DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Response to Amendment
The amendment filed on 31 JULY 2026 has been entered. No changes to the claims were made.
Pending Claims: 1, 19, 21-22, and 61-75
Cancelled Claims: 2-18, 20, and 23-60
Response to Arguments
Applicant’s arguments filed on 31 JULY 2026 have been fully considered.
Applicant argues, regarding instant claims 1 and 66, that Berzofsky fails to teach the limitations “the ratio between the size-exclusion beady slurry and the total protein in the plasma sample is less than or equal to 2.5 µg protein per 1 µL size-exclusion bead slurry” of instant claim 1 and “the ratio between the size-exclusion beady slurry and the total protein in the CSF sample is less than or equal to 5 µg protein per 1 µL size-exclusion bead slurry” of instant claim 66. Berzofsky teaches the introduction of bovine serum albumin (BSA) to a resin rather than a slurry containing the resin and then teaches the incubation of isolated BSA samples at varying concentrations rather than extracellular vesicle containing samples. Therefore, one of ordinary skill in the art would not have assumed that the BSA protein to resin ratios could be applied to total protein in a sample containing extracellular vesicles and instant claims 1 and 66 are allowable (Arguments filed 31 JULY 2026, Page 6 to Page 7, Paragraph 2 and Page 14, Paragraph 3 to Page 15, Paragraph 1). Applicant’s argument is not found persuasive. Firstly, the general teaching of Berzofsky is to the purification of extracellular vesicles (Abstract) and the cited BSA testing is an experiment to test the binding capacity of the resin. The binding capacity is the maximum binding amount of the resin and the stated 450 µg of BSA removed by 100 µL of resin (Paragraph 0135) would be used as an upper bound for total protein removal, as using a higher protein loading amount would result in failing to remove all of the protein desired to be removed. Next, the chromatography resin is taught to be in a slurry in example 2 which describes the resin being stored suspended in 20% ethanol and being added to well plates and then exchanged with Dulbecco’s phosphate-buffered saline (DPBS) prior to the sample being mixed with each well (Paragraphs 0126-0127). The missing element from Berzofsky is the particular ratio of size-exclusion beads to buffer solution, which is taught by Schlegl. Therefore, Berzofsky makes obvious the limitations “the ratio between the size-exclusion beady slurry and the total protein in the plasma sample is less than or equal to 2.5 µg protein per 1 µL size-exclusion bead slurry” and “the ratio between the size-exclusion beady slurry and the total protein in the CSF sample is less than or equal to 5 µg protein per 1 µL size-exclusion bead slurry” and so instant claims 1 and 66 are not allowable.
Applicant argues, regarding instant claims 1 and 66, that the teachings of Berzofsky are too broad because Berzofsky teaches a mixed mode resin composition rather than only a size-exclusion bead resin and an array of ranges for the size exclusion resin cut-off. Therefore, Berzofsky contains insufficient detail and one of ordinary skill in the art would not have been able to determine the type of resin used in the aforementioned BSA experiment and so instant claims 1 and 66 are allowable (Arguments filed 31 JULY 2026, Page 7, Paragraph 3 to Page 9 and Page 15, Paragraph 2 to Page 16, Paragraph 3). Applicant’s argument is not persuasive because the instant claim uses comprising language for the size-exclusion bead resin, which Berzofsky does teach as part of the composition. Furthermore, the size cut-off listing for the size-exclusion beads in Berzofsky is only six different size cut-offs of 1000 kDa, 900 kDa, 800 kDa, 700 kDa, 600 kDa, and 500 kDa (Paragraph 0041) with claim 33 explicitly teaching the use of the 700 kDa cut-off while the size cut-off is taught to remove small unbound molecules (Paragraph 0018) which means that this element is optimizable based upon the sample and the impurities that one is trying to remove said samples. Therefore, Berzofsky teaches the use of the size-exclusion resin with enough specificity for one of ordinary skill in the art to replicate the results and so instant claims 1 and 66 are not allowable.
