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 .
Status of the Application
Applicant’s amendment and remarks submitted on 11/14/2022 and 12/03/2025 are acknowledged.
Claims 20-38 are pending.
Claims 1-19 are canceled.
Claims 20-38 are new.
Claims 20-38 have been examined on the merits.
Applicant’s election of the protease species in the response filed on 12/03/2025 in response to the species requirement set forth in the prior office action is acknowledged.
Upon searching and further consideration, Examiner has rejoined the species of trypsin to be examined with the elected species of protease. The remaining of species requirements is maintained.
Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.03(a)).
Specification
The specification is objected to for not complying with sequence rules. Applicants are required to insert sequence identifiers in front of sequences referred to in the specification (for example: see page 14/lines 1-4; and page 22/lines 21 and 30). Applicant is requested to make the appropriate changes.
Priority
This application, U.S. Application number 17/925238, is a national stage entry of International Application Number PCT/GB2021/051168, filed on 05/14/2021, which claims for foreign priority under 35 U.S.C. 119(a)-(d) to foreign application number UK 2007249.2 filed on 05/15/2020.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 11/14/2022 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97., and has been considered by the examiner.
Drawings
The drawings submitted on 11/14/2022 have been reviewed and are accepted by the Examiner for examination purposes.
Claim Objections
Claim 1 is objected to due to the recitation of “loading a first plurality of microdroplets … wherein each of the first microdroplet contains a microbead …; loading a second plurality of microdroplets … wherein each of the second microdroplet contains an adherent cell …; merging the first plurality of microdroplets and the second plurality of microdroplets to form a plurality of merged microdroplets, each merged microdroplets containing …”. It is noted that plural and singular forms of microdroplet as well as first/second plurality and first/second microdroplet are used interchangeably in the claim. To make the terms to be consistent with each other, the recited phrase should be changed or corrected to: “loading a plurality of first microdroplets … wherein each of the first microdroplets contains a microbead …; loading a plurality of second microdroplets … wherein each of the second microdroplets contains an adherent cell …; merging the plurality of the first microdroplets and second microdroplets to form a plurality of merged microdroplets, each merged microdroplet containing …”. It is noted that dependent claims 22, 26, and 31 recite the same terms, which should be changed accordingly. Appropriate correction is required.
Claim 24 is objected to for not complying with sequence rules. Applicants are required to insert sequence identifiers in front of each of the sequences: Gly-Arg-Gly-Asp-Ser (GRGDS) and Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) recited in the claim. Furthermore, the claim is objected to due to the recitation of “the peptide surface functionalisation comprises one or more … sequences … (GRGDSP)”. It is noted that the amino acid sequences recited in the claim are comprised in a peptide, not in a surface functionalisation. The recited peptide surface functionalisation should be changed to the peptide or the polypeptide. Appropriate correction is required.
Claim 38 is objected to due to the recitation of “each merged microdroplets”. The recited limitation should be corrected to “each of the merged microdroplets” or of “each merged microdroplet”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b), or 112, Second Paragraph
The following is a quotation of 35 U.S.C. 112(b):
(B) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 26-28, 31, 34, and 38 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention.
Claim 26 is indefinite due to the recitation of “to determine the contents of the microdroplets and the number of beads or cells per microdroplet”. There is no sufficient antecedence for the terms “the contents of the microdroplets” and “the number of beads or cells” in the claim. In addition, there are two distinct microdroplets recited in the claim: first microdroplets and second microdroplets. It is unclear whether the recited limitation “the contents of the microdroplets” refers to contents of first microdroplets or to contents of second microdroplets.
Claim 31 is indefinite due to the recitation of “a coupling promoter”. The recited term “coupling promoter” is not defined in the specification. It is unclear whether the term refers to a promoter coupled to cells in the droplets, or it refers to a chemical material that promotes the merging/coupling of the first and second droplets in the claimed method.
Claim 34 is indefinite due to the recitation of “one or more of the following; trypsin, EDTA, protease, or citric acid”. It is noted that the recited alternative term “or" makes the claim indefinite because it is not clear what the Markush group consisting of. For the purpose of examination, the phrase is interpreted as “one or more of trypsin, EDTA, protease, and citric acid”.
Claim 38 recites the limitation “the surface of the treated well plate”. There is no sufficient antecedent basis for this limitation in the claim.
