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 .
Claims 1-6, 8-10 and 12 are pending in this application, Claims 8-10 are acknowledged as withdrawn, Claims 1-6 and 12 were examined on their merits.
The objection to Claim 12 has been withdrawn due to the Applicant’s amendments to the claim filed 06/03/2026.
The rejection of Claims 1 and 12 under 35 U.S.C. § 103 as being unpatentable
over Gibbings et al. (US 2019/0093105 A1) and Konoshenko et al. (2018), Pall (2003)
and Benedikter et al. (2017), all of record, has been withdrawn due to the Applicant’s amendments to the claims filed 06/03/2026.
Claim Interpretation
Claim 1 now requires the contingent limitations, “when the biofluid is urine or CSF, enriching the biofluid down to a threshold volume; when the biofluid is serum, diluting the biofluid with a phosphate-buffered solution (PBS)”.
Consistent with the MPEP at 2111.04, II., the broadest reasonable interpretation of a method (or process) claim having contingent limitations requires only those steps that must be performed and does not include steps that are not required to be performed because the condition(s) precedent are not met. In this case, the biofluid may be blood or plasma, in which case neither contingent limitation would be required.
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.
Claims 1-6 and 12 are newly rejected under 35 U.S.C. § 103 as being unpatentable over Gibbings et al. (US 2019/0093105 A1) and Konoshenko et al. (2018), Pall (2003) and Benedikter et al. (2017), and further in view of Vlassov et al. (EP 3327440 A1), all of record, Choi et al. (WO2019/035057 A2), and Millipore (2018), of record, as necessitated by Applicant’s amendment to the claims filed 06/03/2026.
Gibbings et al. teaches a method for isolating exosomes from a culture media (a
biofluid, giving the term its' broadest reasonable interpretation) comprising enrichment
(increasing the concentration thereof) by tangential flow filtration (TFF) or centrifugal
filtration to eliminate cells, larger vesicles and debris then concentrating by TFF or other
filtration methods or by (immuno)affinity purification using antibodies coupled to beads;
and wherein the ordinary artisan could select a suitable collection or enriching technique (Pg. 7, Paragraph [0105]).
Gibbings et al. does not teach wherein the biofluid is selected from blood, urine, serum, plasma, and cerebrospinal fluid;
or the required number (3) of isolation methods (e.g. tangential flow filtration, centrifugal filtration and immunomagnetic affinity), the specific order of process steps; adding a buffer solution to the biofluid and performing TFF after the addition, then transferring the biofluid to a centrifugal filtration unit, wherein the centrifugal filtration unit includes a membrane having a 10 kDa molecular weight cutoff, applying centrifugal force to the biofluid in the centrifugal filter unit and capturing extracellular vesicles (EV) in the membrane, then transferring the captured EVs to an immunomagnetic affinity container with a plurality of magnetic beads, wherein the beads are magnetic and removing the magnetic beads and eluting/separating the EVs therefrom or wherein the isolated target extracellular vesicles (EV) includes at least 95% of the EVs from the biofluid sample, as now required by Claim 1;
wherein the biofluid is blood, as required by Claim 2;
wherein the biofluid is urine, and enriching the biofluid down to a threshold volume as now required by Claims 1 and 3;
wherein the biofluid is serum, diluting the biofluid with a phosphate-buffered solution (PBS) as now required by Claims 1 and 4;
wherein the biofluid is plasma, as required by Claim 5;
or wherein the biofluid is CSF, and enriching the biofluid down to a threshold volume as now required by Claims 1 and 6;
or wherein the centrifugal force is applied at no greater than 3,000g to the biofluid in the centrifugal filtration unit, as required by Claim 12.
Benedikter et al. teaches the isolation of EVs/exosomes using centrifugal filtration units with a 10 kDa molecular weight cutoff, applying centrifugal force of 4000 g to the biofluid in the centrifugal filter unit and capturing extracellular vesicles (EV) in the membrane (Pg. 10, Lines 40-43).
Pall teaches that TFF involves concentration/enrichment (Pg. 3, Column 2, Lines 7-29) and diafiltration, wherein in continuous diafiltration water or buffer is added to the sample feed (e.g. biofluid) reservoir at the same rate as filtrate is generated keeping volume in the sample reservoir constant (Pg. 4, Column 1, Lines 1-18).