Applicant argues that the results achieved by the methods of instant claims 1 and 66 are unexpected because it would not be expected for the purified extracellular vesicle samples to lack detectable albumin while being high-yield and high-purity because albumin is the most abundant protein in human blood plasma and the second resin mode taught by Berzofsky is known to use antibodies which non-specifically bind albumin and result in a low extracellular vesicle yield. Furthermore, immunoaffinity depletion is significantly more expensive than other separation methods and so the methods of instant claims 1and 66 are a significant improvement over one of the most effective extracellular purification techniques used in the art at the time of filing the instant application. Therefore, instant claims 1 and 66 are allowable (Arguments filed 31 JULY 2026, Page 10 to Page 12, Paragraph 2 and Page 16, Paragraph 4 to Page 18, Paragraph 3). Applicant’s argument is not found persuasive because Applicant is arguing that the elements of high-yield, high purity, and size-exclusion beads which explicitly don’t include anti-body containing resins create unexpected results, none of which are recited in instant claims 1 and 66. Applicant may amend instant claims 1 and 66 to include such limitations, but it is noted that the features upon which applicant relies are not recited in the rejected claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Furthermore, Berzofsky explicitly used bovine serum albumin which is used to approximate human albumin in experimental models. Therefore, instant claims 1 and 66 are not allowable.
Applicant argues that instant claims 19, 21-22, 61-65, and 72-73 are allowable for the same reasons as instant claim 1 above because these claims depend upon instant claim 1 (Arguments filed 31 JULY 2026, Page 12, Paragraph 3 to Page 14, Paragraph 2) and instant claims 67-71 and 74-75 are allowable for the same reasons as instant claim 66 above because these claims depend upon instant claim 66 (Arguments filed 31 JULY 2026, Page 18, Paragraph 4 to Page 19). Regarding Applicant’s argument, instant claims 1 and 66 are not allowable for the reasons described above and so instant claims 19, 21-22, 61-65, and 67-75 are also not allowable.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claims 1, 61-64, and 72-73 are rejected under 35 U.S.C. 103 as being unpatentable over Baranyai T, Herczeg K, Onódi Z, Voszka I, Módos K, Marton N, et al. (2015) Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods. PLoS ONE 10(12): e0145686. doi:10.1371/journal.pone.0145686 (hereinafter Baranyai) in view of Berzofsky et al International Patent Application No. WO 2019133842 A1 (hereinafter Berzofsky) in view of Schlegl et al International Patent Application No. WO 2016156613 A1 (hereinafter Schlegl).
Regarding Claim 1, Baranyai teaches that it is possible to use size exclusion chromatography to isolate exosomes from blood plasma (i.e., a method for purifying extracellular vesicles from a plasma sample, the method comprising) without significant albumin contamination but that the process has a low yield of vesicles (Pages 1-2, Abstract)
wherein the blood plasma is loaded into chromatography columns (i.e., (a) subjecting the plasma sample to a size exclusion chromatography (SEC) column)
and the eluted fractions are collected (i.e., (b) collecting fractions containing the extracellular vesicles from the SEC column; Pages 3-4, Isolation of exosomes from blood plasma with SEC). Baranyai further teaches that adjusting column characteristics such as medium type and adjusting the supplied amount of sample might improve yields of SEC methods (Page 10, Paragraph 2 to Page 11, Discussion).
Baranyai does not teach (c) combining the fractions collected from the SEC column with size-exclusion beads capable of capturing molecules smaller than about 700 kDa to create a mixture, wherein the size-exclusion beads comprise an inactive exterior and a core with an octylamine ligand, wherein the ratio between the size exclusion bead slurry and the total protein in the plasma sample is less than or equal to 2.5 µg protein per 1 µL size exclusion bead slurry, and (d) separating and removing the size-exclusion beads from the mixture, such that a sample of the extracellular vesicles remains, wherein the sample of the extracellular vesicles lacks detectable albumin, thereby purifying the extracellular vesicles from the plasma sample.
