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 .
Claim Status
Claims 1-14, 16-17, and 19-20 are currently pending in this application.
Previous Rejections
Status of the rejections: the previous claim rejections under 112(b) are withdrawn in view of the claim amendments except as maintained below.
Claim Objections
Claim 1 is objected to because of the following informalities: the amendments to claim 1 are illegible due to scanning of a submission with colored text, i.e., colored MS Word tracked changes, particularly the superscripts. See 37 CFR 1.52.
Claim Interpretation
In the claims, the term “about” is considered as encompasses a 10% range of values both less and greater than the subsequent value.
In claim 1, the phrase “incubating the pluripotent stem cells in the bioreactor for a period of time sufficient to yield a fold expansion of about 50 times or greater to give expanded pluripotent stem cells” is interpreted as requiring a fold expansion of about 50 times or greater actually occurs when performing the claimed incubating step, i.e., not from counting any expansion of pluripotent stem cells outside of the bioreactor, such as during a passaging (claim 2).
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/cm2” to “10 X 105 cells/cm2.” However the agitation speed at the “initial” speed is interpreted wherein “initial” means the agitation just prior 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 1 x 105 cells/cm2. 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 below about 0 °C, i.e., frozen.
In claim 8, the term “non-enzymatic passaging solution” is interpreted as encompassing any cell solution that promotes cell detachment from a microcarrier of interest, such as a hypertonic salt water solution or hypertonic saline (see instant [0031]; [0033]; [0058]; [0068]; [0074]-[0076]; [0010]).
Claim Rejections - 35 USC § 112(a) - Written Description (new)
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 1-14, 16-17, and 19-20 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.
The claimed invention as a whole is not adequately described if the claims require essential or critical elements that are not adequately described in the specification and that is not conventional in the art as of applicant’s effective filing date. Possession may be shown by actual reduction to practice, clear depiction of the invention in a detailed drawing, or by describing the invention with sufficient relevant identifying characteristics such that a person skilled in the art would recognize that the inventor had possession of the claimed invention. Pfaff v. Wells Electronics, Inc., 48 USPQ2d 1641,1646 (1998).
In making a determination of whether the application complies with the written description requirement under 35 U.S.C. 112(a) or 35 U.S.C. 112, first paragraph, it is necessary to understand what Applicant is claiming and what Applicant has possession of.
In view of the specification, claim 1 is directed to processes for manufacturing in a bioreactor containing microcarriers more pluripotent stem cells from provided pluripotent stem cells to yield at least about a 50-fold expansion of the cells (45 to 55 fold). It is also noted that fold expansion is interpreted to occur solely during the incubating period and the inoculated amount of cells may be as low as 2 pluripotent stem cells meaning 50-fold expansion occurs after enough cell divisions to produce about 90-100 daughter cells. The claims are broad in that (1) the incubating conditions are unlimited, (2) the minimum seeding density is unlimited, (3) the amount and type of microcarriers is unlimited, and (4) the pluripotent stem cells need not adhere to any microcarrier(s).
Breadth of incubating conditions
Claims 1-14, 16-17, and 19-20 each encompasses any incubating conditions, e.g., any media compositions and yet the recited method purportedly is capable of producing at least about 10 x 105 cells/cm2 from a seeding density of 50-fold less (e.g., approximately less than 1.6 x 104 cells/cm2) wherein all these cells are pluripotent stem cells. The prior art teaches some media/conditions cause loss of pluripotency, i.e., differentiation or cell fate specification, including due to matrix/substrate choice, poor attachment, agglomeration/aggregation, agitation-related stress, and/or persistence of pluripotent stem cell secreted factors and metabolites (Adil et al., Curr Opin Chem Eng 15: 24-35 (2017) at pg. 29, left col., para. 1-2). The prior art also teaches that pluripotency is not maintained at 100% during long term cell culture, e.g., 20% loss of Oct4 expression over 7 days (id.).
Breadth of microcarriers
Claims 1-4, 6-14, 16-17, and 19-20 each encompasses any amount and type of microcarrier. This means these claims encompass wherein there are only two microcarriers, each with a diameter of 30-40 microns or less. 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)). Claim 5 merely limits the minimum size of the microcarriers without ensuring adherent culture conditions.