The remaining claims are rejected for depending from an indefinite claim.
Claim Rejections - 35 USC § 102/103
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale or otherwise available to the public before the effective filing date of the claimed invention.
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 of this title, 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.
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 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.
Claims 20-22, 25-27, 31-33 and 35-38 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by or, in the alternative, under 35 U.S.C. 103 as obvious over Pollack et al. (US 2009/0155902, 2009, cited in IDS).
Pollack et al. teach a method for manipulating a cell (i.e. an adherent cell) in a droplet/microdroplet system by conjugating the cell to a bead/microbead, comprising steps: (i) providing a droplet actuator; (ii) loading sample droplets/microdroplets onto the droplet actuator, the sample droplets comprising cells and a fluid; (iii) loading bead droplets/microdroplets onto the droplet actuator, the bead droplets comprising one or more beads/microbead having affinity for cells of a specific cell type as well as a fluid; (iv) conducting one or more droplet operations to combine/merge the bead droplets with the sample droplets to obtain merged droplets/microdroplets comprising the cells, beads and fluids, thereby permitting cells of the specific cell type to bind to the beads/microbeads; (v) conducting a droplet-based washing protocol to separate the cells bound to the beads from cells not bound to the beads (when there are cells of other cell type(s) not bound to the beads); and (vi) growing cells bound to the beads in merged droplets/microdroplets (Figs. 7-8, Claim 4, paras 0051-0054, 0017-0021, and 0006); wherein the droplet actuator is a micro-actuator comprised in a microfluidic system/chip (first half of para 0019), thus the sample and bead droplets being loaded into a microfluidic space of the system; wherein the one or more droplet operations comprise merging the bead droplets and the sample droplets by transporting these two sets of droplets/microdroplets into contact with each other on the droplet actuator (paras 0020: right col/lines 1-8, and 0054); wherein the droplets/microdroplets are aqueous droplets (i.e. containing a fluid of water) and filler fluid associated with droplets is oil (paras 0018/line 5-6 and 0021/lines 5-6) and the fluid in the droplets includes water, saline solutions, acidic solutions, basic solutions, buffers and/or other reagent involved with biochemical protocol or biological fluids (para 0018: page 2/right col/last 4 lines and page 3/first 4 lines); wherein the manipulation of droplets/microdroplets by the droplet actuator is electrowetting mediated (paras 0019, 0052/lines 3-4, and 0054/lines 1-4; Figs. 7-8); and wherein the beads/microbeads are magnetically responsive or non-magnetically responsive (paras 0017, 0051, 0053, and 0056).
Regarding the step of “agitating each of the merged microdroplets …” recited in the claim 20, the specific embodiment and claims of Pollack et al. do not expressively teach a step of agitating merged microdroplets. However, they teach that conducting droplet operations to merge the bead droplets and sample droplets to obtain merged microdroplets, thereby permitting cells to bind to microbeads; and the droplet operations comprise moving the bead droplets and sample droplets into contact with each other on the droplet actuator for merging these droplets, as indicated above. It is noted that the movement of microdroplets into contact with each other would cause agitation of moved microdroplets and agitation of the microdroplets being merged, and such agitation would be carried over to the merged microdroplets. Thus, it appears that the method of Pollack et al. inherently comprises a step of agitating each of the merged microdroplets, as required by the claim 20. However, even if the droplet movement does not cause agitation of each of merged microdroplets, in which case there is no anticipation, it would at least have been obvious to include agitating each of merged microdroplets as one of the droplet operations in the method of Pollack et al. for promoting cells and microbeads to move and contact with each other, thus the binding/adhering the cells to the microbeads, because Pollack et al. (in para 0020/lines 1-2 and 8-9) specifically teach that their droplet operations comprise agitating droplets on the droplet actuator.
Regarding the claim 21, Pollack et al. teach performing a culturing or assaying process on cells adhered/bound to the microbeads (Claims 16 and 19; paras 0054, 0060, and 0018/last 4 lines).
Regarding the claim 22, the microfluidic space used in the method of Pollack et al. is a part of a microfluidic chip, which is configured to manipulate the bead droplets (reading on the claimed “first droplets”) and sample droplets (reading on the claimed “second droplets”) via electrowetting mediation, as indicated above. Pollack et al. further teach the electrowetting mediation is opto-electrowetting mediated (i.e. optically mediated electrowetting) (para 0019: lines 10-11 from the bottom), thus meeting the claimed limitation.