However, Berzofsky teaches a method of purifying extracellular vesicles (Paragraph 0006) from a biofluid sample that can be plasma (Paragraph 0022) where, prior to the start of the method, the sample can be filtered to remove some unbound molecules (Paragraphs 0021) that comprises mixing a resin composition that traps particles by a size exclusion mechanism with the sample (i.e., (c) combining the fractions collected from the SEC column with size-exclusion beads; Paragraph 0006) where the beads can be modified to capture small unbound molecules with a molecular weight cut off for entry of less than or equal to 700 kDa (i.e., capable of capturing molecules smaller than about 700 kDa to create a mixture; Paragraph 0018),
where the size exclusion resin comprises beads with a porous core comprising at least one affinity ligand and a porous shell wherein the porous shell of the bead is non-functionalized (i.e., wherein the size-exclusion beads comprise an inactive exterior; Paragraph 0029), that the affinity ligand can be tri-octyl amine (i.e., and a core with an octylamine ligand; Paragraph 0046),
wherein about 150 µg and 50 µg of bovine serum albumin is removed completely from a sample after 60 minutes of incubation with 100 µL of the resin (i.e., wherein the ratio between the size exclusion bead slurry and the total protein in the plasma sample is less than or equal to 2.5 µg protein per 1 µL size exclusion bead slurry; Fig. 4, Paragraph 0135),
wherein the unbound molecules are trapped in the resin composition and then the sample is separated from the resin composition to obtain extracellular vesicles at a higher concentration than prior to mixing the resin composition (i.e., and (d) separating and removing the size-exclusion beads from the mixture, such that a sample of the extracellular vesicles remains, thereby purifying the extracellular vesicles from the plasma sample; Paragraph 0006) with explicit description of albumin being one of the unbound molecules (Paragraph 0024) and a graph demonstrating the capture of albumin down to zero or nearly zero (i.e., wherein the sample of the extracellular vesicles lacks detectable albumin; Fig. 4, Paragraph 0135) where it is desirable to remove additional unbound molecules from size exclusion chromatography for scaling up production for therapeutics (Paragraph 0004).
Berzofsky is analogous to the claimed invention because it pertains to a method of purifying extracellular vesicles in a sample (Abstract). It would have been obvious to one of ordinary skill in the art at the time of filing of the instant claimed invention to modify the method of isolating exosomes from blood plasma as taught by Baranyai with the size exclusion beads as taught by Berzofsky because the size exclusion beads would remove additional unbound molecules leftover from size exclusion chromatography for scaling up of production.
Baranyai in view of Berzofsky does not teach wherein the size-exclusion beads are suspended in an equal volume of a buffer to create a slurry.
However, Schlegl teaches the use of a chromatography resin prepared in a 50% slurry (i.e., wherein the size-exclusion beads are suspended in an equal volume of a buffer to create a slurry) with phosphate buffered solution (PBS) with CAPTO® Core 700 resin (i.e., size-exclusion beads) slurry showing a significant reduction of residual impurities (Page 20, Lines 22-33).
Schlegl is analogous to the claimed invention because it pertains to the separation of small biological particles from biological samples (Page 1). It would have been obvious to one of ordinary skill in the art at the time of filing of the instant claimed invention to modify the resin composition made obvious by Baranyai in view of Berzofsky with the phosphate buffered solution slurry as taught by Schlegl because the slurry would significantly reduce residual impurities in the sample.
Regarding Claim 61, Schlegl further teaches the use of a chromatography resin prepared in a 50% slurry with phosphate buffered solution (PBS) (i.e., wherein the buffer is PBS buffer) with CAPTO® Core 700 resin (i.e., size-exclusion beads) slurry showing a significant reduction of residual impurities (Page 20, Lines 22-33).
Regarding Claim 62, Berzofsky further teaches contacting (i.e., mixed) the sample with the resin composition and then separating the sample and the resin composition (i.e., wherein the mixture is mixed prior to separating and removing the size-exclusion beads; Abstract).
Regarding Claim 63, Berzofsky further teaches that the contact time is from 1 hour or less and from 30 minutes or more (i.e., wherein the mixture is mixed for between 30 minutes and 1 hour; Paragraph 0051).
Regarding Claim 64, Berzofsky further teaches in an example that, after mixing the sample and the resin composition, the tubes were spun in a centrifuge and the resin forms a pellet (i.e., wherein the size-exclusion beads are separated from the mixture using centrifugation; Paragraph 0140).
Regarding Claim 72, Baranyai further teaches the use of western blot to determine the level of albumin relative to the level of vesicles in order to assess the efficiency of the isolation (Page 5, Western blot; Pages 7-9, Yield of exosome isolation with SEC and purity of isolates). Berzofsky further teaches that western blotting is not impacted by the methods for downstream analysis (i.e., wherein after separating and removing the size exclusion beads from the mixture, the method further comprises measuring the level of albumin in the sample of the extracellular vesicles by a Western blot assay; Paragraph 0078).
Regarding Claim 73, Berzofsky further teaches wherein about 150 µg of bovine serum albumin is removed completely from a sample after 60 minutes of incubation with 100 µL of the resin (i.e., wherein the ratio between the size exclusion bead slurry and the total protein in the plasma sample is 2.5 µg protein per 1 µL size exclusion bead slurry to 0.6 µg protein per 1 µL size exclusion bead slurry; Fig. 4, Paragraph 0135).