Adherent versus in solution culturing
Claims 1-14, 16-17, and 19-20 each encompasses wherein the cells are in solution and not adherent during the incubating period resulting in the about 50-fold expansion. The prior art teaches pluripotent stem cells can be expanded in solution or adhered to a surface (Adil et al., at Fig. 2). For any implied adherent culture, the claims need to provide not only adherent capable microcarriers suitable for pluripotent stem cells but also enough adherent-effective microcarrier surface area to accommodate the final 50-fold or greater expanded amount of cells, e.g., an estimated minimum of 4-6000 cm2 of total available adherent surface area (assuming an about 50 µm pluripotent cell diameter over 10 x 105 cells/cm2).
In analyzing whether the written description requirement is met for genus claims, it is first determined whether a representative number of species have been described. In the instant case, the specification fails to provide sufficient species of the genus of processes resulting in at least 45-fold expansion across the full-breadth of (1) any incubating conditions, (2) any amount of microcarriers, (3) microcarrier types that prevent pluripotent stem cell adherence, and starting with as few as two pluripotent stem cells.
The incubation conditions are described in a non-limiting manner as generally for human induced pluripotent stem cells (hiPSCs) using a xeno-free and defined medium ([0024]), such as using a bioreactor of any suitable volume ([0037]) containing a nutrient media [0047]), which may or may not comprise any of one 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; any gas, such as oxygen or carbon dioxide; any carbohydrate source; an amino acid(s) including a non-standard amino acid(s); any vitamin; any fatty acid; any lipid; any protein; any peptide, including growth factors and growth inhibitors; and any trace element, inorganic salts, and/or hydrolysate ([0028]-[0029]). There is no limit discussed for the amount of dissolved oxygen in the claims and yet admitted prior art teachings guide that oxygen levels can impact expansion and maintenance of pluripotency, with the instant specification teaching using at least 5-10% dissolved oxygen or even as much as 30%-50% ([0095]-[0096]).
The bioreactor may be adherent or designed for non-adherent culturing ([0037]) and may optionally comprise an impeller(s) and/or a means to remove waste (FIG. 1B; [0038]-[[0041]). Without specifying the bioreactor volume or shape nor the agitation means, the agitation is described as 25-125 rpm as well as increasing over a period of days by at least 5-30 rpms ([0054]). As the agitation is described as including any wave-type rocking motion or any stirred tank bioreactor, such as the disclosed species of agitator that is a dual impeller or any attachment to the rotator shaft 14, the translation of such rpms to different formats is unpredictable without modification. Thus, the description of agitation conditions is very limited and lack guiding nexus to prior art teachings.
For microcarriers, the instant specification describes microcarriers as made of any material and having any shape, such as having a median size from about 50 μm to 350 or a size of at least about 125 μm, with specific examples of SoloHill polystyrene plastic microspheres having a diameter within the range of 90-212 μm and coated with L7™ hPSC Matrix ([0050]; [0073]-[0078]). However, the microcarriers may be uncoated or coated ([0051]) and of smaller sizes, e.g. 81 μm (id.).
In its description, nowhere does the application provide a single working example for a process according to claim 1 that occurs within less than 9 days (FIG. 33), wherein the microcarriers are not porous plastic spheres coated for pluripotent stem cell adhesion, wherein the medium used is not a supplemented Lonza L7™ TFO2 medium, wherein the seeding density is less than 0.02 x 106 cells/mL. There is no working example wherein the agitation speed is described as being increased.
Instead, the instant application provides a single working example for claim 1 based on a three-liter (3L) bioreactor (Eppendorf BioBLU 3c) inoculated either with cell clusters or individualized pluripotent stem cells ([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 comprising L7™ hPSC Matrix-coated plastic microcarriers, all of which is then incubated at 37 °C, e.g., for 9-16 days apparently at an agitation rate of 50 RPM until greater than 2 x106 cells/mL total were produced.
The specification fails to provide any species of microcarrier beyond polystyrene microspheres coated with extracellular matrix promoting pluripotent stem cell adhesion and having a diameter within 90-212 μm. The specification 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 oxygen, carbon dioxide, carbohydrate, amino acid(s) including any non-standard amino acids; vitamins, fatty acid, lipid, growth factors, or growth inhibitors in such a medium. The presence of certain growth factors (e.g., BMP4) or insufficient bFGF levels can dramatically alter pluripotency while certain growth inhibitors may completely arrest expansion. Yet, the ingredients in the L7™ TFO2 medium and hPSC L7™ supplement are not described.
