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 .
DETAILED ACTION
Claim Status
Claims 1-14, 17, and 19-26 are currently pending in this application.
Claim Objections
Claim 26 is objected to because of the following informalities: claim 26 recites “a seeding density about,” which would be clearer if rewritten as “a seeding density of about” and aligning with wording of claim 1; and also claim 26 appears to have a typographical error of “X” in lieu of the multiplication symbol ‘×’ or ‘x’, as in claims 1 and 25. Appropriate correction is required for consistency.
Previous Rejections
Status of the rejections: the previous claim rejections under 112(a) and 103 are withdrawn except as specifically maintained herein; and the previous claim rejections under 112(b) and 112(d) are withdrawn in view of the claim amendments.
Claim Interpretation
In the claims, the term “about” is considered as encompassing a 10% range of values both less and greater than the subsequent value in view of instant [0020].
In claim 1, the agitation is interpreted as a continuous agitation at the initial speed until the increased speed when the cell density reaches about “1 x 105 cells/mL” to “20 x 105 cells/mL.” However the agitation speed at the “initial” speed (a “first speed) is interpreted wherein “initial” means the agitation just prior to and continuous with the increased “second” speed; thus, any optional agitation(s) prior to the “initial” speed agitation and/or after the second speed agitation may be discontinuous and at any speed so long as all agitation speeds prior to the second speed are less than or equal to the initial speed (see claim 19).
In claim 1, under a broadest reasonable interpretation the contingent step conditioned on “when the cell density reaches” a certain threshold is not limiting when the condition is not met, i.e., when the cell density never surpasses about 5 x 105 cells/mL. See MPEP 2111.04(II). As the seeding density can be any amount less than about 0.2 x 106 cells/mL so long as a plurality of stem cells are inoculated, a 50-fold or more expansion may occur without reaching the cell density threshold in some situations. Similarly, this contingent step is not limiting when the condition is preemptively met upon the inoculating step, i.e., when the cell seeding density is initially at about 0.2 x 106 cells/mL, as this does not qualify as “reaching” “during the incubation” allowing for a change from an “initial” speed to a second speed at the moment of inoculating. See MPEP 2111.04(II). As the seeding density can be any amount less than about 0.2 x 106 cells/mL so long as a plurality of stem cells are inoculated, a 50-fold or more expansion may occur without reaching the cell density threshold in some situations.
In claim 3, the phrase the pluripotent stem cells “are inoculated into the bioreactor as cryopreserved pluripotent stem cells” is interpreted as meaning at the moment before inoculating, the pluripotent stem cells are at temperature at least equal to or below 0 °C, i.e., frozen or lyophilized.
Claim Rejections - 35 USC § 112(a) - Written Description (modified)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-7, 9-14, 17, and 19-25 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
In view of the specification, claim 1 is directed to processes for producing new cells via cell division in a bioreactor containing microcarriers by expanding (presumably in a cell culture medium) pluripotent stem cells (“manufacturing” via incubating) to yield at least “about” a 50-fold expansion of cells (45 to 55 fold). It is also noted that fold expansion is interpreted to occur solely during the incubating period(s) and the inoculated amount of cells may be as low as two pluripotent stem cells, meaning 50-fold expansion occurs after enough cell divisions to produce as little as 90-100 daughter cells, or as high as 0.22 x 106 cells/mL based on the maximum inoculated seeding density, meaning the claimed process yields over 1 million cells (e.g., 1.1 x 106 cells/mL), and in view of dependent claim 2, may include an unlimited number of passagings.
Claim 1 is broad in that (1) the incubating conditions are unlimited (but may include intervening passaging(s) (see dependent claim 2)), (2) the amount and type of microcarriers is unlimited (see e.g., dependent claims 5-6 and 22), and (3) the pluripotent stem cells need not adhere to any microcarrier(s) except for as implied in dependent claim 8. Thus, claims 1-7, 9-14, 17, and 19-25 broadly encompass a 45-fold or greater expansion using pluripotent stem cell-nonadherent microcarriers without a single passage during the incubation.
Incubating Conditions: Breadth of microcarriers (MC)
Claims 1-7, 9-14, 17, and 19-25 each encompasses any amount and/or type of microcarrier regardless of any ability to interact with the pluripotent stem cells.
The term “microcarrier” is interpreted in view of the claim set merely as any microscopic materials capable of supporting a cell adhesion in suspension culture but not necessarily capable of adherence specifically to pluripotent stem cells based on claim 6 and the description in the instant specification related thereto discussed below.
In view of dependent claim 6 and the principle of claim differentiation, the microcarriers of claims 1-5, 7-14, 17, and 19-25 may lack a matrix coating with a molecule that is utilized by a pluripotent stem cell to increase in mass (i.e., live, grow or otherwise add biomass) as defined by the instant application at [0028]. For purposes of this rejection, the term nutrient growth matrix is interpreted as encompassing (but not limited to) an extracellular matrix (ECM) coating on the surface of each microcarrier (e.g., Matrigel, vitronectin, or laminin-derivatives). Thus, the microcarriers of claim 1, and thus of claims 2-5, 7, 9-14, 17, and 19-25 may lack such a coating recited in claim 6, and merely represent microscopic materials capable of supporting cell adhesion in suspension culture as cell culture microcarriers (e.g., only for non-pluripotent cells), such as non-functionalized or uncoated beads known in the prior art because the prior art teaches such uncoated beads do not support sufficient adhesion for pluripotent stem cell growth in culture (see e.g., Rodrigues et al., Biotechnol J. 14: e1800461 (2019) at pg. 1; Adil et al. (2017) at Fig. 2(b)). Further, claim 6 encompasses other nutrient matrix coatings that are not sufficient to support pluripotent stem cell adhesion during the incubating.
In view of claim 2, an “incubating” period may comprise a “passaging,” which may include separating expanded cells from the microcarriers and the bioreactor, i.e., harvesting and transferring to a new container and optionally washing (see instant [0111]) so long as the process is closed or automated. Thus, while microcarriers need be present in the inoculated bioreactor, the incubating period sufficient to yield 45-fold expansion need not always have those initial microcarriers in the bioreactor during the entire incubating period(s) (e.g., after the first passage), and need not have any microcarriers during most of the incubating period (e.g., if the first passage is performed before a majority of the incubation period and microcarriers are removed and new ones not re-introduced).
The prior art teaches expanding cultured pluripotent stem cells over 50-fold, e.g., in agitated suspension cultures without any microcarriers; however serial passaging was required as each individual expansion was less than 50-fold (e.g., 5 to 13-fold each session) and thus passaging was required for cumulative expansion, e.g., over 49-fold (see e.g., Zweigerdt et al., Nat Protoc 6: 689-700 (2011); Abecasis et al., J Biotechnol 246: 81-93 (2017); Kwok et al., J Tissue Eng Regen Med 12: e1076-87 (2018)).
The prior art also teaches expanding pluripotent stem cells over 45-fold using adherent 3D-microcarrier culture in static or agitated suspension culture (Ting et al., Stem Cell Res 13: 202-13 (2014) at Table 1; Badenes et al., PLoS One 11: e0151264 (2016) at Fig. 1B; Badenes et al., JCTB 92(3):492-503 (2017) at abstract, Fig. 1 and 5, pg. 500; Levinson et al. (2018), of record; at Fig. 4, pg. 265, right col., to pg. 266, 1st para.); but while not disclosing an agitation speed change except for intermittent agitation in Badeness et al. and as taught by Abecasis et al. as discussed in the art rejections below. Thus, the general process of claim 1 would be considered by the skilled artisan in view of the prior art as achievable merely by following various prior art protocols with multiple passagings or, alternatively without any passaging, by selecting a microcarrier with suitable adhesion to the pluripotent stem cells, e.g., in view of the prior art generally and Levinson specifically.
