DETAILED ACTION
Applicant’s amendment and Arguments/Remarks received on 03 February 2026 have been entered. Claims 1-7, 9, 11-12, 14-15, 17-21, and 23-25 were previously pending in the application. No claims have been cancelled, and no new claims have been added by Applicant. Claims 1-7, 9, 11-12, 14-15, 17-21, and 23-25 are currently pending in the application. Claims 1 and 20 are independent claims.
The following election of species remains in effect in the instant application:
Vitrification medium components: a. Lysing agent;
Vitrification agents: c. a sugar;
Lysing agent detergents: b. 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (e.g., TritonTM X-100).
Claims 20-21 and 23-25 remain withdrawn from consideration as being directed to a nonelected species, there being no allowable generic or linking claim.
Claims 1-7, 9, 11-12, 14-15, and 17-19 are currently pending and under examination in the instant application. An action on the merits follows.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/US2021/019887, filed 26 February 2021, which claims priority to U.S. Provisional Application 62/982,856, filed 28 February 2020.
Thus, the earliest possible priority for the instant application is 28 February 2020.
Information Disclosure Statement
The information disclosure statements filed 02 October 2025 and 21 October 2025 have been considered by the Examiner. Examiner notes the filing of IDS Size Fee assertions for the IDSs filed 02 October 2025 and 21 October 2025, as required under 37 CFR 1.98, indicating that no IDS size fee is required under 37 CFR 1.17(v) at this time.
Specification
The objection to the specification of the disclosure for reciting trade names and/or marks without the appropriate symbols and/or generic terminology is withdrawn in view of the amendment to the specification.
Claim Objections
The objection to amended claim 11 for an apparent typographical error is withdrawn in view of the amendment to claim 11 deleting the word “of” from line 1.
Claim Rejections - 35 USC § 112(b)
The rejection of amended, previously presented, and original claims 1-7, 9, 11-12, 14-15, and 17-19 under 35 U.S.C. 112(b) as failing to particularly point out and distinctly claim the subject matter which the inventor(s) regards as the invention for multiple issues of indefiniteness is withdrawn.
Note that Applicant argues that the limitation of claim 4 reciting “wherein the biological sample comprises whole blood, plasma, or serum” is intended to encompass wherein the biological sample comprises both cells, as recited in independent claim 1, and wherein the biological sample comprises whole blood, plasma, or serum, including, for example, cells to which fetal bovine serum has been added or plasma which has not been fully separated from any accompanying blood cells. As such, claim 4 is being interpreted to encompass any biological samples which both comprise cells and comprises whole blood, plasma, or serum. Note additionally that use of the term “or” in previously presented claim 4 has been interpreted such that the biological sample comprises one of the options listed without excluding the inclusion of other options within the biological sample given that a biological sample comprising whole blood additionally necessarily comprises both plasma and serum.
Claim Rejections - 35 USC § 103
The rejection of amended, previously presented, and original claims 1-7, 9, 11-12, 14-15, and 17-19 under 35 U.S.C. 103 as being unpatentable over Chakraborty et al. [2008, Biotechnology & Bioengineering, 100(4), 782-796]; in view of Solivio et al. [2016, Scientific Reports, 6:24186, 1-14]; Koley & Bard et al. [2010, Proceedings of National Academy of Science, 107(39), 16783-18787]; van de Ven et al. [2009, Journal of Biomedical Optics, 14(2), 021012, 1-10]; Yamamoto et al. [2019, Analytical Biochemistry, 580, 21-29]; and Xu et al. [2015, Methods, 87, 11-25], is maintained. Applicant's amendments to the claims and arguments have been fully considered but have not been found persuasive in overcoming the rejection for reasons of record as discussed in detail below.
Applicant amended the claims to address issues of indefiniteness, but has not amended the claims to alter the scope sufficiently to overcome a finding of obviousness under 35 U.S.C. 103 over Chakraborty in view of Solivio, Koley, van de Ven, Yamamoto and Xu.
Applicant argues that:
Solivio does not teach the use of or provide a motivation to use Tween 20 as a lysing agent, in that Solivio does not use Tween 20 as a lysing agent and teaches a lyoprotectant matrix to stabilize proteins and not cells;
Solivio provides no expectation of success adding Tween 20 to the method of Chakraborty because Solivio does not teach the effectiveness of Tween 20 to stabilize proteins, let alone cellular components such as nucleic acids, and provides mixed teachings on the effectiveness of Tween 20 even for simple proteins; and
Solivio teaches away from using whole blood, plasma, or serum as a biological sample because Tween 20 is ineffective at stabilizing albumin and haptaglobulin and specifically that Tween 20 destabilizes haptaglobulin.
However, this is not agreed.