The specification lacks any description of a specific temperature, atmospheric setting, or pH goal used during the incubating, such as to achieve the requisite cell expansion, e.g., over an incubation period of 9-14 days. Typically, a cell culture medium comprises sufficient amount of a carbon source (e.g., a carbohydrate), a nitrogen source (e.g., certain amino acids), and a phosphate source (e.g., inorganic phosphate salt); however for 50-fold expansion of mammalian pluripotent stem cells there may be unique requirements specific to this cell type.
The described species lacks a sufficient nexus to the breadth of the genus of any incubating conditions and bioreactor. The described species also lacks a sufficient nexus to the breadth of any microcarrier as well as the scope encompassing both non-adherent and adherent culture conditions.
As claim 13 requires at least about 80% pluripotent stem cell recovery from the fluidized bed, only the single working example provides empirical evidence of such but depending on using the Eppendorf BioBLU 3c bioreactor and kSep(400.50, Sartorius) automated setup. The instant application is silent as to any other effective process to achieve such despite various non-limiting open-ended descriptions of closed and/or automated bioreactor systems.
The skilled artisan could not rely upon the disclosure such that the instant specification would sufficiently describe that Applicant was in possession of a process having a predictable effect of expansion of about 50 times or greater over the entire scope of the claims. While there is evidence that providing sufficient adherent surface area among suitable microcarriers may allow for expansion to cell densities beyond 11 X 105 cm2 there is a lack of evidence in the prior art and instant application that the method of claim 1 achieves such expansion and maintenance of pluripotency state over the full scope of the claims, including when limited by claims 2-14, 16-17, and 19-20 for the same reasons explained above.
35 USC § 112(a) – Scope of Enablement (new)
Claims 1-14, 16-17, and 19-20 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 and the incubating conditions promote pluripotent stem cell growth and preserve pluripotency; 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 is unlimited in the incubating conditions and the minimum seeding density.
Enablement is considered in view of the Wands factors (MPEP 2164.01 (a)). The court in Wands states that "Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue experimentation. The key word is 'undue.' Not 'experimentation;" (Wands, 8 USPQ2d 104). Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. "Whether undue experimentation is needed is not a single, simple factual determination, but rather is a conclusion reached by weighting many factual considerations." (Wands, 8 USPQ2d 1404).
The factors to be considered when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation required is “undue” include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
Furthermore, the USPTO does not have laboratory facilities to test if an invention will function as claimed when working examples are not disclosed in the specification. Therefore, enablement issues are raised and discussed based on the state of knowledge pertinent to an art at the time of the invention. And thus, skepticism raised in the enablement rejections are those raised in the art by artisans of expertise.
All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Nature of the invention:
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 (1) the incubating conditions are unlimited, (2) the minimum seeding density is unlimited, (3) the amount and type of microcarriers is unlimited, and (4) the pluripotent stem cells need not adhere to any microcarrier(s) during the incubation period.
The state of the art:
The prior art teaches methods of culturing pluripotent stem cells with microcarriers to achieve over 50-fold expansion and producing cell densities of 1 x106 per mL using a stirred-bioreactor system, e.g., using Cytodex 1 microbeads (190 µm diameter) with an anionic surface at over 1 g/mL in a 2L bioreactor with 60 rpm agitation via a 40 mm blade impeller or 60 mm stirring bar and mTeSR medium at 37 C with 5% CO2 and O2 at least above 5-7% (Olmer et al., Tissue Eng Part C Methods 18: 772-84 (2012) at pg. 778, right col., last para., to pg. 779, right col., 1st para.; Fig. 3 (e.g., achieving 5 x 105 to 2 x 106 cell/mL by day 7 from a seeding density of 4–5 105 cells/mL)). The prior art also teaches achieving over 50-fold expansion using Cultispher-G (porous gelatin beads of 130-180 µm diameter) in an automated stirred bioreactor system, such as using NutriStem hPSC media (Levinson et al., Biochemical Engineering Journal 132: 262-9 (2018) at pg. 264, right col., 2nd para., to pg. 266, 1st para.; Fig. 2-4; Abstract).
