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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 18 FEBRUARY 2026 has been entered.
Claim Status
Rejected Claims: 1, 19, 21-22, and 61-75
Cancelled Claims: 2-18, 20, and 23-60
Response to Amendment
The amendment filed on 18 FEBRUARY 2026 has been entered.
In view of the amendment to the claims, the amendment of claims 1, 21, and 66 and the addition of new claims 72-75 have been acknowledged.
In view of the amendment to claim 21, the objections to the claims have been withdrawn.
In view of the amendment to claims 1 and 66, the rejections under 35 U.S.C. 103 have been modified.
Response to Arguments
Applicant’s arguments filed on 18 FEBRUARY 2026 have been fully considered.
Applicant argues, regarding instant claims 1 and 66, that the previous prior art does not teach the limitations 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 or wherein the ratio between the size exclusion bead slurry and the total protein in the plasma 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 allowable (Arguments filed 18 FEBRUARY 2026, Page 7 to Page 9, Paragraph 3 and Page 13, Paragraph 4 to Page 15).
Regarding Applicant’s argument, the Examiner has re-evaluated Berzofsky and found that Berzofsky teaches 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 (Fig. 4, Paragraph 0135) and that this teaching makes obvious the total protein to size exclusion bead slurry ratio as described in instant claims 1 and 66, therefore instant claims 1 and 66 are not allowable.
Applicant argues that the method of the instant claims 1 and 66 generate unexpected results of the sample of extracellular vesicles lacking detectable albumin and are therefore allowable (Arguments filed 18 FEBRUARY 2026, Page 9, Paragraph 4 to Page 11, Paragraph 2 and Page 16 to Page 17, Paragraph 2).
Regarding Applicant’s argument, Berzofsky teaches that the use of the same or similar size exclusion beads as described in the instant claims is capable of removing 90% or greater bovine serum albumin from a sample, with several samples appearing to remove all or nearly all of the bovine serum albumin (Fig. 4, Paragraph 0135). Therefore, the intended results of removing albumin are expected to one of ordinary skill in the art at the time of filing the instant claimed invention and therefore instant claim 1 is not allowable.
Applicant argues that claims 19, 21-22, 65, and 67-71 are allowable for the same reasons that instant claims 1 and 66 are allowable as described above because these claims depend upon claims 1 and 66 (Page 11, Paragraph 3 to Page 13, Paragraph 3 and Page 17, Paragraphs 3-8).
Regarding Applicant’s arguments, instant claims 1 and 66 are not allowable and so the dependent claims 19, 21-22, 65, and 67-71 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 its 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
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/A.A.G./ Examiner, Art Unit 1777
/IN SUK C BULLOCK/ Supervisory Patent Examiner, Art Unit 1772
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