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Baranyai in view of Berzofsky in view of Schlegl as applied to claim 1 above, and further in view of Hof et al US Patent Application No. 20170052154 A1 (hereinafter Hof).
Regarding Claim 19, Baranyai further teaches that the chromatography columns have matrices of Sepharose 2B and Sepharose CL-4B (Pages 3-4, Isolation of exosomes from blood plasma with SEC) which are matrices of agarose with crosslinking of 2% and 4%, respectively, and acknowledges that adjusting column characteristics such as medium type might improve yields of SEC methods (Page 10, Paragraph 2 to Page 11, Discussion).
Baranyai in view of Berzofsky in view of Schlegl does not explicitly teach wherein the size exclusion chromatography column comprises a stationary phase comprising a 6% cross-linked agarose size exclusion chromatography base matrix.
However, Hof teaches that agarose resins are typically known to contain 2-6% crosslinked agarose by weight for use in size exclusion chromatography because the agarose porous channels are large enough to allow biomolecules to pass through (i.e., wherein the size exclusion chromatography column comprises a stationary phase comprising a 6% cross-linked agarose size exclusion chromatography base matrix; Paragraph 0097).
Hof is analogous to the claimed invention because it pertains to chromatography columns to separate biomolecules (Abstract). Hof teaches a range of 2%-6% crosslinked agarose for size exclusion chromatography, while Baranya teaches the use of 2% and 4% crosslinked agarose matrices and that it is known to vary the medium to optimize yield. Baranyai in view of Berzofsky in view of Schlegl in view of Hof does not explicitly teach a 6% cross-linked agarose size exclusion chromatography base matrix. However, a prima facie case of obviousness exists for claimed ranges that overlap or lie inside ranges disclosed by prior art (In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976))(See MPEP 2144.05(I)). It would have been obvious to one of ordinary skill in the art at the time of filing of the instant claimed invention to have selected the 6% cross-linked agarose when experimenting with the range made obvious by Baranyai in view of Berzofsky in view of Schlegl in view of Hof.
Claims 21-22 are rejected under 35 U.S.C. 103 as being unpatentable over Baranyai in view of Berzofsky in view of Schlegl as applied to claim 1 above, and further in view of Frechet et al US Patent No. 5316680 A (hereinafter Frechet).
Regarding Claim 21, Baranyai further teaches that only albumin was tracked as far as contaminants are concerned, and so there are likely other soluble serum proteins present (Page 11, Paragraph 2, Discussion). Berzofsky further teaches that the filtering of the sample may be desired to remove some of the unbound molecules prior to the first step of the method (i.e., prior to combining with the size-exclusion beads capable of capturing molecules smaller than about 700 kDa; Paragraph 0021).
Baranyai in view of Berzofsky in view of Schlegl does not teach wherein the sample is further subjected to a cation exchange chromatography resin after the size exclusion chromatography column.
However, Frechet teaches size exclusion chromatography combined with ion exchange used consecutively (i.e., further subjected to an ion exchange chromatography resin after the size exclusion chromatography column; Col. 6, Line 53 to Col. 7, Line 6) where the ion chromatography separates ionic species, typically low molecular weight cations (i.e., a cation exchange chromatography resin; Col. 2, Lines 26-32) for the purpose of separating molecules that have different sizes and polarities from samples with multiple different molecules (Col. 5, Lines 27-34).
It would be obvious to one of ordinary skill in the art to modify the method made obvious by Baranyai in view of Berzofsky in view of Schlegl with the prefilter chromatography as taught by Frechet because it would reduce the amount of unbound molecules from the sample by size and polarity prior to purifying the extracellular vesicles.
Regarding Claim 22, Baranyai in view of Berzofsky in view of Schlegl in view of Frechet makes obvious the method of claim 21. Frechet further teaches that the fixed ions of the ion exchange are from acidic groups such as carboxyl and sulfonate (i.e., wherein the cation exchange chromatography resin comprises a stationary phase comprising a functional group selected from the group consisting of sulfonate; Col. 2, Lines 15-25).
Claim 65 is rejected under 35 U.S.C. 103 as being unpatentable over Baranyai in view of Berzofsky in view of Schlegl as applied to claim 64 above, and further in view of James KT, Cooney B, Agopsowicz K, Trevors MA, Mohamed A, Stoltz D, Hitt M, Shmulevitz M. Novel High-throughput Approach for Purification of Infectious Virions. Sci Rep. 2016 Nov 9;6:36826. doi: 10.1038/srep36826. PMID: 27827454; PMCID: PMC5101806 (hereinafter James).