Regarding the claim 25, the cells in the method of Pollack et al. are adherent cells in their native adherent state, which grow on the support of beads (para 0054).
Regarding the claims 26 and 27, Pollack et al. teach inspecting the microdroplets prior to the merging by sorting the droplets, and discarding droplets not having a desired cell counts (paras 0038, 0039/lines 1-4, and 0040).
Regarding the claim 31, the recited limitation “coupling promoter” is not defined in the specification. Examiner notes that any component that facilitates merging of the first and second droplets can be considered as a coupling promoter. As such, even the water comprised in the aqueous droplets of Pollack et al. can be considered as a coupling promoter, because it provides an aqueous phase that facilitates contacting and merging of the bead/first droplets and cell/second droplets of Pollack et al. Thus, the teachings of Pollack et al. meet the claimed limitation.
Regarding the claims 32 and 33, Pollack et al. teach cell-containing droplets are provided with a fluid comprising cell growth/culture medium for culturing cells (paras 0008/line 4-5, 0054-55); and Pollack et al. further teach introducing a reagent droplet (i.e. a carrier phase) comprising a lysis reagent into the microfluidic space for treating cells in cell-containing droplets and further performing analysis on the cells, such as nucleic acid amplification, affinity-based or enzymatic assay, and sequencing (paras 0048/lines 5-6 and last 3 lines, and 0018/last 6 lines) (Note: the lysis reagent of Pollack et al. can be considered as a releasing agent recited in the claim 32 because it lyses and releases adherent cells from beads and the instant claim does not require intact cells are released from beads). Given Pollack et al. teach that a fluid in cell-containing droplets comprising cell-bound beads can be readily depleted and exchanged/replaced with a reagent fluid in reagent droplets (paras 0056-57), it would have been obvious to deplete the cell growth medium from the microdroplets containing cells adhering to microbeads and then exchange and replace it with a lysis reagent from the reagent droplets/carrier phase in the method of Pollack et al., such that the cells adhered to the beads can be released and lysed for performing analysis on the cells.
Regarding the claim 35, Pollack et al. teach incubating the merged microdroplets at a controlled temperature and monitoring growth of cells bound to the microbeads of each of the merged microdroplets (para 0054, Fig. 8).
Regarding the claim 36, Pollack et al. teach performing an on-chip reporter assay on merged microdroplets by using material extract from the cells with a droplet-based protocol, for determining gene expression, presence of proteins produced, and enzymatic activity of the proteins of the cells (para 0060).
Regarding the claims 37 and 38, Pollack et al. teach that cells in the merged microdroplets bind to the microbeads because the beads have an affinity for cells of a particular cell type (para 0054/lines 8-11); and cells-containing droplets are transported/deposited into a cell culture reservoir (i.e. a receptacle) or a well for culturing cells (para 0055/lines 1-6). Examiner notes that it is a common practice in the art to deposit cells into a well of a treated well plate (e.g. sterile well plate) for depositing or culturing cells. As such, the claims would have been obvious over Pollack et al.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
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 of this title, 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.
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 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.
Claims 23 and 24 are rejected under 35 U.S.C. 103 as obvious over Pollack et al. (US 2009/0155902, 2009, cited in IDS), as applied to Claims 20-22, 25-27, 31-33 and 35-38, further in view of Ingber et al. (WO 94/25487, 1994, cited in IDS).
The teachings of Pollack et al. are described above.
Regarding Claims 23 and 24, Pollack et al. do not teach that a surface of the microbeads is coated with a peptide comprising the amino acid sequence RGD to form a surface functionalization for facilitating cell adhesion. However, Pollack et al. further teach the beads are coated on surface and they have an affinity to cells of a particular cell type and specifically adhere/bind to the cells (page 2: left col/last line and right col/first line; paras 0052/lines 9-11 and 0054/lines 9-11).
