation is performed at a centrifugal force of 2000-3000 g for 10 min (DUC included a series of low-velocity cycles at 400, 800, 2000 and 15,000× g using a Sorvall centrifuge (RC 6 Plus Centrifuge, Fisher Scientific GmbH, Schwerte, Germany) with a fixed angle rotor, each for 30 min at room temperature. 2.2.1. Preparation of Classic PEVs, page 3);
S4: performing a third centrifugation on the second supernatant obtained in S3, and taking a third supernatant; wherein the third centrifugation is performed at a centrifugal force of 15,000× g for 30 min (DUC included a series of low-velocity cycles at 400, 800, 2000 and 15,000× g using a Sorvall centrifuge (RC 6 Plus Centrifuge, Fisher Scientific GmbH, Schwerte, Germany) with a fixed angle rotor, each for 30 min at room temperature. 2.2.1. Preparation of Classic PEVs, page 3));
S5: filtering the third supernatant obtained in S4 by using a filter membrane to obtain a filtrate (The supernatant after 15,000× g was filtered (0.45 µm), 2.2.1. Preparation of Classic PEVs, page 3).
S6: performing an ultracentrifugation on the filtrate obtained in S4, and discarding a fourth supernatant to obtain a precipitate; wherein the ultracentrifugation is performed at a centrifugal force of 100000-140000 g for 60-90 min (The supernatant after 15,000× g was filtered (0.45 µm) and centrifuged at 120,000× g for 60 min at 4 °C using a Sorvall ultracentrifuge (MTX 150 Micro-Ultracentrifuge, Fisher Scientific GmbH, Schwerte, Germany) with a S50-A fixed angle rotor. 2.2.1. Preparation of Classic PEVs, page 3); and
S7: mixing the precipitate obtained in S6 with a second PBS solution, performing a fourth centrifugation after a resuspension, and taking a fifth supernatant to obtain a cucumber-derived classical PEVs (It was resuspended in a small volume of PBS and vortexed vigorously for about 20 min. 2.2.1. Preparation of Classic PEVs, page 3).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date to have utilized the buffer solution PBS and an additional filtering step before S4 and S6 using a membrane filter as taught by Abraham to the method of Eilam to derive the claimed invention. Doing so provides one of ordinary skill in the art to extract ribosome from cucumber cubes instead of cucumber leaves (Abraham, Abstract).
The combination does not teach the difference in centrifuge speed and time for some of the steps.
Regarding S2, S4, and S7, Eilam teaches that first centrifugation is performed at a centrifugal force of 2000-3000 g for 10 min and the fourth centrifugation is performed at 20,000g for 10 min. Abraham, after incorporation, discloses the third centrifugation is performed at 15,000 g for 30 min. While Eilam does not explicitly teach a range that overlaps with the claimed range of 20-30 min for S2 and 14000-16000 g for 15-30 min for S7 and Abraham does not explicitly teach a range that overlaps with claimed range of 9000-100000 for 60-90 min for S4, it would have been obvious to one of ordinary skill in the art before the effective filing date to have derived the claimed centrifuge speed and running time for S2, S4, and S7 by routine experimentation and optimization based on Eilam’s and Abraham’s disclosure. It is the Examiner’s position that the disclosed values are close enough that one of ordinary skill in the art before the effective filing date of the invention would have expected the same properties. Case law holds that a prima facie case of obviousness exists where the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have the same properties. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 227 USPQ 773 (Fed. Cir. 1985).
Regarding S3, Eilam discloses second centrifugation is performed at a centrifugal force of 20,000 g for 20 min (Differential centrifugation, page 317), which is different from 5000-6000 g for 60-90 min. However, RCF x times are similar (400,000 g-min vs 300,000-540,000 g-min). As pelleting depends on other external variables such as particle size/density, medium viscosity, and rotor geometry, it is Examiner’s position that it would have been obvious to one of ordinary skill in the art before the effective filing date to derive the exact centrifugation speed and time of S3 by routine experimentation and optimization based on disclosure from Eilam. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In reAller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding the specific weight ratio in S1, Eilam discloses a volume ratio of the cucumber leaves to the first buffer solution is 1-2 (…with one to two times their [cucumber leaves] their volume of tris-magnesium buffer… Preparation of Ribosomes and Polysomes, para. 1, page 317), and Abraham discloses the final weight of cucumber per 1000 mL of solution is 629g/1000 mL of solution (2.2.1. Preparation of Classic PEVs, page 3). It is Examiner’s position that it would have been obvious to one of ordinary skill in the art before the effective filing date to derive the exact weight ratio of S1 by routine experimentation and optimization based on Eilam’s and Abraham’s disclosure. Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." See MPEP 2144.05 and In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claim 2, Modified Eilam discloses the claimed invention as discussed above in claim 1. Abraham, after incorporation, discloses the first and second PBS solution have a concentration of 0.01 M (Phosphate buffered saline (PBS, pH 7.4 at 25 ◦C) composed of 100 mmol/L phosphate and 10 mmol/L ethylenediamine tetra acetic acid (EDTA) was used as extraction buffer. 2.1. Materials, page 3).