Konoshenko et al. teaches the use of magnetic beads with antibodies to isolate exosomes from a liquid and separating the exosomes from the magnetic beads by elution (Pg. 18, Column 1, Lines 52-59).
Vlassov et al. teaches that exosomes may be isolated from biological fluids (Pg.
2, Paragraph [0007]] wherein the biological fluid may be: culture media, serum, plasma,
blood, urine or CSF (Pg. 3, Paragraph [0013]).
Choi et al. teaches that PBS is a suitable buffer for dilution of a sample prior to TFF (Pg. 13, Lines 1-4).
Millipore teaches that TFF systems have a minimal working volume required to
prevent air from getting into the system (Pg. 1, Lines 6-9).
It would have been obvious to those of ordinary skill in the art before the effective filing date of the claimed invention to modify the exosome isolation method of Gibbings et al. so that a step of tangential flow filtration is performed on an exosome containing biofluid, wherein buffer is added to the sample reservoir (thus to the biofluid) during TFF, as taught by Pall, followed by a step of centrifugal filtration at 4000g using centrifugal filtration units with a 10 kDa molecular weight cutoff, applying centrifugal force to the biofluid in the centrifugal filter unit and capturing extracellular vesicles (EV) in the membrane, as taught by Benedikter et al., followed by affinity purification, because the reference teaches these are suitable means for purifying and isolating exosomes and it would have been obvious to combine them all into a single method for the same purpose. Further, the selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.
See the MPEP at 2144.04, IV., C. Those of ordinary skill in the art would have been motivated to make this modification in order to have the purest, isolated exosomes from a sample. There would have been a reasonable expectation of success in making this modification because Gibbings et al. already teaches that exosomes can be isolated using centrifugal filtration, TFF and affinity purification, and a sequential performance of at least two of the methods.
It would have been further obvious to those of ordinary skill in the art before the effective filing date of the claimed invention to modify the exosome isolation method of Gibbings et al., Pall and Benedikter et al. to further utilize beads which are magnetic and removing the magnetic beads from the biofluid and eluting the exosomes therefrom, as taught by Konoshenko et al. because Gibbings et al. is generally drawn to affinity purification of exosomes by bead-bound antibodies and Konoshenko et al. provides a specific type of bead suitable for exosome isolation. Those of ordinary skill in the art would have been motivated to make this modification in order to further isolate desired exosomes from a liquid. There would have been a reasonable expectation of success in making this modification because at least Gibbings et al. is drawn to the use of affinity purification to isolate exosomes and Konoshenko et al. teaches that magnetic affinity purification is suitable for exosome purification.
It would have been further obvious to those of ordinary skill in the art to modify the method of Gibbings et al., Pall, Benedikter et al. and Konoshenko et al. of isolating
exosomes from culture media to apply the method to other biofluids such as those taught by Vlassov et al. because all of the samples are art-recognized sources of exosomes. See the MPEP at 2144.06, I. Those of ordinary skill in the art would have been motivated to make this modification due to the desire to isolate exosomes from a particular biofluid of interest. There would have been a reasonable expectation of success in making this modification because all of the biofluids are art-recognized equivalent sources from which exosomes may be isolated.
With regard to Claim 1, it would have been further obvious to those of ordinary skill in the art before the instant invention to isolate target EVs to include at least 95% of the EVs from the sample because while the Gibbings et al. reference is silent with regard to the percent purity of EVs isolated from the biofluid concentration of the DNAse in the composition, the determination of the optimal percent purity of EVs isolated by the method by routine experimentation and optimization of result effective variables is not inventive. In this instance, the concentration of EVs isolated from the biofluid will directly correlate with the percentage of EVs isolated from the sample. That is, a more efficient EV isolation method will isolate a higher percentage of EVs available from the biofluid.
Those of ordinary skill in the art before the effective filing date on the instant invention would have been motivated to make this modification in order to obtain the highest percentage of the desired EVs from the initial biofluid.
There would have been a reasonable expectation of success in making this modification because the Gibbings et al. reference already provides a method for isolating exosomes from a culture media/biofluid and the determination of result effective variables by routine optimization and experimentation is within the purview of those of ordinary skill in the art.
Absent any teaching of criticality by the Applicant concerning the percentage of EVs isolated from the biofluid, it would be prima facie obvious that one of ordinary skill in the art would recognize this limitation as an optimizable variable which can be met as a matter of routine optimization (see MPEP § 2144.05 (II)(B). Those of ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to make this modification in order to obtain the highest amount of desired EVs from the initial biofluid. There would have been a reasonable expectation of success in making these modifications because Gibbings et al. is drawn to a method for isolating desired EVs (exosomes) from a biological fluid.