However, the prior art is silent as to processes wherein there is both no passaging and no pluripotent stem cell-adherent microcarrier, such as wherein the microcarriers are uncoated or coated with a nutrient matrix that does not provide sufficient adhesion (e.g., contrast with instant claim 26 reciting “configured to adhere pluripotent stem cells” during the incubating). The instant written description is analyzed for filling this gap in the prior art.
In analyzing whether the written description requirement is met for genus claims, it is first determined whether a representative number of species of microcarrier conditions have been described for the above described scope.
For microcarriers, the instant specification describes microcarriers as made of any material and having any shape with specific examples of polystyrene microspheres coated with an ECM mimicking matrix ([0050]; [0073]-[0078]). However, the microcarriers may be uncoated or coated ([0051]) and, according to the instant description, the process may be designed for adherent or non-adherent culturing ([0037]; [0005]). The specification notes that “coating microcarriers in nutrient matrix may enable hPSC attachment to the microcarriers” but only provides a single representative species of such a microcarrier, “large” sized microcarrier coated with L7™ hPSC Matrix, without describing this trademarked product’s composition.
The instant written description is vague and at a high level of generality regarding representative processes of claim 1, except for in two instances and a procedure describing closely related processes achieving 50-fold expansion regardless of agitation speed change. The application provides its closest working example for claim 1 based on a stirred-tank bioreactor inoculated either with cell clusters or individualized pluripotent stem cells ([0075]-[0078]; FIG. 8-9 and 12-32) and comprising adherent L7™ hPSC Matrix-coated plastic microcarriers but no mention of any agitation speed change. The written description also includes empirical results from a spinner flask format without agitation speed change nor any description of such a closed or automated system ([0073]-[0074]), and mentions laminin-coated microcarriers without further process details ([0114]).
The detailed description lacks processes comprising nonadherent microcarriers achieving 45-fold or greater expansion. The specification notes that if L7™ hPSC Matrix-coated microcarriers were not included during incubating, that no expansion was detected (FIG. 7; [0093]). Thus, the microcarrier description and adherent conditions are nebulous and lacking a guiding nexus to prior art teachings, thus leaving these details to the imagination of the skilled person. In particular, the instantly described process species lack a sufficient nexus to the breadth of the genus of unlimited incubating conditions and seeming ambivalence of microcarrier adherence even in the absence of any passaging.
The prior art teaches of numerous different commercial microcarriers developed for large scale culture of mammalian cells, such as Cytodex® 1/3, DE-52/3, and SoloHill®. The prior art teaches some microcarriers do not provide pluripotent stem cell adhesion (see e.g., Chen et al., Stem Cell Res 7: 97-111 (2011) at abstract; Adil et al., at Fig. 2(b) US20120009645A1 at Examples, noting ROCK inhibitor requirement for microcarrier adherence) while also suggesting culturing with non-adherent leukocyte inhibitory factor (LIF) releasing microcarriers (e.g., PLGA slow release StemBeads®) to maintain pluripotency in the absence of feeder cells (Ferguson et al., Stem Cell Res 33: 69-78 (2018) at pg. 70, left col.; 74, right col.; Tsuji et al., Dev Dyn 237: 2129-38 (2008) at pg. 2130-2132; Fig. 2).
Achieving a specific expansion of at least 45-fold is deemed unpredictable in view of the prior art and instant description over the entire claims scope in view of the prior art and the limited instant description, particularly nonadherent microcarriers combined with zero passagings during incubation.
Thus, the skilled artisan could not rely upon the instant disclosure such that the specification sufficiently described that Applicant was in possession of a process having a predictable effect of cell expansion of about 50-fold or greater over the entire scope of claims 1-7, 9-14, 17, and 19-25 due to the scope of microcarriers. There is a lack of evidence in the prior art and instant application that the process of claim 1 achieves such expansion over the full scope of nonadherent microcarriers (e.g., lacking a coating) and excluding any passaging during incubation, including when limited by claims 2-7, 9-14, 17, and 19-25 for the same reasons explained above regarding the unlimited microcarrier culturing conditions. Subsets of claim 6’s scope are supported by the written description either wherein multiple passagings are required or the matrix coating is configured to promote adherence to the pluripotent stem cells under the incubation conditions used.
Response to Arguments
Applicant’s response filed 7/27/26 was fully considered but only found partially persuasive regarding the 112(a) rejections of record.
Previously, the term expanded pluripotent stem cells was interpreted to mean wherein all 50-fold or greater expanded cells were “pluripotent stem” cells. Based on Applicant’s remarks it is clear that some of the cells in the expanded population may not be pluripotent and/or stem cells (or perhaps not even alive) but that the number/percentage of pluripotent stem cells is not limited in any of the claims so long as there is a plurality of viable pluripotent stem cells (e.g., two such cells). Note, if applicant acts as his or her own lexicographer to specifically define a term of a claim contrary to its ordinary meaning such as a “microcarriers” coated with any fluid, compound, molecule, or substance that can increase the mass of a bioproduct; the written description must clearly redefine the claim term and set forth the uncommon definition so as to put one reasonably skilled in the art on notice that the applicant intended to so redefine that claim term.
Applicant argues that a skilled artisan would understand the inventor to be in possession of the process of claim 1, as not every nuance need have a written description, especially for features conventional in the prior art, with prior art known structure-function relationships or that are not critical to the claimed process. While this is true in general, in the instant case the description is lacking such structure-function relationships specifically for achieving about 50-fold expansion with the overly broad genus of microcarriers (see e.g., dependent claim 6) and optionality of passaging step(s) (see e.g., dependent claim 2). Furthermore, there is no written description exception for auxiliary claim elements deemed by applicant as conventional in the prior art as the claim is viewed as a whole incorporating all the claim elements together, such as to achieve an about 50-fold or greater expansion with variables considered conventional in other contexts.
Firstly, it is not clear to the examiner which elements are considered conventional in the art by the Applicant. Second, it is noted that the “test for written description … is the same whether the claim element is essential or auxiliary to the invention.” Boston Scientific Corp. v. Johnson & Johnson, 647 F.3d 1353, 1365 (Fed. Cir. 2011). Applicant is kindly invited to point out what features are considered critical or not. As noted in the modified rejection above, what features of the claimed process are critical to the invention remains mysterious, such as the purpose of the presence of the microcarriers, which may be uncoated or coated with a broad swath of “nutrients” noted in the specification, and thus encompassing functionally non-adherent conditions for a pluripotent stem cell.
Applicant also argues (pg. 13-15, 20) the application has multiple working examples, (1) the 3L stirred tank bioreactor discussed in the non-final office action as well as using a (2) 1L bioreactor and (3) spinner flask and (4) exemplified with three different cell types), however upon further consideration the examiner finds no actual working examples incorporating all the claim limitations of claim 1 or 26. Instead, the 3L stirred tank process description at [0075]-[0078] is considered closest to the claimed invention as a whole and the spinning flask and other data as supporting evidence as detailed above.
As detailed above, the claims are broad in that the incubating conditions can vary greatly, for example, regarding microcarrier function (adherent or not), intervening passaging and restoring or replacing the microcarriers, and as to the presence/absence of pluripotent stem cell aggregates (e.g., in solution), and yet somehow an about 50-fold cell expansion is achieved regardless. The instant written description, however, is limited to representative evidence based on processes solely comprising adherent microcarriers and not showing a completely closed or automated system nor any impact on fold-expansion of an agitation speed increase ([0073]-[0078]). However applicant is invited to furnish evidence to the contrary. It is the opinion of this office action, that the about 50-fold expansion result is not described with sufficient particularity across all these variables even in view of the prior art, and especially, over the scope of an improvement over the prior art due to some detailed setting (novel fine tuning of the prior art agitation rates/changes and/or inoculation density).