In response to Applicant’s arguments against the references individually, it is noted that the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Further, the Examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In addition, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Specifically, regarding Applicant’s argument 1), note that although Applicant claims that the biological sample comprises cells, the claims do not require that the storage of the biological sample preserve the viability or structures of the cells or any particular components of the cells. Nor do the claims require that the biological sample be preserved with any particular function or suitable for any particular use. Further, cells comprise proteins, and as such, a biological sample comprising cells necessarily comprises proteins. Accordingly, a process for storage of a biological sample comprising cells is a process for storage of proteins at least within those cells. Further, among the various possible uses of a biological sample comprising cells is the subsequent extraction and use of proteins from the sample for various assays in which protein integrity is desirable. Note also that Applicant’s arguments against the 112b rejection of claim 4 indicate that the biological sample is meant to encompass samples, such as plasma, which have residual levels of cells, and as such, the claims encompass biological samples wherein the cells themselves are not the desired component to be preserved and later used.
Solivio was cited for teaching a process for storage of a biological sample, the process comprising: providing a biological sample (e.g., sera isolated from human whole blood, as recited in claim 4) [page 11 ¶ 5]; contacting the biological sample with a vitrification medium (e.g., a lyoprotectant cocktail) comprising a sugar vitrification agent (e.g., dextran and trehalose, an elected species) and a detergent lysing agent (e.g., Tween 20) to form a vitrification mixture [page 10 ¶ 3, page 11 ¶ 6]; and vitrifying the vitrification mixture to generate a storage-stable sample [page 11 ¶ 6]. As such, Solivio teaches a cryopreservation medium for a biological sample which comprises both a vitrification agent and a lysing agent.
Although Solivio does not teach that the surfactant Tween 20 is a lysing agent, the ability to lyse cells is an inherent property of Tween 20. Additionally, Solivio also teaches that Tween 20 is a biocompatible surfactant that is often added to protein formulations in order to prevent damage during purification, transportation, freeze/drying, and spray drying, which acts by impeding surface or air-interface adsorption, which can result in protein unfolding and aggregation [page 7 ¶ 7]. The amphipathic structure of the surfactant/detergent Tween 20 that impedes surface or air-interface adsorption is also the structural characteristic that contributes to the cell permeabilization functions of surfactants, as taught by Koley.
Koley was cited for teaching that detergents are widely used in biology for protein extraction from cell membranes, in the crystallization of proteins, as stabilizing and denaturing agents, and as membrane permeabilizing agents [column 1 ¶ 1]. Koley also teaches that nonionic surfactants, such as TritonTM X-100, lyse cells to extract protein and other cellular organelles or to permeabilize the living cell membrane for transfection [column 1 ¶ 1]. Koley further teaches that the polar head group of the surfactant disrupts the hydrogen bonding present within a cell’s lipid bilayer, leading to disruption of cellular structure and permeabilization of the cell membrane [column 1 ¶ 1]. Therefore, Koley teaches that surfactants both stabilize proteins and permeabilize cells.
Further, both Chakraborty, Koley, and van de Ven were cited for providing further teachings and motivation to add the permeabilizing agent TritonTM X-100 to the preservation medium of Chakraborty for storing biological samples comprising cells.
As discussed in the prior action, Chakraborty teaches the need for trehalose to enter the cells of the biological sample to protect the cells from desiccation stress [abstract, column 1 ¶ 2- column 2 ¶ 2]. Chakraborty teaches that the specific cells used in their study, namely macrophages, have the capacity to take up solutes from the extracellular milieu by fluid phase endocytosis, which was taken advantage of for the delivery of trehalose into the intracellular space in their study [column 3 ¶ 4- column 4 ¶ 1]. However, Chakraborty also teaches that trehalose does not naturally permeate mammalian cells, nor do mammalian cells produce trehalose, so that a variety of techniques have been explored to find an efficient mechanism to introduce sugars, like trehalose, into the interior of mammalian cells, including transfection, engineered pores, activation of native channels, microinjection, and endocytosis [column 2 ¶ 2].
Therefore, given the teachings of Chakraborty to use a vitrification medium for non-cryogenically vitrifying biological samples comprising mammalian cells, wherein the mammalian cells maintain viability upon rehydration; the teachings of Chakraborty that mammalian cells require the trehalose to cross the cell membrane to enter the interior of the cell; and the additional teachings of Chakraborty of various methods for introducing sugars, like trehalose, into the interior or mammalian cells, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to include a step or reagent for facilitating the entry of trehalose into the mammalian cell interior in a method of vitrifying a mammalian cell using the vitrification agent trehalose for cell types which do not naturally endocytose solutes as macrophages do.
Additionally, Koley was cited for teaching TritonTM X-100 to permeabilize the membrane of a mammalian (e.g., HeLa) cell to the hydrophilic molecule ferrocyanide (MW 368 g/mol) [abstract], which is similar in size to a molecule of the hydrophilic disaccharide trehalose (MW 342 g/mol). Koley also teaches that the concentration of TritonTM X-100 used to permeabilize the cell to the ferrocyanide (e.g., 0.17 mM TritonTM X-100) did not affect cell viability [abstract].
van de Ven was cited for teaching that TritonTM X-100 can be used to permeabilize a variety of live mammalian cells (e.g., HeLa cells, transformed GM847 cells, primary HDF cells, and primary MCF-10A cells) to deliver molecules ranging from 1 kDa to 150 kDa, such that the permeabilization is reversible and treated cells continue to proliferate and show metabolic activity during the restoration of membrane integrity [abstract]. Van de Ven teaches that TritonTM X-100 is a promising permeabilizing agent for efficient and reproducible delivery of agents into live mammalian cells [abstract].