In an admitted prior art teaching(s), the instant specification states previously a seeding density of 0.2 x 106 cells/cm2 was taught as optimal with as low as 0.01 ×106 cells/cm2 demonstrated as possible in the instant empirical data ([0053]), microcarriers of a size of 90-150 µm was taught as optimal with as large as 212 µm demonstrated as possible in the instant empirical data ([0050), and a certain amount of dissolved oxygen was required for optimal expansion, such as 20 or 30% with evidence of as low as 5% being acceptable ([0096]).
The prior art is silent as to using an adherent bioreactor with merely unoccupied microcarriers present, e.g., microcarriers too small for pluripotent stem cell attachment or in amounts totaling insufficient surface area for adherence of the entire 50-fold expanded cell population. The prior art is silent as to many incubating conditions, including using media comprising a growth inhibitor or lacking at least 5% oxygen, lacking any carbohydrate, lacking essential amino acids and certain vitamins, trace element, and inorganic salts. The prior art is silent as to incubating conditions involving pluripotent stem cell agglomeration/aggregation but teaches aggregation induces pluripotent stem cell differentiation (Lipsitz et al., Biotechnol Bioeng 115: 2061-6 (2018) at pg. 2061). The prior art is also silent as to very high (e.g., above 60 rpm) and low agitation levels, teaches away from 30 rpm or less in some setups (Olmer et al., at pg. 776, right col., 1st para.), and that some agitation conditions can cause loss of pluripotency (Leung et al., Tissue Eng Part C Methods 17: 165-72 (2010) at abstract).
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:
The instant application provides a single working example for claim 1 based on a three-liter (3L) bioreactor (Eppendorf BioBLU 3c) inoculated either with cell clusters or individualized pluripotent stem cells ([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 comprising L7™ hPSC Matrix-coated plastic microcarriers, all of which is then incubated at 37 °C, e.g., for 9-16 days, and eventually producing 6-15 x109 concentrated expanded cells, presumably with more than 80-90% of which maintain pluripotency. The initial agitation rate was 50 RPM but no increased agitation is noted. Neither any amount of microcarrier nor any surface area available for pluripotent stem cell adherence (e.g., total specific surface area due to porosity) is clearly provided for the sole working example, but it is implied that 20 grams of polystyrene (SoloHill) were used (see [0073]). The incubation period was ended when 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).
From the empirical data of the single working example, it is not necessarily predictable that a 50-fold or greater expansion of pluripotent stem cells would be achieved wherein enough cells maintain pluripotency and viability to meet all the limitations of claim 1. Many of the incubating conditions are critical to predictability, including a minimum oxygen level, precise media compositions, seeding density, microcarrier type and amount of microcarrier surface area, as well as minimum and maximum agitations. However applicant is invited to furnish evidence the contrary.
Applicant’s own arguments in the previous response stresses the importance of a minimum agitation in improving cell expansion specifically of human induced pluripotent stem cells in suspension (citing instant [0107] and [0132]) (response pg. 8, first full para.) and yet the claims open-endedly recite any agitation speed.
For claim 13, both the concentrating and cryopreserving are restricted to being a closed and/or automated process wherein 80% cell recovery is achieved in a continuous centrifugation device that can perform in about 15 minutes or less. This has only been demonstrated in single working example using both an Eppendorf BioBLU 3c bioreactor and kSep(400.50, Sartorius). The instant application lacks any guidance on how to adapt this to other bioreactors or spinning flask systems.
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 (1) the incubating, (2) the microcarrier type and amount, and (3) no minimum seeding density. Furthermore, the claims are narrow in that a fold expansion of about 50 times or greater must be achieved as well as an implied capability of reaching about 1 x 105 to 10 x 105 cells/cm2.
Thus, neither the instant application nor the prior art describes a method enabled over the full scope of claim 1 and there is little to no guidance as to how to adapt the single working example to the broad in the scopes of (1) any incubating conditions (e.g., media) and (2) any microcarrier type and amount. This leaves the unguided skilled artisan only with the teachings of the closest prior art.
The quantity of experimentation needed to make and/or use the invention:
Extensive experimentation would be required to determine how to achieve 50-fold expansion of pluripotent stem cells across their full scope of claim 1. 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.
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. Given the lack of working examples, the limited guidance provided in the specification, the lack of guidance in the prior art, and the broad scope of the claims with regard to several features simultaneously, undue and unreasonable experimentation would have been required.