The prior art teaches while some microcarriers are suitable (gelatin or beads coated with a MEF composition, Matrigel, laminin and vitronectin, collagen, or a cationic surface charge (e.g., trimethyl ammonium or poly-L-lysine coated), other microcarriers do not allow for any mammalian cell adhesion without further modification, e.g., certain coatings, like uncoated polyvinyl alcohol (PVA) or poly(ethylene glycol) (PEG) hydrophobic microspheres (see e.g., Adil et al., at Fig. 2(b), but note, not even all microcarriers suitable for mammalian cells work specifically for all pluripotent stem cells).
The unpredictability regarding written description is specifically regarding the scope encompassing nonadherent microcarrier conditions (e.g., uncoated or due to choice of “nutrient” matrix coating or media compositions) without sufficient description of representative species in view of the teachings of the prior art and requirement to produce at least a 45-fold expansion without passaging. This is a problem of claim breadth in view of the written description, while the skilled worker would recognize possession of certain prophetic working examples and could optimize variations to achieve the requisite expansion result level while varying various parameters to some extent, such as by using nonadherent conditions combined with passagings or by using adherent conditions and no passaging, the microcarrier description and adherent conditions are nebulous and lacking a guiding nexus to prior art teachings over the full scope regarding processes lacking passagings but not requiring microcarrier adhesion.
35 USC § 112(a) – Scope of Enablement (modified)
Claims 1-7, 9-14, 16-17, and 19-25 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because while the specification in view of the prior art is enabled for wherein during the incubating the pluripotent stem cells are adherent to the microcarriers; the specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to perform the full scope of claim 1 which encompasses any microcarrier and unlimited incubating conditions while encompassing not passaging during the incubation period(s) required to achieve at least an about 50-fold expansion. All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Claim 1 is directed to a process for manufacturing in a bioreactor containing microcarriers more pluripotent stem cells to yield at least about a 50-fold expansion from the starting amount of cells. The claims are broad in that the pluripotent stem cells need not adhere to any microcarrier(s) during the incubation period except as implied in dependent claim 8 or as expressly required in claim 26.
The prior art teaches methods of culturing pluripotent stem cells with microcarriers to achieve over an about 50-fold expansion and producing cell densities of 1 x106 per mL using ECM-coated gelatin/polystyrene microbeads (Ting et al. (2014) at Table 1; Badenes et al. (2016) at Fig. 1B; Badenes et al. (2017) at abstract, Fig. 1 and 5, pg. 500; Levinson at pg. 264, right col., 2nd para., to pg. 266, 1st para.; Fig. 2-4; Abstract).
The prior art is silent as to using microcarriers for 45-fold or more pluripotent stem cell expansion under nonadherent conditions and without a passaging, e.g., using potentially adherent microcarriers under nonadherent conditions, e.g., in the absence of ROCK inhibitor noted as essential for this by US20120009645A1 (FIG. 1 and 4; [0473]-[0474], [0200], [0099]). Therefore, the disclosure provided by the applicant in view of prior art must encompass a wide area of knowledge to a reasonably comprehensive extent so that one of the ordinary skills in the art would be able to practice the invention without any undue or reasonable burden being on such artisan.
The amount of direction and guidance and working examples provided by Applicant:
As noted above, the instant application provides a single closest working example for claim 1 based on a 3L Eppendorf bioreactor comprising L7™ hPSC Matrix-coated plastic microcarriers ([0075]-[0078]; FIG. 8-9 and 12-30). The seeded cell amount was 0.02-0.04×106 cells/mL (e.g., 62-204 ×106 cells) into Lonza L7™ TFO2 medium supplemented with xeno-free hPSC L7™ supplement and, apparently based on applicants comments in the response filed July 27, 2026, at an agitation rate of 50 RPM until greater than 2 x106 cells/mL total were produced. The only example of a closed and/or automated process comprising concentrating the cells relies on a kSep device (400.50, Sartorius), and there is no guidance regarding a closed or automated system that monitors cell density levels during the incubating and triggers an agitation speed increase upon reaching a cell density set point.
As noted above, the process may be designed for adherent or non-adherent culturing ([0037]; [0005]); however detailed guidance in the instant application is limited to processes comprising adherent microcarriers (MC) ([0073]-[0119]) and lacks any detailed description of an MC-free, aggregate-based 3D pluripotent stem cell culture process or improvement of such over the prior art. Importantly, there is no working example wherein the agitation speed is described as being increased or whether this has any effect on the process, such as within certain agitation speed ranges. Moreover, the application fails to provide any species of incubation conditions beyond the proprietary defined medium Lonza L7™ TFO2 medium supplemented with xeno-free hPSC L7™ supplement, which fails to describe the presence of growth factors or growth inhibitors in such a medium. The presence of certain agents (e.g. ROCK inhibitor or ROCKi) or growth factors (e.g., BMP4) or insufficient bFGF levels can dramatically alter yields and pluripotency (e.g., Y27632 is noted as required at instant [0005] for aggregate-based culture) while certain growth inhibitors may completely arrest expansion. Yet, the ingredients in the L7™ TFO2 medium and hPSC L7™ supplement are not described in the instant disclosure. Although a detailed procedure is described at [00122] with agitation speed changes, this procedure lacks empirical evidence as to achieving the limitation of claim 1 of about 50-fold expansion, and thus is prophetic regarding all instant claims.
Thus, the microcarrier and adherent condition choices are left to the discretion and expertise of the unguided skilled person and the state of the prior art, such as to whether/how to perform adherent or non-adherent culturing when departing from the working embodiment of an L7™ hPSC Matrix coating on a microcarrier or wherein the seeding density is less than 0.02 x 106 cells/mL.
The quantity of experimentation needed to make and/or use the invention:
Extensive experimentation would be required to determine how to achieve about 50-fold expansion of pluripotent stem cells across their full scope of claim 1. In one sense, there is no working embodiment shown for agitation speed increase with a closed and/or automated system, but increasing/decreasing agitation speed during cell expansion was routinely used in the prior art, such as based on real-time information tracking culture conditions, e.g., concerning oxygen and pH changes indirectly related to or as a proxy for cell density (see e.g., Xu and Chen, J Biotechnol 231: 149-59 (2016) at pg. 151, left col., 2nd para.; Table 1, noting a range of 260-450 impeller rpm). Regardless, the adherent conditions, or lack of, are deemed critical in light of the optionality of passagings.
Claims 1-7, 9-14, 17, and 19-25 each encompasses any microcarriers and incubating conditions (e.g., microcarrier coating types) and yet the recited process purportedly is capable of producing at least about 1 million cells (~1.1 x 106 cells/mL) from a seeding density of 50-fold less (~0.2 x 106 cells/mL) wherein at least some of the 1 million cells are pluripotent stem cells. These claims each encompass wherein the cells are in solution and not adherent during the entire incubating period resulting in the about 50-fold expansion, which is known in the prior art using aggregates but not without multiple passagings as noted above. Additionally, the claims also encompass wherein the cells are adherent to microcarriers only during a subset of the entire incubating period, e.g., due to passaging involving separating the cells from the microcarriers as noted above.
By using serial passaging, cumulative expansion can achieve very large fold increases, such as 241-, 256- or 279-fold based on 5 to 14-fold per individual incubation (see e.g., Badenes et al., JCTB 92(3):492-503 (2017) at abstract; US20160340633A1 at [0126], [0197], [0076], FIG. 13), perhaps only limited biologically by the cell type’s passage limit. If passaging is excluded, teachings of 45-fold or greater expansion in the prior art appears limited to only wherein the microcarriers adhere to the pluripotent stem cells and provide sufficient surface area for such expansion.