Regarding Claim 65, Baranyai in view of Berzofsky in view of Schlegl does not teach wherein the centrifugation is about 600g, 700g, 800g, or 900g.
However, James teaches the purification of a resin bead slurry by subjecting the sample to centrifugation at 800 x g for 10 minutes after adding the slurry to the sample (i.e., wherein the centrifugation is about 800g; Supplementary Materials and Methods, (C) Purification Capto700 Slurry, Pages 14-15) for the purpose of efficiently removing confounding cellular contaminants in a high-throughput manner (Abstract).
James is analogous to the claimed invention because it pertains to high-throughput chromatography with resin beads being added to a sample (Abstract). It would have been obvious to one of ordinary skill in the art at the time of filing of the instant claimed invention to modify the centrifugation step made obvious by Baranyai in view of Berzofsky in view of Schlegl to be performed at 800g as taught by James because it would efficiently remove confounding cellular contaminants in a high-throughput manner.
Claims 66-70 and 75 are rejected under 35 U.S.C. 103 as being unpatentable over Berzofsky in view of Schlegl.
Regarding Claim 66, Berzofsky teaches a method of purifying extracellular vesicles from unbound molecules (i.e., a method for purifying extracellular vesicles; Paragraph 0006) from a biofluid sample that can be cerebrospinal fluid (i.e., from a cerebrospinal fluid (CSF) sample, the method comprising; Paragraph 0022)
that comprises mixing a resin composition that traps particles by a size exclusion mechanism with the sample (i.e., (a) combining the CSF sample with size-exclusion beads; Paragraph 0006) where the beads can be modified to capture small unbound molecules with a molecular weight cut off for entry of less than or equal to 700 kDa (i.e., capable of capturing molecules smaller than about 700 kDa to create a mixture; Paragraph 0018),
where the size exclusion resin comprises beads with a porous core comprising at least one affinity ligand and a porous shell wherein the porous shell of the bead is non-functionalized (i.e., wherein the size-exclusion beads comprise an inactive exterior; Paragraph 0029), and that the affinity ligand can be tri-octyl amine (i.e., and a core with an octylamine ligand; Paragraph 0046),
wherein about 250 µg, 150 µg, and 50 µg of bovine serum albumin is removed completely from a sample after 60 minutes of incubation with 100 µL of the resin (i.e., wherein the ratio between the size exclusion bead slurry and the total protein in the CSF sample is less than or equal to 5 µg protein per 1 µL size exclusion bead slurry; Fig. 4, Paragraph 0135),
wherein the unbound molecules are trapped in the resin composition, and then the sample is separated from the resin composition to obtain extracellular vesicles at a higher concentration than prior to mixing the resin composition (i.e., and (d) separating and removing the size-exclusion beads from the mixture, such that a sample of the extracellular vesicles remains, thereby purifying the extracellular vesicles from the CSF sample; Paragraph 0006) with explicit description of albumin being one of the unbound molecules (Paragraph 0024) and a graph demonstrating the capture of albumin down to zero or nearly zero (i.e., wherein the sample of the extracellular vesicles lacks detectable albumin; Fig. 4, Paragraph 0135) where it is desirable to remove additional unbound molecules from size exclusion chromatography for scaling up production for therapeutics (Paragraph 0004).
Berzofsky does not teach wherein the size-exclusion beads are suspended in an equal volume of a buffer to create a slurry.
However, Schlegl teaches the use of a chromatography resin prepared in a 50% slurry (i.e., wherein the size-exclusion beads are suspended in an equal volume of a buffer to create a slurry) with phosphate buffered solution (PBS) with CAPTO® Core 700 resin (i.e., size-exclusion beads) slurry showing a significant reduction of residual impurities (Page 20, Lines 22-33).
Schlegl is analogous to the claimed invention because it pertains to the separation of small biological particles from biological samples (Page 1). It would have been obvious to one of ordinary skill in the art at the time of filing of the instant claimed invention to modify the resin composition as taught by Berzofsky with the phosphate buffered solution slurry as taught by Schlegl because the slurry would significantly reduce residual impurities in the sample.