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, 16-17, and 19-20 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 a cell density in the units cells/mL (volume) for inoculating and then a subsequent cell density in the units “cells/cm2” (area) for cells after incubating and agitating with microcarriers. As neither the claim nor the instant specification defines what area is being considered for “cells/cm2”, e.g., bioreactor surface area and/or microcarrier surface area, the term “/cm2” is ambiguous and indefinite. Further, a claim may be rendered indefinite by reference to an object that is variable. (MPEP §2173.05(b)). Here, the determination of an undefined area in “cm2” is an indefinite variable as it may depend on if the cells are in suspension or adherent, the bioreactor surface area and working volume, the height of liquid associated with the surface(s), number of cell layers, total scaffold area, if any, and cell distribution over the area or surface(s). Claims 2-14, 16-17, and 19-20 are included in this rejection for depending on indefinite claim 1. Moreover, the contingent step conditioned on when the cell density reaches at least about 10 X 105 cells/cm2 is incoherent as the initial cell density is limited in different units to being no greater than about 200 x 105 (0.2 x 106) cells/mL, which does not appear to ensure the cell density at the inoculating step is below the contingent amount required for both claim limitations to be coherent with each other.
Claim 6 is indefinite for referring to a “growth” matrix with regard to a coating for a microcarrier. The term “growth 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. 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 an “extracellular matrix”, “culture matrix” or “cell culture matrix.”
Response to arguments
Applicant’s response filed 10/3/25 argues that the term “growth matrix” is understood by those skilled in the art but without providing any evidence. A review of the prior art indicates that while those skilled in the art understand the terms extracellular matrix, culture matrix, and matrix-coated in the field of cell culture, the term “growth” is not ordinarily present. For example, Lonza L7™ microcarriers are described as coated with the L7™ human pluripotent stem cell (hPSC) Matrix, a proprietary, chemically defined recombinant protein and commercially available product (see Baghbaderani et al., PLoS One 11:e0161229 (2016) at pg. 3, 1st para.).
Claim Rejections - 35 USC § 112(d) (new)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 16 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 16 broadens (or incoherently alters) the scope of claim 1 to wherein the initial speed is increased after about 1 to 5 days to a second speed in contradiction to when the cell density reaches about 1 X 105 to 10 X 105 cells/cm2.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103 (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, and 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Levinson (Levinson et al., Biochemical Engineering Journal 132: 262-9 (2018); IDS ref.) 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, Levinson teaches closed automated manufacturing processes for culturing stem cells to high densities comprising placing a plurality of 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)), including at a seeding density less than 0.2 x 106 cells/mL (< 5E+05 iPSCs); 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., 90-fold) to produce expanded pluripotent stem cells (pg. 264, right col., 2nd para., to pg. 266, 1st para.; Fig. 2-4; Abstract). 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 dosage (pg. 265, left col., para. 5, to pg. 266, 1st para.).
Although Levinson does not teach a single method comprising both expanding the pluripotent stem cells about 50 times or greater and then 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 1 x 105 cells/cm2 to about 10 x 105 cells/cm2, 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 in using a BioBLU bioreactor and Lonza’s allogeneic platform, there is still motivation to optimize agitation parameters to ensure such level of expansion or even exceed it.
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 16, although Levinson teaches the process comprising agitation of the cells adhered to microcarriers and logarithm growth begins between 5-10 days in culture (Fig. 4).
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 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.
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.
Claims 1-4, 6-7, 11, and 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Levinson in view of Gupta and Xu as applied above, and further in view of Oh (Oh et al., US20140315300A1; IDS ref.).
Regarding claim 3, Levinson, Gupta and Xu does not expressly teach the inoculating step comprising cryopreserved pluripotent stem cells.
However Oh teaches thawing pluripotent stem cells onto microcarriers provides quick adherence (Example 17; FIG. 11; [0521]; FIG. 4) as well as 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 large bioreactor ([0189]).
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 Levinson 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 quickly bind the cells to the microcarriers which as taught by Oh is promoted by this type of inoculation.
Claims 1-2, 4-7, 11, and 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Levinson in view of Gupta and Xu as applied above, and as evidenced by Twal (Twal et al., Ann Biomed Eng 42: 1470-81 (2014)).
Regarding claim 5, although Levinson does not expressly teach the particle size of the microcarriers used in their methods, the microcarrier taught by Levinson (Cultispher-G) inherently has a particle size of about 125 µm (112-137 µm) or greater as evidenced by Twal teaching Cultispher-G particles have an average diameter of 130–380 μm (pg. 1471, left col., last para.).