The prior art notes that “accurate understanding of how stem cells interact with microcarriers still remains unknown” and thus requires empirical testing each time (Sart et al., Biotechnol Prog 29: 1354-66 (2013) at abstract; Table 4; pg. 1359 last para., to 1361, with porosity, surface chemistry, size, and coatings having effects on proliferation and potency).
Without evidence, it is not reasonably predictable applicant possessed the method over the full scope of claim 1, which is broad in the scopes of microcarrier type, incubating conditions, and optionality of passaging. From the empirical data of the closest working examples (3L stirred tank or stirred flask) as viewed in light of the prior art, it is not necessarily predictable that a 45-fold or greater expansion of pluripotent stem cells would be achieved when adherent conditions are not required and there are no passagings. However applicant is invited to furnish evidence the contrary.
Thus, the instant application in view of the prior art fails to describes with predictability a method enabled over the full scope of claim 1 and there is little to no guidance as to how to adapt the closest working examples without passaging (3-L, 1-L or spinning flask) to the broad scopes of (1) any incubating conditions (e.g., media components) and (2) any type of microcarrier (e.g., adherent or not and/or uncoated or coated) encompassed by the claims. The science of pluripotent stem cell culture had not evolved such that by Nov. 25, 2019, without guidance or working examples in the specification, one of skill in the art could predictably perform claim 1 across its full scope without undue and unreasonable experimentation.
While the prior art of Zweigerdt et al., Abecasis et al., Kwok et al., Ting et al., Badenes et al., Xu, and Levinson provides a wealth of information and guidance regarding implementing claim 1 over much of its features, the prior art is silent as to achieving about 50-fold expansion with non-adherent/uncoated microcarriers and no passagings. This specific expansion requirement is unpredictable in this context in view of the prior art and the limited guidance and evidence presented in the application. However, a subset of claim 6 is enabled either wherein there is either multiple passagings as taught by the prior art or the matrix coating is configured to promote adherence to the pluripotent stem cells in the incubating period(s) under the culture conditions of the incubating step(s).
In summary, the claims are rejected under 35 U.S.C. 112(a) because the specification does not reasonably provide enablement to a person skilled in the art, to which it pertains or with which it is most nearly connected, to perform the claimed process across its full breadth except for claims 8 and 26. Given the limited guidance provided in the specification, the lack of guidance in the prior art, lack of working examples and empirical evidence, and the broad scope of the claims with regard to adherent conditions yet not requiring a passaging(s), undue and unreasonable experimentation would have been required.
Response to Arguments
Applicant’s response filed 7/27/26 was fully considered but only found partially persuasive. As detailed above, the claims are broad in that the incubating conditions can vary greatly, for example, regarding microcarrier function (adherent or not), initial seeding density, intervening passaging and restoring/reloading or replacing the microcarriers, and as to the presence/absence of pluripotent stem cell aggregates and/or a ROCK inhibitor, and yet somehow an about 50-fold cell expansion is considered predictably achievable. It is the opinion of this office action, that the about 50-fold expansion result is not described (in full view of the prior art) with sufficient particularity across all these variables regarding the combination of adherent conditions and passagings scopes.
In view of the prior art, it appears relatively trivial to accomplish an about 50-fold or greater cumulative expansion by using multiple serial passaging and solely applying prior art methods, e.g., with a constant agitation rate. However if there are no passagings, then the prior art only provides guidance for adherent conditions suitable for the pluripotent stem cell type being used. And yet, the scope as interpreted for the microcarrier genus encompasses diverse microcarriers, such as uncoated or coated with “any fluid, compound, molecule, or substance that can increase the mass of a bioproduct,” such as a ROCK inhibitor or growth factor like LIF as noted above, none of which ensures pluripotent stem cell adhesion or adherent culture conditions.
Applicant argues the critical inventive concept is the change in agitation speed, a “tuning” of agitation rates at a particular moment during the expansion (i.e., at “1 x 105 cells/mL” to “20 x 105 cells/mL”) but yet there is no evidence provided that this parameter effects achieving 50-fold expansion at all, which upon further consideration was already available to the skilled artisan before the earliest effective filing date via prior art taught processes using either adherent or non-adherent conditions, albeit using different agitation approaches from those claimed, e.g., single speed or increasing speed based on time not any precise cell density measurement. Thus, from the prior art alone, the skilled artisan can design and perform subsets of the process of claim 1 without undue experimentation, such as following the teachings of Abecasis or Kwok and merely including an insignificant nonadherent microcarrier, e.g., a LIF slow-release microcarrier or a labeled Cytodex1 bead to monitor mixing (see e.g., Olmer et al. 2012 at pg. 773, Fig. 1). Regarding dependent claim 2, the prior art Levinson teaches this was already achievable using adherent microcarrier culturing methods without passaging and regardless of any agitation speed increase.
The most comprehensive description of agitation conditions appears to be at instant [00122] (5.14-5.20, 5.22, 8.4, 9.5), which can be viewed in light of disclosures at ([0055]-[0057]); however, the structure-function relationship of these to the fold-expansion is not clear. Nothing at instant [00122] establishes any empirical relationship between achieving about 50-fold expansion and a particular incubator setting(s) or process design, e.g., agitation speed/schedule, inoculated cell density between zero and 0.02 x 106 cells/mL, and/or the use of passagings. Thus, the skilled artisan is lacking guidance as to how to adapt these disclosures to the full scope of the claimed process for achieving the recited result, e.g., microcarrier genus combined with the scope of optional passaging.
Claim Rejections - 35 USC § 112(b), (new)
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 1-14, 17, and 19-26 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 applications subject to pre-AIA 35 U.S.C. 112, the applicant) regards as the invention.
Claim 1 recites the phrase “a fold expansion of about 50 times or greater to give expanded pluripotent stem cells,” which in light of Applicant’s remarks on the record includes (pg. 12, Response dated 7/27/26) expansion into any type of cell as pluripotency need not be maintained. This renders indefinite for ambiguity, either, (1) what must be 50 times greater compared to an inoculated number of pluripotent stem cells, the number of pluripotent stem cells or total cells and/or (2) what the phrase “expanded pluripotent stem cells” refers, e.g., a mixed cell population of pluripotent stem cells and other cell type(s) or some isolated/purified subpopulation thereof lacking non-pluripotent stem cells. Claims 2-14, 17, and 19-25 are included in this rejection for depending from claim 1.
Claim 6 is indefinite for referring to a “nutrient” matrix with regard to a coating for a microcarrier. The term “nutrient matrix” is not defined by the claim and the instant specification does not provide any definition or any limitation to its structure(s) and/or properties beyond the implied presence of nutrition for some organism. Thus for purposes of examination, the claim is construed to merely limit the microcarriers to having any coating. It would be remedial to amend the term to instead recite a eukaryotic/vertebrate “cellular extracellular matrix”, “culture matrix” or “cell culture matrix.” Claim 22 is included in this rejection for depending from indefinite claim 6.
Claim 21 recites the term “recombinant, xeno-free, defined medium,” however the modifier “xeno-free” is incoherent and unclear as a relative term. To be considered “xeno-free” is only relative to something else, such as the cells, but the cells are not limited in the claims to any species or type, thus rendering what is “xeno” or not as indefinite, such as any given animal derived ingredient.
Claim 22 recites the term “recombinant, xeno-free, defined matrix,” however the modifier “xeno-free” is incoherent and unclear as a relative term. To be considered “xeno-free” is only relative to something else, such as the cells, but the cells are not limited in the claims to any species or type, thus rendering what is “xeno” or not as indefinite, such as any given animal derived ingredient.