Regarding Claim 67, Schlegl further teaches the use of a chromatography resin prepared in a 50% slurry with phosphate buffered solution (PBS) (i.e., wherein the buffer is PBS buffer) with CAPTO® Core 700 resin (i.e., size-exclusion beads) slurry showing a significant reduction of residual impurities (Page 20, Lines 22-33).
Regarding Claim 68, Berzofsky further teaches contacting (i.e., mixed) the sample with the resin composition and then separating the sample and the resin composition (i.e., wherein the mixture is mixed prior to separating and removing the size-exclusion beads; Abstract).
Regarding Claim 69, Berzofsky further teaches that the contact time is from 1 hour or less and from 30 minutes or more (i.e., wherein the mixture is mixed for between 30 minutes and 1 hour; Paragraph 0051).
Regarding Claim 70, Berzofsky further teaches in an example that, after mixing the sample and the resin composition, the tubes were spun in a centrifuge and the resin forms a pellet (i.e., wherein the size-exclusion beads are separated from the mixture using centrifugation; Paragraph 0140).
Regarding Claim 75, Berzofsky further teaches wherein about 250 µg of bovine serum albumin is removed completely from a sample after 60 minutes of incubation with 100 µL of the resin (i.e., wherein the ratio between the size exclusion bead slurry and the total protein in the plasma sample is 5.0 µg protein per 1 µL size exclusion bead slurry to 2.5 µg protein per 1 µL size exclusion bead slurry; Fig. 4, Paragraph 0135).
Claim 71 is rejected under 35 U.S.C. 103 as being unpatentable over Berzofsky in view of Schlegl as applied to claim 70 above, and further in view of James.
Regarding Claim 71, Berzofsky in view of Schlegl does not teach wherein the centrifugation is about 600g, 700g, 800g, or 900g.
However, James teaches the purification of a resin bead slurry by subjecting the sample to centrifugation at 800 x g for 10 minutes after adding the slurry to the sample (i.e., wherein the centrifugation is about 800g; Supplementary Materials and Methods, (C) Purification Capto700 Slurry, Pages 14-15) for the purpose of efficiently removing confounding cellular contaminants in a high-throughput manner (Abstract).
It would have been obvious to one of ordinary skill in the art at the time of filing of the instant claimed invention to modify the centrifugation step made obvious by Berzofsky in view of Schlegl to be performed at 800g as taught by James because it would efficiently remove confounding cellular contaminants in a high-throughput manner.
Claim 74 is rejected under 35 U.S.C. 103 as being unpatentable over Berzofsky in view of Schlegl as applied to claim 70 above, and further in view of Soares Martins T, Catita J, Martins Rosa I, A. B. da Cruz e Silva O, Henriques AG (2018) Exosome isolation from distinct biofluids using precipitation and column-based approaches. PLoS ONE 13(6): e0198820. https://doi.org/10.1371/journal.pone.0198820 (hereinafter Martins).
Regarding Claim 74, Berzofsky further teaches that western blotting is not impacted by the methods for downstream analysis (i.e., wherein after separating and removing the size exclusion beads from the mixture, the method further comprises measuring the level of albumin in the sample of the extracellular vesicles by a Western blot assay; Paragraph 0078).
Berzofsky in view of Schlegl does not explicitly teach wherein after separating and removing the size exclusion beads from the mixture, the method further comprises measuring the level of albumin in the sample of the extracellular vesicles by a Western blot assay.
However, Martins teaches characterization methods for the isolation of exosomes from cerebrospinal fluid samples by size exclusion chromatography involving Western blot analysis (Pages 1-2, Introduction; Page 5, Western blot analysis) where albumin is an explicitly chosen contaminant (i.e., wherein after separating and removing the size exclusion beads from the mixture, the method further comprises measuring the level of albumin in the sample of the extracellular vesicles by a Western blot assay; Fig. 7, Page 10, Paragraphs 1-2, Exosome purity and immunoblotting analysis) for the purpose of analyzing the purity of exosome preparation methods (Page 1, Abstract).
Martins is analogous to the claimed invention because it pertains to the isolation of exosomes from cerebrospinal fluid (Pages 1-2, Introduction). It would have been obvious to one of ordinary skill at the time of filing of the instant claimed invention to modify the method made obvious by Berzofsky in view of Schlegl to measure exosome and albumin concentration of purified samples as taught by Martins because the measurement would determine the purification effectiveness of the exosome purification method.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/A.A.G./ Examiner, Art Unit 1772
/Ryan B Huang/ Primary Examiner, Art Unit 1772