Claims 1-2, 4, 6-8, 11, and 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Levinson in view of Gupta and Xu as applied above, and further in view of Derakhti (Derakhti et al., Mater Sci Eng C Mater Biol Appl 103:109782 (2019)).
Regarding claim 8, Levinson , Gupta and Xu does not describe any solution used for separating stem cells from microcarriers; however, Derakhti teaches while enzymatic separation of stem cells from microcarriers is an option, non-enzymatic detachment is preferred because using enzymes like trypsin are damaging (pg. 10, right col., para. 2-3). 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 Levinson to use a non-enzymatic passaging solution to separate the pluripotent stem cells from the microcarriers. One of ordinary skill in the art would be motivated as taught by Derakhti teaching this approach is preferred to avoid introducing enzymatic damage.
Claims 1-2, 4, 6-11, and 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Levinson in view of Gupta and Xu and Derakhti as applied above, and further in view of Bardy (Bardy et al., Tissue Eng Part C Methods 19: 166-80 (2013)).
Regarding claim 9, the combination of Levinson, , Gupta, Xu, and Derakhti does not expressly teach a filtering step comprising the sieving action of a mesh that restricts the microcarriers to separate the expanded cells from the microcarriers.
However Bardy teaches methods of microcarrier suspension cultures for high density expansion of pluripotent stem cells wherein the microcarriers are separated from expanded cells by passage through a 40-µm mesh (BD Falcon) after a dissociation step (pg. 169, right col., 3rd para.; Abstract).
Thus regarding claims 9-10, 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 Levinson to use a non-enzymatic passaging solution to separate the expanded pluripotent stem cells from the microcarriers as taught by Derakhti and then to filter the cells away from the microcarriers by passage through a 40-µm mesh. One of ordinary skill in the art would be motivated to recovery the dissociated cells and Bary provides a routine method of cell harvesting by filtration using a 40-µm mesh.
Claims 1-2, 4, 6-7, 11-13, and 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Levinson in view of Gupta and Xu as applied above, and further in view of Ko (Ko and Bhatia, BioPharm International. 25(11) (2012)) and Pattasseril (Pattasseril et al., Bioprocess Int. 11: 38-47 (2013)).
Regarding claim 12, Levinson, Gupta and Xu does not expressly teach wherein the flow rate of the continuous centrifugation step allows for formation of a fluidized bed in about 15 minutes or less.
However Ko teaches the kSep™ continuous centrifuge technology as taught by Levinson (at Table 4; pg. 265, right col., last para., to pg. 266, 1st para.) is capable of forming a fluidized bed comprising cells within the first few minutes (pg. 8, Determination of initial feed flow rate (FBC)) and recovering over 90% cell viability (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 Levinson already using a kSep™ continuous centrifuge to choose a setting capable of forming a fluidized bed within 15 minutes as taught by Ko because one of ordinary skill in the art would be motivated to perform the method as quickly as possible. 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, Levinson 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 Levinson using the kSep™ continuous centrifuge system taught by Levinson which is capable of greater than 80% cell recovery from a fluidized bed as taught by Ko and Pattasseril. One of ordinary skill in the art would be motivated by Pattasseril teaching this system provides one of the best recovery efficiency of the commercially available continuous centrifugation systems.
Claims 1-2, 4, 6-7, 11, 14, and 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Levinson in view of Gupta and Xu as applied above, and further in view of Liu (Liu and Chen, Curr Protoc Stem Cell Biol 31 (2014)).
Regarding claim 14, Levinson, Gupta and Xu does not expressly teach a recovery of 70% or more pluripotent stem cells after cryopreservation.
However Liu teaches routine protocols for cryopreservation and recovery of cryopreserved pluripotent stem cells capable of maintaining viability of at least 70% or more the cells (Fig. 4C; pg. 13, 2nd para.; pg. 6-9; Fig. 2-3). 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 taught by Levinson and Xu using a cryopreservation and recovery protocol known in the art to recover at least 70% viable cells after pluripotent stem cell cryopreservation and thawing. One of ordinary skill in the art would be motivated by Liu teaching recovery methods achieving at least about 80% viability.