Claim 24 recites an agitation speed in “rpm,” which although is understood to a person of ordinary skill in the art as an abbreviation for “revolutions per minute,” it is ambiguous and unclear as to what is revolving according to the speed claim limitations, such as the cells, microcarriers, medium, an unrecited impeller of the stirred tank, all of the aforementioned, and/or something else (e.g., general bioreactor agitation) (see instant [0054], [0049]).
Claim 26 recites the phrase “concentrating expanded pluripotent stem cells” while also reciting “cryopreserving the expanded pluripotent stem cells,” which is ambiguous as to the antecedents for both and their relationship, if any. Are the concentrated pluripotent stem cells the same as the “expanded pluripotent stem cells,” and, are the cryopreserved expanded pluripotent stem cells ones which are required to have been concentrated?
Response to Arguments
Applicant’s response filed 7/27/26 (pg. 23) argues that the term “nutrient matrix” is understood by those skilled in the art, especially in light of the specification’s description of this term as “any fluid, compound, molecule, or substance that can increase the mass of a bioproduct, such as anything that may be used by an organism to live, grow or otherwise add biomass” ([0028]). However the metes and bounds of any compound or substance coating defined solely functionally as providing for increasing the mass of a bioproduct is indefinite without definition of what constitutes “nutritional” and satisfies the term “matrix,” suggesting a cell support ([0028]-[0029]). For example, what does capable of increasing the mass of a stem cell, must the nutrient matrix include an energy source or would just a vitamin, cholesterol, trace element, or non-essential amino acid suffice as being used by the cell to live/survive and thus eventually add biomass upon growth? How is a microcarrier “coated” with a fluid that functions as a matrix in the context of a bioreactor expanding cells? In view of [0051] and [0073], the microcarrier coating (nutrient matrix) may be made from a cell culture medium (e.g., Hematopoietic Progenitor Growth Medium) or be the L7™ human pluripotent stem cell (hPSC) Matrix (referred to as “recombinant, xeno-free and defined” as its intended use is specifically for human-derived cells). It is not clear how coating a microcarrier with a medium results in a “matrix” as typically used in the art of cell culturing referring to a polymer or hydrogel. Also, L7 hPSC Matrix lacks an energy source, vitamin, cholesterol, or individual amino acid, etc., so it is not clear how L7 hPSC Matrix serves as a “nutrient” matrix, except beyond providing a proteinaceous substrate as possibly a source of amino acids.
In response, if applicant argues this term is commonly known and understood in the prior art, submission of technical publications using the same term could be sufficient evidence. Note, if applicant acts as his or her own lexicographer to specifically define a term of a claim contrary to its ordinary meaning, the written description must clearly redefine the claim term and set forth the uncommon definition so as to put one reasonably skilled in the art on notice that the applicant intended to so redefine that claim term. Process Control Corp. v. HydReclaim Corp., 190 F.3d 1350, 1357 (Fed. Cir. 1999).
Claim Rejections - 35 USC § 103 (modified and new)
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 4, 6-7, 11, 17, 21-23, and 25-26 are rejected under 35 U.S.C. 103 as being unpatentable over Levinson (Levinson et al., Biochemical Engineering Journal 132: 262-9 (2018)) in view of Gupta (“Parameter optimisation for expansion of pluripotent stem cells” (Doctoral dissertation, Monash University, 2014) and Xu (Xu and Chen, J Biotechnol 231: 149-59 (2016)).
The claims are interpreted as provided in previous sections.
Regarding claim 1 or 26, Levinson teaches closed automated manufacturing processes for culturing stem cells to high densities comprising placing a plurality of pluripotent stem cell-adherent microcarriers (e.g., digestible carriers such as Cultispher-G (Percell Biolytica)) in a bioreactor (e.g., a single-use vessel perfusion bioreactor system (BioBLU by Eppendorf); inoculating the bioreactor with pluripotent stem cells (e.g., allogeneic induced pluripotent stem cells (iPSC)), incubating the pluripotent stem cells with agitation in the bioreactor (e.g., a stirred tank) for a period of time sufficient to yield an over 50-fold expansion (e.g., stem cells expanded 90-fold or 50–100 fold in 10 days on Cultispher-G) to produce expanded pluripotent stem cells (pg. 264, right col., 2nd para., to pg. 266, 1st para.; Fig. 2-4; Abstract). Levinson teaches a seeding density less than 0.2 x 106 cells/mL (Fig. 4, note Y-axis teaching far less than 0.5 x 106 iPSC cells/mL, such as approximately 0.1 to 0.2 x 106 cells/mL). Levinson also teaches harvesting stem cells for use in cell therapies after further downstream processing steps, including concentrating (e.g., 20 to 50 -fold) and cryopreserving such expanded stem cells via routine downstream processing via a closed-automated system, such as for producing final therapeutic cell products at a desired quantity (pg. 265, left col., para. 5, to pg. 266, 1st para.).
Although Levinson does not teach a single method comprising expressly concentrating and cryopreserving the expanded cells, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to combine all of the above mentioned teachings of Levinson into a single, closed automated process using a stirred bioreactor system seeded with pluripotent stem cells. One of ordinary skill in the art with the goal of producing a large number of pluripotent stem cells would be motivated by Levinson already demonstrating this is possible using an automated closed manufacturing process comprising expanding pluripotent stem cells adhered to Cultispher-G microcarriers in a stirred (agitating) BioBLU perfusion bioreactor and would be motivated to perform such a method further comprising concentrating and cryopreserving the resulting expanded pluripotent stem cells in an automated closed system as a matter of routine downstream processing for storing the expanded cells as also taught by Levinson generally for any harvested cell product manufactured in a bioreactor at scale (pg. 265, left col., para. 5, to pg. 266, 1st para.). Motivation comes from Levinson teaching such downstream processing is required to make the final cell product at a target dose (concentrated), for long-term storage (cryopreserved), and sterility (closed system), such as for medical applications. Moreover, Gupta teaches improved recovery of pluripotent stem cells cryopreserved while attached to microcarriers (pg. 21).
Although Levinson does not expressly teach such a method comprising increasing the agitation speed from an initial speed when the cell density reaches about 5 to 20 x 105 cells/mL, Levinson teaches logarithmic growth of iPSCs on microcarriers over 5-17 days of culture depends on seeding density (Fig. 4). Additionally, Gupta teaches optimizing various parameters for expansion of pluripotent stem cells in bioreactors with microcarriers, including agitation parameters (pg. 20-22), e.g., because pluripotent stem cells (iPS cells) can be sensitive to narrow agitation ranges (pg. 77, 4th para.; pg. 65-73). Gupta teaches that an initial minimum agitation speed is required for proper microcarrier suspension (pg. 77, 5th para.) and 25 rpm is preferred to no agitation for improved maintenance of pluripotency (Fig. 4.10; pg. 78, last para.), but the agitation should be kept below a level causing significant microcarrier breakage or cell death (pg. ii, 1st para.; pg. 62). Further, Xu teaches when culturing mammalian cells in stirred-tank perfusion bioreactors that the agitation rate is typically increased gradually to improve oxygen transfer as increasingly higher cell densities are reached in later stages and to avoid foam formation, e.g., impeller rpm of 260-450 over a series of days for a 3 liter bioreactor volume (pg. 151, left col., 2nd para.; Table 1).