While the references listed above do not specifically teach the limitations of Claims 1, 3 and 6 and 1&4 respectively; that when the biofluid is urine or CSF, enriching the biofluid down to a threshold volume; and when the biofluid is serum, diluting the biofluid with a phosphate-buffered solution (PBS), as taught by Choi et al. above, one of ordinary skill in the art would recognize that the selection of whether to concentrate or dilute a biofluid is a result-effective optimizable variable dependent on the volume of the sample.
For example, a large volume sample could be concentrated prior to TFF to reduce the processing time required for a large volume sample and a low volume sample could be diluted to ensure enough sample for the process and prevention of air introduction into the system, see Millipore (2018). This is motivation for someone of ordinary skill in the art to practice or test the sample concentration/enrichment or dilution parameters widely to find those that are functional or optimal to isolate EVs from a biofluid by TFF which then would be inclusive or cover the instantly claimed range. Absent any teaching of criticality by the Applicant concerning the concentration/enrichment or dilution of particular biofluid samples, it would be prima facie obvious that one of ordinary skill in the art would recognize this limitation as an optimizable variable which can be met as a matter of routine optimization (see MPEP § 2144.05 (II)(B). Those of ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to make this modification in order to obtain the desired EVs. There would have been a reasonable expectation of success in making these modifications because Pall teaches that TFF involves sample dilution with a buffer and Millipore teaches that TFF requires a certain sample volume to function.
While the references listed above do not specifically teach the limitation of Claim 12, that the centrifugal force applied to the biofluid in the centrifugal filtration unit at not more than 3,000g, one of ordinary skill in the art would recognize that the selection of centrifugation speed for a centrifugal filtration unit to isolate exosomes from a biofluid is a result-effective optimizable variable.
Benedikter et al. teaches the isolation of EVs/exosomes using centrifugal filtration units with a 10 kDa molecular weight cutoff, applying centrifugal force of 4000 g to the biofluid in the centrifugal filter unit and capturing extracellular vesicles (EV) in the membrane (Pg. 10, Lines 40-43). This is a general teaching utilizing a similar method and centrifuge speed as claimed. This is motivation for someone of ordinary skill in the art to practice or test the centrifugation speeds widely to find those that are functional or optimal to isolate EVs from a biofluid which then would be inclusive or cover the instantly claimed range. Absent any teaching of criticality by the Applicant concerning the centrifugation speed, it would be prima facie obvious that one of ordinary skill in the art would recognize this limitation as an optimizable variable which can be met as a matter of routine optimization (see MPEP § 2144.05 (II)(B). Those of ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to make this modification in order to obtain the desired EVs. There would have been a reasonable expectation of success in making these modifications because Benedikter et al. teaches the isolation of EVs/exosomes using centrifugal filtration units at a similar centrifugation speed.
With regard to Claim 7, Gibbings et al. teaches enrichment (concentration) of exosomes in a biofluid by TFF, the finding as obvious the use of an additional centrifugal filtration step, and the use of an immunoaffinity bead isolation step. Konoshenko et al. teaches the isolation of exosomes using magnetic immunoaffinity beads.
Thus, as the prior art performs the same method steps as claimed, the ordinary
artisan would expect a similar result as claimed with respect to the percentage of
isolated exosomes from the biofluid sample.
Response to Arguments
Applicant’s arguments, see Remarks, filed 06/03/2026, with respect to the objection and rejection withdrawn above have been fully considered and are persuasive.
The remaining arguments have been considered insofar as they apply to the pending rejections.
The Applicant argues that Gibbings fails to disclose isolation of EVs from the claimed biofluids and that the reference includes a cell and cell debris removal step which does not read on the instant TFF which removes impurities such as proteins and nucleic acids or centrifugal filtration step which does not require prior centrifugal cell debris removal steps not required by the claims. Applicant asserts that the reference does not teach a step of removing proteins, nucleic acids and other contaminants (Remarks, Pg, 6, Lines 26-30 and Pg. 7 and Pg. 8, Lines 1-15 and Pg. 9, Lines 3-15).
This is not found to be persuasive for the following reasons, as discussed above, Vlassov et al. teaches that exosomes may be isolated from biological fluids (Pg.