It would have been obvious to coat the microbeads of Pollack et al. with a RGD-containing peptide for modifying surface functionalization of the beads for facilitating cell adhesion, thus arriving at the beads having an affinity to a particular cell type required by the method of Pollack et al. for binding cells of the particular cell type in merged microdroplets. This is because it is a common practice in the art to coat beads for modifying their surface functionalization and it is well known in the art that microbeads coated with a RGD-containing peptide adhere/bind to cells of a particular cell type. Furthermore, the RGD-coated microbeads have an advantage of exhibiting less non-specific clumping during magnetic pelleting. In support, Ingber et al. teach using microbeads coated a RGD-containing peptide for binding adherent cells of a specific cell type, i.e. eukaryotic cells such as mammalian, insect, or plant cells, for isolating adhesion complex (FACs) formed between the RGD and cells from the surface of the beads, wherein the RGD-containing peptide/protein provide an attachment site for the beads to bind/ligate integrins of the cells (page 4/lines 13-22, page 12/lines 10-32, page 10/lines 8-13, page 26/lines 6-13, page 30/lines 19-21). Ingber et al. further teach RGD-coated microbeads are chosen because they exhibit less non-specific clumping during magnetic pelleting (pages 11/lines 15-18, and 27/lines 9-11).
Examiner notes that the mammalian, insect, and plant cells taught by Ingber et al. are cells in their inactive adherent state, meeting the limitation of Claim 25.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 28-30 and 34 are rejected under 35 U.S.C. 103 as obvious over Pollack et al. (US 2009/0155902, 2009, cited in IDS), as applied to Claims 20-22, 25-27, 31-33 and 35-38, further in view of Pollack-2 et al. (US 2015/0329891, 2015, cited in IDS).
The teachings of Pollack et al. are described above.
Regarding Claims 28-30, Pollack et al. do not expressively teach discarding microdroplets not having a desired bead count, or merging and splitting, respectively, two or more microdroplets not having a desired bead count (below and at a threshold of bead count) for increasing and decreasing the cell count. However, Pollack et al. teach merging and splitting the droplets for changing contents of the droplets (e.g. changing a concentration or number of cells or beads), as described above and also see paras 0007, 0020, 0022, and 0046.
It would have been obvious to sort the bead microdroplets and exclude or discard the droplets not having a desired bead count prior to conducting the merging step in the method of Pollack et al. for ascertaining the bead droplets having a desired bead count are used for forming merged droplets, thus facilitating proper droplet operation and binding of cells to beads, wherein the bead droplets are either merged to increase the bead count (when the bead count is below a threshold level) or split to increase the bead count (when the count is at a threshold level), because it is well known in the art that sorting bead droplets allows bead droplets having a desired bead count to be selected and used in the merging step, and bead numbers in the droplets need to be maintained at a desired level for conducting proper droplet operations. In support, Pollack-2 et al. teach a method of manipulating droplets on a droplet actuator for separating and analyzing target substance, comprising a step of merging a bead-containing droplet with a target substance-containing sample droplet, wherein the target substance include cells (abstract, paras 0007 and 0022); and a step of sorting bead-containing droplets before the merging step to determine the bead content of the droplets, wherein droplets having a desired bead count are selected and droplets not having any beads or a desired bead count (a predetermined number of beads) are excluded (paras 0092: last 6 lines; and 0094). Furthermore, the techniques for merging droplets and splitting a single droplet are well established in the art, which would allow two or more bead droplets to be merged into a single droplet with an increased bead count as well as a bead droplet to be split into two or more daughter droplets with a decreased bead count, as supported by Pollack et al. described above as well as by Pollack-2 et al. (see paras 0006/lines 10-11, and 0024/lines 1-8).
Regarding Claim 34, Pollack et al. do not expressively teach that the releasing agent (lysis reagent) is a protease. However, it would have been obvious to apply a protease as the releasing agent (lysis reagent) in the method of Pollack et al. for lysing and releasing the cells from the microbeads and further analyzing cell components, because it is a common practice in the art to use the protease as a lysis reagent, as supported by Pollack-2 et al., who further teach using a lysis reagent for lysing target substance including cells and virus, wherein the lysis reagent is Proteinase K (paras 0099 and 0155/lines 2-5).
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Conclusion
No claim is in condition for allowance.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to Qing Xu, Ph.D., whose telephone number is (571) 272-3076. The examiner can normally be reached on Monday-Friday from 9:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath N. Rao, can be reached at (571) 272-0939. Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to the receptionist whose telephone number is (571) 272-1600.
/Qing Xu/
Patent Examiner
Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656