Claims 1-2, 4, 6-7, 11, 16-17, and 19-20 are rejected under 35 U.S.C. 103 as being unpatentable over Levinson in view of Gupta and Xu as applied above, and further in view of Correia (Correia et al., Stem Cell Rev Rep 10: 786-801 (2014)).
Regarding claim 19, although Levinson teaches the process comprising agitation of the cells adhered to microcarriers, neither Levinson nor Xu expressly teaches 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 BioBLU bioreactor method of Levinson by testing and selecting a discontinuous agitation profile within the first 24 hours after inoculation when optimizing yields, such as to promote cryopreservation recovery, microcarrier loading, and minimize initial shearing before the cell population undergoes an average first cell division, i.e., with the first 24 hours.
Regarding claim 20, Levinson teaches wherein the process uses a perfusion bioreactor (BioBLU by Eppendorf) (pg. 265, right col., 5th para.; Fig. 5). 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 3/5/26 have been fully considered and are partially persuasive; however upon further consideration, a new ground of rejection is made in view of Gupta.
Applicant traverses the previous 103 rejection by arguing the prior art of record fails to unambiguously teach a precise inoculation seeding density of 0.2 x 106 cells/mL or less. As “or less” has no minimum amount, the claims can be rendered obvious by any teaching of a seeding density below 0.2 x 106 cells/mL. Note, an overlapping range between the prior art and a claim provides a prima facie case of obviousness (see MPEP 2144.05).
Applicant traverses the previous 103 rejection by arguing the prior art of record fails to teach an increasing agitation speed precisely when the cell density reaches about 1 X 105 cells/cm2 to about 10 X 105 cells/cm2. However as noted in both the previous rejection and new rejection above, increasing agitation was well-known in the prior art for any type of cell culture as cell densities increases to ensure sufficient oxygenation, nutrition, waste removal, and foam avoidance for continued proliferation. Moreover, a gradual increase in agitation meets the claim limitation so long as a speed step up of any kind occurs between about 1-10 x 105 cells/cm2 despite applicant’s arguments to the contrary (pg. 9), especially in view of claim 19 denoting claim 1 encompasses discontinuous agitations throughout the incubating.
Applicant traverses the previous 103 rejection by arguing that even if motivated to combine teachings in Levinson and Badenes, the prior art presents too many factors to consider simultaneously to arrive at an optimal increasing agitation rate based on monitoring cell density aligned with 1-10 x 105 cells/cm2. This arguments raises questions of 112(a) deficiencies in the claims as noted in the new rejections above. Further, it is the opinion in this office action that it is within the skills of one of ordinary skills to optimize all these parameters sufficiently and simultaneously to meet all the claim limitations, at least for a single embodiment (e.g., a BioBLU stirred bioreactor seeded with human iPSC). One of ordinary skill in the art would be aware of teachings in the prior art about increasing cell densities relation to poor cell expansion, such as due to oxygen, nutrition and waste accumulation, and the known solution of increasing agitation as taught by Xu. Gupta provides detailed guidance on optimizing various parameters, importantly agitation speeds and seeding densities to guide such empirical optimizations.
Furthermore, based on the instant claim language, the contingent condition of need never occur. Thus, the prior art need not teach any reason to increase from an initial agitation speed to a second speed to render claim 1 obvious, such as wherein the process is optimized for seeding at less than 20 x 104 cells/cm2. Then the claimed method is prima facie obvious absent any increase in speed at a cell density time point equivalent to about 1 x 105 cells/cm2 to about 10 x 105 cells/cm2 because this contingency never occurs. Regardless, the rejection above puts forth the obviousness of arriving at such a wide range of cell density (about 1-10 x 105 cells/cm2) as a trigger for increasing agitation speed, such as the logic of setting a timing with the cells anticipated entry into logarithmic growth phase or just after such entry or instead by blind empirical optimization without this specific logic, i.e., trial and error.
Although applicant argues no reasonable expectation of success to find such a precise timing range. However, 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 find an overlapping timing using routine optimization. However as the precise density does not appear to be critical, as the prior art teaches 50-fold expansion in the absence of any agitation increase, this limitation is not considered to be critical for meeting any other claim limitations, i.e., a recited requisite result of performing the process (e.g., an about 50 times or greater fold expansion).
Conclusion
No claim is allowed.
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/ERIC J ROGERS/Examiner, Art Unit 1638
/Tracy Vivlemore/Supervisory Primary Examiner, Art Unit 1638
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