Therefore, given the teachings of Koley that low concentrations of TritonTM X-100 permeabilize the membrane of mammalian HeLa cells to hydrophilic compounds similar in size to trehalose, and the teachings of van de Ven that low concentrations of TritonTM X-100 permeabilize the membranes of several mammalian cell types to a variety of molecules which would otherwise be excluded from entry into the cellular interior, thereby facilitating the intracellular delivery to live cells, an ordinarily skilled artisan would have been motivated to utilize the detergent TritonTM X-100 in a vitrification medium comprising the hydrophilic sugar trehalose to promote the cellular uptake of the trehalose prior to vitrification to allow the trehalose to be present on both sides of the cell membrane, thereby facilitating the vitrification process.
Regarding Applicant’s argument 2), Solivio was not relied on to teach a reasonable expectation of success for stabilizing particular cellular components, such as nucleic acids. Note also that the claims as written do not require the stabilization of any particular cellular content, the preservation of any particular cellular structures or functions, nor the suitability of the samples for any particular use.
As discussed in the prior action, Chakraborty was cited for teaching a process for storage of a biological sample, the process comprising providing a biological sample comprising one or more cells therein (e.g., mammalian cells); contacting the biological sample with a vitrification medium comprising a sugar trehalose (e.g., an elected vitrification agent) to form a vitrification mixture; and vitrifying the vitrification mixture to generate a storage-stable sample [abstract]. Chakraborty further teaches that trehalose offers protection by i) effectively replacing hydrogen-bonded water molecules on the surface of a folded protein without changing the protein conformation, due to the unique placement of the hydroxyl groups on the trehalose molecule; ii) preventing cytoplasmic leakage during rehydration by binding with the phospholipid heads of the lipid bilayer; and iii) forming a room temperature glass at low water content to reduce the molecular mobility, preventing degradative biochemical reactions that lead to deterioration of cell function and ultimately cell death [column 2 ¶ 2- column 3 ¶ 1]. Chakraborty further teaches that the vitrification mixture comprising the sugar trehalose is able to increase cellular viability following vitrification compared to cells vitrified without the trehalose due to osmotic protection provided by the trehalose [column 12 ¶ 1, Figure 3-4].
Therefore, Chakraborty is teaching that the trehalose sugar provides effective protection to cellular components sufficient to preserve cellular viability, which necessarily requires preservation of nucleic acids as well as proteins within the cells. The motivations and teachings of Chakraborty, Solivio, Koley, and van de Ven discussed above provide the motivation to add a surfactant lysing agent, such as TritonTM X-100, to the preservation medium of Chakraborty to permeabilize cells and allow for the use of the preservation medium to cryopreserve non-phagocytic cells.
Regarding Applicant’s argument 3), note that Solivio was cited for teaching an example of a combination of a lyoprotectant medium comprising both a vitrification agent and a lysing agent. To the extent that the activity of Tween 20 on albumin and haptaglobulin are relevant, note that Solivio teaches that the optimal concentration (0.1%) of Tween 20 for post-desiccation recovery is much lower than that used in the experiments wherein albumin aggregation was observed [page 7 ¶ 8]. Therefore, Solivio is not teaching away from the use of Tween 20 surfactant for preservation, but merely teaching to avoid excessively high concentrations.
As discussed above, Chakraborty, Koley, and van de Ven provide teachings and motivations to include the elected cell permeabilizing lysis agent TritonTM X-100 in the vitrification medium of Chakraborty to facilitate the vitrification agent trehalose crossing the cell membrane for entry into the cells. As also discussed above, Chakraborty teaches that the trehalose present internally within a cell is sufficient to preserve viability of the cell following vitrification. Therefore, Chakraborty teaches that the vitrification agent is able to stabilize the cellular structures essential for viability, which include both proteins and nucleic acids.
Further, Chakraborty teaches wherein the biological sample comprises serum in that fetal bovine serum was added to the culture media of the cells [column 4 ¶ 2, column 4 ¶ 4-column 5 ¶ 2].
Therefore, Applicant’s amendments and arguments do not overcome a finding of obviousness under 35 U.S.C. 103 over Chakraborty in view of Solivio, Koley, van de Ven, Yamamoto and Xu, and the rejection is maintained.
Conclusion
No claim is 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 Dr. KATIE L PENNINGTON whose telephone number is (703)756-4622. The examiner can normally be reached M-Th 8:30 am - 5:30 pm, Friday 8:30 am - 12:30 pm CT.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634
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