Regarding claim 3, Modified Eilam discloses the claimed invention as discussed above in claim 2. Abraham, after incorporation, discloses only ultracentrifugation step is performed at 4 degrees Celsius. Eilam discloses all the preparation and extraction procedures were conducted at 0 degrees Celsius (This preparation and all subsequent handling of extract or ribosomes were made at about 0 C unless otherwise stated. Preparation of Ribosomes and Polysomes, page 317). While Eilam does not teach a range that overlaps with 3-5 deg C, the ranges are close enough that one skilled in the art would have reasonable expectations that the compositions would have the same properties. The claimed lower level of the range is 3 deg C which is 3 deg C difference from the 0 deg C disclosed by Eilam. Therefore, a prima facie case of obviousness exists where the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have the same properties. See Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 227 USPQ 773 (Fed. Cir. 1985). Without showing unexpected results, the claimed range and range disclosed by Eilam are close enough that one skilled in the art would have a reasonable expectation of success. Therefore, it is Examiner’s position that it would have been obvious to one of ordinary skill in the art before the effective filing date to have derived the claimed range by routine optimization and experimentation based on Eilam’s disclosure.
Regarding claim 4, Modified Eilam discloses the claimed invention as discussed above in claim 1. Neither Eilam nor Abraham explicitly disclose the squeezing is performed for 1-5 min. As the squeezing time is dependent on the amount of sample, with more cucumber requires more time to squeeze, it is Examiner’s position that it would have been obvious to one of ordinary skill in the art to derive the exact squeezing time by routine experimentation and optimization. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In reAller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claim 5, Modified Eilam discloses the claimed invention as discussed above in claim 4. Neither Eilam nor Abraham provides an explicit numerical ratio for precipitate obtained in S6 and second PBS to be 1 microgram to 1-5 microliters. Abraham discloses the pellets are “resuspended in a small volume of PBS” (2.2.1. Preparation of Classic PEVs, page 3). It is Examiner’s position that it would have been obvious to one of ordinary skill in the art to derive the exact weight ratio by routine experimentation and optimization. Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." See MPEP 2144.05 and In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claim 6, Modified Eilam discloses the claimed invention as discussed above in claim 5. Abraham, after incorporation, discloses the filter membrane has a pore size of 0.45 microns (The supernatant after 15,000× g was filtered (0.45 µm) and centrifuged at 120,000…2.2.1. Preparation of Classic PEVs, page 3). However, Abraham discloses a 0.22-micron filter is used for filtering the PBS buffer (After adding the protease inhibitor cocktail, filtered (0.22 µm) PBS was added to reach a final volume of 1000 mL. 2.2.1. Preparation of Classic PEVs, page 3). While Abraham does not teach a range that overlaps with 0.22-micron for the filtering step before S6, the ranges are close enough that one skilled in the art would have reasonable expectations that the compositions would have the same properties. The claimed lower level of the range is 3 deg C which is 3 deg C difference from the 0 deg C disclosed by Eilam. Therefore, a prima facie case of obviousness exists where the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have the same properties. See Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 227 USPQ 773 (Fed. Cir. 1985). Without showing unexpected results, the claimed range and range disclosed by Abraham are close enough that one skilled in the art would have a reasonable expectation of success.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
CN113528505A discloses a rapid extraction and amplification method of cucumber gene DNA based on membrane adsorption (Abstract).
Isolation of Plant Polysomal mRNA by Differential Centrifugation and Ribosome Immunopurification Methods discloses differential centrifugation procedure to isolate ribosome from plant (Conventional Isolation of Polysomes).
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MICKEY HUANG whose telephone number is (571)272-7690. The examiner can normally be reached M-F 9:30-5:30 PM ET.
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/M.H./Examiner, Art Unit 1758
/MARIS R KESSEL/Supervisory Patent Examiner, Art Unit 1758
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