Thus, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to optimize the BioBLU stirred tank perfusion bioreactor method of Levinson for adherent pluripotent stem cell expansion on microcarriers in view of teachings of Xu by shifting from an initial agitation rpm setting to an increased agitation setting upon/after logarithmic growth after the expanding cells transition to a log growth phase to improve oxygenation and final cell densities. Further, it would have been prima facie obvious to one of ordinary skill in the art before the time of filing with the goal of generating as large a population of pluripotent stem cells as possible to optimize various parameters of the method taught by Levinson as results effective variables (see MPEP 2144.05(II)) to concomitantly optimize the inoculation amount and the agitation conditions using routine experimentation within the skills of one of ordinary skill in the art as taught by Gupta to arrive at an agitation speed increase timing at about 1 x 105 to about 10 x 105 cells/cm2 of total microcarrier surface area. It is noted that optimization may be impacted by other variables, including the medium composition, cell line/type, the microcarrier type, the matrix coating on the microcarrier (if any), use of shear protectants, oxygenation, waste removal, etc. as taught by Gupta (pg. 12-15, 21-23, 25; and 76-78) as well as the exact ratio of seeding density per available microcarrier attachable surface area as mentioned above. Furthermore, it is noted that while Levinson already teaches achieving over 50 times expansion of pluripotent stem cells using a BioBLU bioreactor and Lonza’s allogeneic platform, there is still motivation to optimize agitation parameters to more predictably ensure such 50-fold level of expansion or to further exceed it (e.g., beyond 70-fold).
Regarding claim 2, Levinson teaches using a single-use stirred tank vessel (BioBLU by Eppendorf) without any passaging step once culturing incubation is begun until achieving approximately 90-fold expansion of iPSCs (pg. 265, right col., 5th para.; Fig. 4).
Regarding claim 4, in the teachings of Levinson teaches there is no mention of any methods requiring the use of a 2D incubation process prior to inoculating the bioreactor. Thus, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to combine all of the teachings of Levinson noted above wherein there is no 2D incubation process prior to inoculation of the bioreactor, such as inoculating directly commercially available GMP-grade iPSCs shipped in a cryopreserved form or cells recovered and grown in suspension first.
Regarding claim 6, Levinson teaches commercially available microcarriers with different coatings (pg. 265, 1st para.) and demonstrated a method of greater than 50-fold pluripotent stem cell expansion detailed above specifically using precoated growth matrix-coated microcarriers (Cultispher-G) for adherent pluripotent stem cell culture (pg. 265, right col., para. 5., Fig. 4).
Regarding claim 7, Levinson teaches harvesting of cultured cells (pg. 265, right col., last para.).
Regarding claim 11, Levinson teaches performing concentrating by continuous centrifugation as part of downstream processing, such as using a kSep™ continuous centrifuge (Table 4; pg. 265, right col., last para., to pg. 266, 1st para.).
Regarding claim 17, Levinson teaches the process comprising agitation of the cells adhered to microcarriers comprises logarithm growth beginning between 5-10 days in culture (Fig. 4). Xu teaches as cell culture densities increase during cell culture such that an increasing stirrer speed is necessary just to maintain a homogenous dissolved oxygen concentration and transfer rate (pg. 151, left col., 2nd para.; Table 1). Thus, any optimization approach by one of ordinary skill in the art would investigate a ramp up of agitation speed over the days of culturing in line with the cell doubling rate in view of Xu. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform the BioBLU bioreactor method of Levinson using a gradually increasing agitation speed, which could be optimized to increase from an initial speed after about 1 to 5 days in order to maintain desired oxygen levels and homogeneity.
Regarding claim 21, Xu teaches using a xeno-free defined medium, such as BRASTEM (end of pg. 48, pg. 16).
Regarding claim 22, Gupta teaches using microcarriers having a xeno-free, defined tropoelastin matrix coating of recombinant human tropoelastin of the form SHELΔ26A (pg. 63).
Regarding claim 23, Levinson teaches a 10-day or longer protocol (Fig. 4; pg. 265, right col.).
Regarding claim 25, as set forth above for claim 1, the combination of Levinson, Gupta and Xu teaches routine optimization of seeding density, while Levinson alone teaches seeding densities around 0.01-0.05 x 106 cells/mL (Fig. 4). Note, a prima facie case of obviousness exists where claimed ranges overlap ranges disclosed in the prior art (MPEP 2144.05).
Claims 1, 4-10, 14, and 21-25 are rejected under 35 U.S.C. 103 as being unpatentable over Abecasis (Abecasis et al., J Biotechnol 246: 81-93 (2017)).
Regarding claims 1 and 25, Abecasis teaches an automated manufacturing processes for 3D aggregate culturing of pluripotent stem cells (ChiPSC4) to high densities comprising a stirred tank bioreactor (Eppendorf DasGip cellferm-pro) by inoculating the bioreactor with pluripotent stem cells at 2.5 to 5 × 105 cells/mL, applying an initial agitation (stirring) at 80 rpm and then increasing the rate to a second speed of 90 rpm for 6 days, such as at a density of about 5 × 105 cells/mL (as the maximum inoculated amount after 6 hours could only increase by 1-3%), and wherein cell passaging is performed to achieve over 50-fold expansion (e.g., 1100-fold in 11 days of culturing with 3 passagings from a seeding of 2.5 × 105 cells/mL) (Abstract; pg. 83, 86-87, 91-92; Fig. 2). Abecasis teaches freezing the expanded cells after concentrating by centrifugation with liquid nitrogen and kept at −80 °C for later analysis (pg. 85, right col., 3rd para.).
Abecasis does not teach placing a plurality of microcarriers into the bioreactor along with the pluripotent stem cells or that the seeding density should be kept below 0.2 × 106 cells/mL while the speed increase is timed to densities of about 5-20 × 105 cells/mL. However the microcarriers are not required to serve any purpose in the claimed method as noted above.
Although Abecasis does not teach the precise seeding density, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to modify the above mentioned automated process using a stirred bioreactor system taught by Abecasis but seeded with human pluripotent stem cells at variable amounts anchored around 2.5 to 5 × 105 cells/mL while increasing speed at about 5 × 105 cells/mL as already taught by Abecasis for achieving high levels of expansion (e.g., 1100-fold) such that seeding density was optimized using routine experimentation to arrive at an inoculating seeding density of 2.2 × 105 cells/mL or less while maintaining the speed increase at about 5 × 105 cells/mL and more than 6 hours after initiation. As of 2019, optimizing cell culture conditions of seeding density in stirred tank bioreactor cultures were routine and within the skills of the skilled artisan. The skilled artisan using routine experimentation would arrive at a reduction from 0.25 × 106 cells/mL (2.5 × 105 cells/mL) to 0.22 × 106 cells/mL (2.2 × 105 cells/mL) or less with a reasonable expectation of achieving about 50-fold expansion or more in view of the higher levels already achieved with very similar seeding densities. One of ordinary skill in the art with the goal of producing a large number of pluripotent stem cells would be motivated to do so because Abecasis itself teaches optimizing inoculum concentrations (Fig. 1-1) as a results-effective variable using routine experimentation (pg. 83, left col., 3rd para.). Absent evidence to the contrary, the precise seeding density is considered irrelevant to achieving 50 fold expansion or greater.
Note, it has been held where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are close enough that one skilled in the art would have expected them to have the same properties. See M.P.E.P. §2144.05(I).
Regarding claim 4, in the teachings of Abecasis there is no mention of any methods requiring the use of a 2D incubation process prior to inoculating the bioreactor with the human pluripotent stem cells. Thus, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform a method taught by Abecasis wherein there is no 2D incubation process prior to inoculation of the bioreactor, such as inoculating directly commercially available iPSCs shipped in a cryopreserved form (ChiPSC4 cell line, P18) or such cells recovered and grown in suspension without any 2D incubation. One of ordinary skill in the art would be motivated to use routine prior art methods for an already art-recognized purpose when Abecasis provides no teaching away or incompatibility with such methods and with a reasonable expectation of achieving about 50-fold expansion or more in view of the higher levels already achieved by methods silent as to this aspect.