2, Paragraph [0007]] wherein the biological fluid may be: culture media (such as taught by Gibbings), serum, plasma, blood, urine or CSF (Pg. 3, Paragraph [0013]). Thus, the combination of Gibbings with Vlassov makes obvious the claimed limitations. Further, Gibbings teaches that exosomes can be isolated from a biofluid (culture media) by first enriching with TFF (Pg. 7, Paragraph [0105]) which would not require a cell/cell debris removal step and would inherently remove proteins, nucleic acids and other contaminants (even though these limitations are not found in the instant claims). The reference teaches that a centrifugal filtration step can be performed prior to another step of TFF or other filtration methods but does not teach that a cell/cell debris removal step is required in the method. Further, the instant “comprising” language does not preclude such a cell/ cell debris removal step.
The Applicant argues that both Gibbings and Benedikter require a debris removal step not required in the instant claims (Remarks, Pg. 8, Lines 16-28).
This is not found to be persuasive for the reasoning provided above, with regard to Gibbings, that is, Gibbings teaches that exosomes can be isolated from a biofluid (culture media) by first enriching with TFF (Pg. 7, Paragraph [0105]) which would not require a cell/cell debris removal step and would inherently remove proteins, nucleic acids and other contaminants (even though these limitations are not found in the instant claims) which contain open comprising language and do not specify that no debris removal step is performed.
The Applicant argues that neither Pall or Konoshenko remedy the alleged deficiencies of Gibbings and Benedikter (Remarks, Pg. 8, Lines 29-31 and Pg. 9, Lines 1-2).
This is not found to be persuasive for the reasoning provided above. The Examiner notes that Pall was cited only for its’ teaching that TFF involves concentration/enrichment (Pg. 3, Column 2, Lines 7-29) and diafiltration, wherein in continuous diafiltration water or buffer is added to the sample feed (e.g. biofluid) reservoir at the same rate as filtrate is generated keeping volume in the sample reservoir constant (Pg. 4, Column 1, Lines 1-18) while Konoshenko was cited only for its’ teaching of the use of magnetic beads with antibodies to isolate exosomes from a liquid and separating the exosomes from the magnetic beads by elution (Pg. 18, Column 1, Lines 52-59).
The Applicant argues that the method of Gibbings would be insufficient to achieve the claimed results as Gibbings discloses a filter with pores larger than 100 nm and typically of 0.22 µm which would fail to retain exosomes with diameters of less than 100 nm. Applicant opines the claimed method is suitable for purifying EVs with diameters larger or smaller than 100 nm (Remarks, Pg. 9, Lines 16-21).
In response to Applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which Applicant relies (i.e., retaining exosomes of any particular size) are not recited in the rejected claim(s). 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).
The Applicant argues that the teachings of Konoshenko would have discouraged the ordinary artisan from combining the references as suggested by the Examiner. Applicant notes that while Konoshenko discloses the use of immunomagnetic affinity separation of EVs, the disclosure would have allegedly encouraged density gradient separation because affinity separation is less efficient in blood and other body fluids and density gradient separation has advantages in such a separation. Applicant concludes the ordinary artisan would have been motivated to use density gradient separation in isolating exosomes from the claimed bodily fluids (Remarks, Pg. 9, Lines 22-31 and Pg. 10, Lines 1-27).
This is not found to be persuasive for the following reasons, as Applicant is no doubt aware, non-preferred embodiments such as the immunomagnetic affinity separation of EVs taught by Konoshenko are still prior art. See the MPEP at 2123, I. and II. There is no “teaching away” in the reference from using immunomagnetic affinity separation of EVs, merely that one method may work better over another in certain situations.
The Applicant argues that Vlassov does not remedy the alleged deficiencies of Gibbings, Benedikter, Pall and Konoshenko (Remarks, Pg. 10, Lines 29-33 and Pg. 11, Lines 1-5).
This is not found to be persuasive for the reasoning provided above. The Examiner notes that Vlassov was cited only for its’ teaching that exosomes may be isolated from biological fluids (Pg. 2, Paragraph [0007]] wherein the biological fluid may be: culture media, serum, plasma, blood, urine or CSF (Pg. 3, Paragraph [0013]).
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to PAUL C MARTIN whose telephone number is (571)272-3348. The examiner can normally be reached Monday-Friday 12pm-8pm EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Sharmila G Landau can be reached at (571) 272-0614. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/PAUL C MARTIN/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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