Regarding claims 5-6 and 22, in the teachings of Abecasis there is no mention of any microcarriers nor their size or coating. But as noted above, the microcarriers are considered irrelevant to the methods taught by Abecasis. Thus, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform a method taught by Abecasis wherein there is a token amount of microcarriers of a size of 125 µm or greater and/or coated with a fluid (e.g., culture medium) and having no effect on the expansion, concentrating, cryopreserving, nor the maintenance of pluripotency. Similarly, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform a method taught by Abecasis wherein there is a de minimums amount of microcarriers coated with a recombinant, xeno-free, defined matrix and having no effect on the expansion, concentrating, cryopreserving, nor the maintenance of pluripotency, etc.
Regarding claim 7, Abecasis teaches harvesting the expanded pluripotent stem cells, i.e., by concentrating via centrifugation, such as for cryopreservation for a future analysis (pg. 85, right col., 3r d para.).
Regarding claims 8-10, Abecasis teaches using a nylon mesh as a mechanical separation method for cultured human pluripotent stem cell aggregates in a solution during a passaging (pg. 87; Table 2). Thus, when any token amount of microcarrier having no functional consequence was present, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform a method taught by Abecasis wherein a nylon mesh is used to purify the expanded cells and would inherently remove some microcarriers of certain size ranges (e.g., < 70 nm). One of ordinary skill in the art would be motivated to avoid using enzymatic dissociation protocols as taught by Abecasis as unfavorable for scaling up large batches in bioreactors (pg. 87, left col., last para.) and to specifically use meshes with higher pore size (e.g., mesh size of 70 nm) is advantageous for promoting higher viable cell recoveries and fold-expansion factors (pg. 87, right col., 1st para.).
Regarding claim 14, as Abecasis teaches the same active method steps as in claim 14, the same result is reasonable expected to occur regardless of any appreciation in the prior art of this recovery percentage.
Regarding claim 21, Abecasis teaches using the xeno-free, defined medium Cellartis® DEF-CSTM Xeno-Free culture medium (pg. 86).
Regarding claim 23, Abecasis teaches achieving over 50-fold expansion in 6 days while continuing the incubating for 11 days using the continuous expansion protocol (FIG. 5, pg. 87).
Regarding claim 24, Abecasis teaches an initial first agitation (stirring) in a stirred tank bioreactor at 80 rpm (which is over about 25 rpm) and then increasing the rate to a second speed of 90 rpm (which is at least 5 rpm greater than 80 rpm) (pg. 83).
Claims 1, 3-10, 14, and 21-25 are rejected under 35 U.S.C. 103 as being unpatentable over Abecasis as applied above and further in view of Oh (Oh et al., US20140315300A1).
Regarding claim 3, Abecasis does not expressly teach the inoculating step comprising cryopreserved pluripotent stem cells, such as cryopreserved without any intervening 2D culturing prior to inoculation.
However Oh teaches using frozen cells generally when introducing cells into a new culture system, e.g., from 2D monolayer culture to suspension culture or spinner flask to a large bioreactor ([0189]). Thus, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to modify a method taught by Abecasis to introduce cryopreserved pluripotent stem cells directly in the inoculating step as taught by Oh. One of ordinary skill in the art would be motivated to apply routine prior art methods for an already art-recognized purpose.
Claims 1, 4-14, and 21-25 are rejected under 35 U.S.C. 103 as being unpatentable over Abecasis (Abecasis et al., J Biotechnol 246: 81-93 (2017)) as applied above and further in view of Levinson (Levinson et al., Biochemical Engineering Journal 132: 262-9 (2018)), Ko (Ko and Bhatia, BioPharm International. 25(11) (2012)) and Pattasseril (Pattasseril et al., Bioprocess Int. 11: 38-47 (2013)).
Regarding claims 11-12, although Abecasis teaches concentrating using centrifugation, Abecasis does not teach wherein the centrifugating is by a continuous centrifugation device.
However Levinson teaches concentrating expanded pluripotent stem cells (iPSC) by continuous centrifugation as part of downstream processing in a bioreactor system (BioBLU by Eppendorf), such as using a kSep™ continuous centrifuge (Table 4; pg. 265, right col., last para., to pg. 266, 1st para.), which is capable of forming a fluidized bed comprising cells within the first few minutes and recovering over 90% cell viability as taught by Ko (pg. 8, Determination of initial feed flow rate (FBC); pg. 9, Table I). Furthermore, Pattasseril teaches the kSep™ continuous centrifuge system is capable of purifying cell at >80% cell recovery, which was one of the best performing commercially available such automated systems at the time (pg. 45, left col., last para.).
It would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to modify the method of Abecasis to incorporate a kSep™ centrifugation device into the bioreactor system for continuous centrifuge concentrating expanded cells as taught by Levinson and Ko as a known prior art technique used for its prior art purpose. Further, as a kSep™ continuous centrifugation device is capable of forming a fluidized bed within 15 minutes as taught by Ko, one of ordinary skill in the art would be motivated to perform the method as quickly as possible and thus use such a setting. Moreover one of ordinary skill in the art would be motivated to use the kSep system taught by Levinson because Pattasseril teaches this system provides one of the best recovery efficiencies of the commercially available continuous centrifugation systems.
Regarding claim 13, Abecasis does not expressly teach cell recovery from a fluidized bed after the continuous centrifugation is 80% or greater.
However as noted above teaches kSep™ continuous centrifuge technology is capable of recovering over 90% cell viability (pg. 9, Table I). Furthermore, Pattasseril teaches the kSep™ continuous centrifuge system as taught by Levinson (at Table 4; pg. 265, right col., last para., to pg. 266, 1st para.) uses automated counterflow centrifugation to allow the cells to remain in suspension while supernatant and residuals are cleared with >80% cell recovery, which was one of the best performing commercially available such automated systems at the time (pg. 45, left col., last para.). Thus, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform the method of Abecasis using the kSep™ continuous centrifuge system taught by Levinson and Ko which is capable of greater than 80% cell recovery from a fluidized bed as taught by Pattasseril. One of ordinary skill in the art would be motivated by Pattasseril teaching this system provides one of the best recovery efficiencies of the commercially available continuous centrifugation systems.
Claims 1, 4-10, 14, 17, and 21-25 are rejected under 35 U.S.C. 103 as being unpatentable over Abecasis (Abecasis et al., J Biotechnol 246: 81-93 (2017)) as applied above and further in view of Xu (Xu and Chen, J Biotechnol 231: 149-59 (2016)).
Regarding claim 17, Abecasis does not teach wherein a third agitation speed is used that is increased from the second speed after about 1 to 5 days of incubating.
However Xu teaches when culturing mammalian cells in stirred-tank perfusion bioreactors that the agitation rate is typically increased gradually to improve oxygen transfer as increasingly higher cell densities are reached in later stages and to avoid foam formation, e.g., impeller rpm of 260-450 over a series of days for a 3 liter bioreactor volume (pg. 151, left col., 2nd para.; Table 1).
Regarding claim 17, Xu teaches as cell culture densities increase during cell culture such that an increasing stirrer speed is necessary just to maintain a homogenous dissolved oxygen concentration and transfer rate (pg. 151, left col., 2nd para.; Table 1). Thus, any optimization approach by one of ordinary skill in the art would investigate a ramp up of agitation speed over the days of culturing in line with the cell doubling rate in view of Xu. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to perform the Eppendorf bioreactor method of Abecasis using a gradually increasing agitation speed, which could be optimized to increase from an initial speed after about 1 to 5 days in order to maintain desired oxygen levels and homogeneity during human pluripotent stem cell culture with a reasonable expectation of success.
Claims 1, 4-10, 14, and 19-25 are rejected under 35 U.S.C. 103 as being unpatentable over Abecasis as applied above, and further in view of Correia (Correia et al., Stem Cell Rev Rep 10: 786-801 (2014)).
Regarding claim 19, Abecasis does not teach wherein an agitation is applied in a discontinuous fashion within the first 24 hours after inoculation.
However Correia teaches a stirred bioreactor culture protocol for pluripotent stem cells comprising a discontinuous (intermittent) agitation profile improved cell yields compared to a continuous agitation protocol (Abstract; Fig. 3a). Thus it would have been prima facie obvious to one of ordinary skill in the art before the effective time of filing to optimize the Eppendorf bioreactor method of Abecasis by selecting a discontinuous agitation profile within the first 24 hours after inoculation for optimizing yields, such as to promote cryopreservation recovery.
Regarding claim 20, Abecasis teaches the bioreactor used in the methods use a perfusion bioreactor (pg. 83). Therefore the claimed invention as a whole is prima facie obvious before the earliest effective filing date in the absence of evidence to the contrary.
Response to Arguments
Applicant's arguments filed 7/27/26 regarding obviousness have been fully considered but not found completely persuasive as laid out in the modified and new rejections above.
Applicant traverses (pg. 26) arguing that the specificity of the seeding density and increased agitation trigger would not be merely stumbled upon by routine experimentation in view of the combined prior art references. However as noted above, achieving 50-fold expansion was capable by prior art methods without microcarriers and over various seeding densities and agitation settings. Thus, the result of the process of claim 1 was achievable with a reasonable expectation of success in view of the prior art and the exact seeding density or agitation speed change timing is not considered essential for the 50-fold or greater expansion. Rather, these represent adjustable parameters known in the prior art which are not yet demonstrated by Applicant to impact the method in any specific manner and thus appear auxiliary to the claimed result. Thus, routine adjustment and experimentation before the filing date could arrive at various seeding densities and agitation settings and still achieve 50-fold expansion, including ones either included or excluded from a range recited in claim 1.
As noted in the previous Office action, applicant has not yet established the criticality of any specific limitation to the seeding density or agitation at a particular cell density during the incubation, with only original claim 18 being so limited when claiming the invention but original claim 1 being unlimited by this regard. If this range is critical, the ordinary skilled worker would reasonably be expected to find an overlapping cell density using routine optimization of expansion results. However as the precise density does not appear to be critical and as the prior art teaches 50-fold expansion in the absence of any agitation increase, this agitation limitation is not considered to be critical for meeting any other claim limitation (e.g., an about 50 times or greater fold expansion).
Applicant also traverses by arguing (pg. 24-25) Levinson does not teach a seeding density within the range recited in claim 1 or is too vague in Fig. 4. It is the opinion of this Office action that Fig. 4 shows two different seeding densities, both much less than 0.5 x 106 and approximately 0.01-0.05 x 106 cells/mL based on the Y-axis scale. Furthermore, the seeding density is routinely optimized in the prior art for various results-effective variables, such as expansion level, and Fig. 4, despite its vague precision, clearly teaches a starting point less than 0.2 x 106. Moreover in an admitted prior art teaching(s), the instant specification states previously a seeding density of 0.2 x 106 cells/cm2 was taught ([0052]).
Applicant also traverses by arguing (pg. 25) Levinson does not teach achieving over a 50-fold expansion as recited in claim 1. Firstly, only a 45-fold expansion is required by claim 1 due to the term “about.” Second, using an appropriate estimate of the cell densities graphed in Fig. 4 of Levinson, even at conservative values, clearly shows a pluripotent stem cell expansion of over 50-fold (e.g., the lowest seeding density data point (Seed density A) starts at around 0.06 x 106 cells/mL and ends over 3.7 x 106, which is approximately a 61-fold expansion by conservative estimation.
Levinson also notes expanding 50-100 fold other stem cells with the same microcarrier culture setup (pg. 265). As to the sentence stating “, also”; a reasonable interpretation is Levinson et al. saying they have expanded bmMSC using the microcarrier stirred tank platform and have expanded 90-fold iPSCs using the same platform, which is adherent microcarrier culture as well (too or also). Applicants assertion of a “clear” reference to two separate phenomena is considered but not accepted as dispositive.
A more precise analysis of the data presented in Fig. 4 suggests the “Seeding density A” line begins at about 5 x 104 cells/mL and ends at about 3.9 x 106 resulting in calculating a fold expansion of about 70-fold, while the “Seeding density B” line begins at about 2 x 105 cells/mL and ends at about 3.6 x 106 showing only an 18-fold expansion.
It is noted that Levinson et al. is Applicant’s own publication. Thus, any confusion as to what Levinson ambiguously discloses could be resolved by a declaration from the Applicant (see response pg. 24-25). For example regarding Fig. 4, applicant is free to dispositively assert via declaration that the disclosure of Levinson is too vague to establish a seeding density less than 0.2 x 106 cells/mL, such as by Fig. 4 therein failing to clearly show an approximate seeding density less than 0.2 x 106 cells/mL resulting in a final cell density over 45-fold greater. Similarly regarding the text of Levinson, applicant is free to dispositively assert via declaration the unambiguous meaning of the statement "[w]e have also demonstrated the platform's ability to expand iPSCs approximately 90-fold, also as adherent culture on microcarriers (Fig. 4)." As noted in the instant rejection above, both of these disclosures of Levinson are interpreted contrary to applicant’s response arguments.
Applicant is reminded of their duty to disclose information material to patentability. See MPEP §2001 and 37 C.F.R. 1.56. The individuals covered by 37 CFR 1.56 have a duty to bring to the attention of the examiner, or other Office official involved with the examination of a particular application, information within their knowledge as "material to patentability" of the application in question. As set forth by the court in Armour & Co. v. Swift & Co., 466 F.2d 767, 779, 175 USPQ 70, 79 (7th Cir. 1972): [T]he applicant has the burden of presenting the examiner with a complete and accurate record to support the allowance of letters patent. See MPEP §2001.06(b). The examiner kindly acknowledges the disclosure of Levinson in an IDS and is merely asking Applicant to help create an accurate record as to what Levinson discloses.
The instant specification begins ([0005]-[0006]) with a problem to be solved, it would be beneficial to provide a microcarrier-based culture system process for hPSC expansion that provide large surface areas and surface area to volume ratios for adhesion and growth during expansion for improved scalability, flexibility in using defined extracellular matrices, and allow maintenance of homogenous culture condition while generating “large numbers of high quality hPSCs” and/or “microcarrier-based expansion that uses xeno-free culture conditions”, such as a cGMP compliant end-to-end process where the “cells are expanded in a closed, automated and controlled single-use stirred tank bioreactor.” “It would be an additional benefit if an end-to-end platform that solved one or more of the above problems while also achieving a high expansion fold of >50 using the platform within 9-14 days of culture. Furthermore, it would be advantageous if the expanded hPSCs demonstrate high quality of self-renewal and pluripotency, and/or are capable of differentiation to all three germ layers.” However, the presently claimed invention has claim limitations indicating solving the stated problems of using (1) inoculating cryopreserved cells in dependent claim 3 and/or (2) xeno-free conditions for such microcarrier expansion in the combination of dependent claims 21-22, but does not purport to solve any other of the stated problems of the introduction, such as regarding the quality hPSC or efficiency (i.e., within 9-14 days). Also, it is noted that none of these limitations are in the independent claims (1 or 26), and claim 23 does not ensure the time is 14 days or less as it lacks an upper time limit, encompassing any time greater than 9 days and unlimited passagings.
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 ERIC J ROGERS whose telephone number is (571)272-8338. The examiner can normally be reached Monday - Friday 9:00-6:00.
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/ERIC J ROGERS/Examiner, Art Unit 1638
/JAMES D SCHULTZ/Supervisory Patent Examiner, Art Unit 1631
