Variable-diameter bioreactors

Abstract

To provide variable-diameter bioreactors.SOLUTION: A variable-diameter bioreactor vessel is provided that includes a first vessel section having a first diameter configured to hold liquid media and biologic material, and a second vessel section having a second diameter that is greater than the first diameter, such that the liquid media can be increased from a first volume to a second volume within the vessel.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 354,216, filed June 24, 2016, the contents of which are incorporated herein by reference.

[0002] The present disclosure relates to bioreactors, and more particularly, to the production of biological materials. [Background technology]

[0003] Typically, the production of bio-based materials is accomplished using a train of bioreactors. The train of bioreactors consists of multiple bioreactors that scale up from a small seed reactor to full production scale. The typical bioreactor is configured such that the liquid height is a dimension larger than the vessel diameter. That is, a typical bioreactor has a height to width aspect ratio greater than 1:1, whereas a typical reactor of small volume (1 / 20th of the working volume) has a very small aspect ratio (liquid height to vessel width). This small aspect ratio is known to make aeration and mixing difficult, and ultimately can lead to cell growth difficulties and undesirable cell death. A typical bioreactor is designed and sized to scale up from an inoculum in a seed reactor of incrementally increasing volume to a culture volume sufficient for production of the desired product in a production bioreactor. A typical bioreactor is designed with a fixed size, dished head and bottom. Bioreactors are generally constructed of stainless steel tanks, but may have disposable liners, disposable bags, etc.

[0004] Thus, production-scale bioreactor processes suffer from the footprint of large bioreactor trains, high cleaning costs, unfavorable delay times when switching between reactors in a train, and wasted seeding time. Each seed bioreactor involves a transfer from one bioreactor to another, introducing the culture to different conditions than at the end of the previous bioreactor. This typically creates a "lag phase" effect, where cell growth stalls for a period of time before achieving exponential growth again. At large scale, this typical process requires a large number of reactors, resulting in an increased facility footprint and increased set-up work, which in turn increases production time and production costs. For example, a train of bioreactors where a production scale of 20,000 liters (L) is desired may consist of a 200 L inoculum bioreactor (referred to as the N-3 stage), followed by a 1000 L seed bioreactor (referred to as the N-2 stage), followed by a 5000 L seed bioreactor (referred to as the N-1 stage), and finally a 20,000 L capacity bioreactor (referred to as the N stage). This train of different reactors results in more clean-in-phase (CIP) cycles and associated CIP systems, more steam-in-place (SIP) sterilizations, bioreactor start-up stages, increased utility costs (water, steam, waste), complex plant scheduling and activities to perform on the stages, and increased contamination risks. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 9,670,446 [Patent Document 2] U.S. Application Serial No. 15 / 613,954 [Patent Document 3] U.S. Patent No. 9,670,466 [Patent Document 4] U.S. Patent Application Serial No. 15 / 455,836 [Patent Document 5] Publication No. WO / 2017 / 072201 A2 [Patent Document 6] U.S. Patent Application Serial No. 15 / 612,769 [Patent Document 7] U.S. Provisional Patent Application No. 62 / 451,470 [Patent Document 8] U.S. Publication No. 2012 / 0077429 [Patent Document 9] U.S. Publication No. 2009 / 0305626 [Patent Document 10] U.S. Patent No. 9,388,373 [Patent Document 11] U.S. Patent No. 8,771,635 [Patent Document 12] U.S. Patent No. 8,298,054 [Patent Document 13] U.S. Patent No. 7,629,167 [Patent Document 14] U.S. Patent No. 5,656,491 [Patent Document 15] U.S. Publication No. 2016 / 0097074 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, improved production-scale bioreactor processing equipment, systems and methods are desirable. [Means for solving the problem]

[0007] The present invention provides a variable diameter bioreactor vessel configured for producing biologically derived materials.

[0008] The present invention also provides a variable diameter bioreactor vessel configured for producing mammalian cells.

[0009] The variable diameter bioreactor vessel can include a first vessel section having a first diameter configured to hold liquid medium and biological feedstock, and a second vessel section having a second diameter greater than the first diameter, such that the liquid medium can be expanded from a first volume to a second volume within the vessel. In some embodiments, the first vessel section can have an aspect ratio greater than 0.3:1. In some embodiments, the second vessel section can have an aspect ratio greater than 0.3:1. In some embodiments, the liquid medium includes an inoculum. The first vessel section can be configured to be an initial inoculum stage bioreactor. The second vessel section can be configured to be a growth stage or seed bioreactor. The variable diameter bioreactor vessel can further include at least one agitator. In certain aspects, the bioreactor can further comprise at least one of an agitator shaft, an agitator such as an impeller, a sparger, a probe port, a fill port, a condenser, a vent filter, a bubble breaker plate, a sample port, a level probe, and a load cell. In certain aspects, the variable diameter bioreactor vessel can be configured to grow mammalian, insect, plant, avian, or microbial cells.

[0010] In another aspect, the variable diameter bioreactor system includes a bioreactor vessel having a first diameter and a second diameter such that the vessel diameter varies with the vessel height, an agitator disposed within the bioreactor vessel such that a desired agitation is possible at a given liquid height in the bioreactor vessel, and a control system operable to expand the bioreactor vessel from a first volume to a second volume. In an aspect, the first vessel section has an aspect ratio greater than 0.3:1 and the second vessel section also has an aspect ratio greater than 0.3:1. The first vessel section can be an initial inoculum stage bioreactor. The second vessel section can be a growth stage vessel section. The variable diameter bioreactor system can also include a sparger, a probe port, a fill port, a condenser, a vent filter, a bubble breaker plate, a sample port, a level probe, and / or a load cell. In an aspect, the variable diameter bioreactor system is configured to produce mammalian cells.

[0011] In other aspects, a method of producing a fermentation product includes inoculating a bioreactor with a growth medium and an inoculum at a first volume, and, after completion of the inoculation step, adding additional growth medium to the bioreactor to expand the volume of the bioreactor to a second volume. In some aspects, the method can further include, after completion of the growth step, adding additional growth medium to the bioreactor to expand the volume of the bioreactor to a third volume. In some aspects, the inoculum is mammalian cells. In other aspects, the bioreactor can have a minimum aspect ratio of 0.3:1.

[0012] In one aspect of the disclosure, a variable diameter bioreactor vessel configured to produce mammalian cells includes a vessel configured to hold a liquid medium and a biological source, the vessel designed such that a base of the vessel section is narrower than a top of the vessel, and the vessel is adapted to allow the liquid medium and the biological source to be expanded within the vessel from a first volume to a second volume.

[0013] In another aspect of the disclosure, a variable diameter bioreactor vessel configured to produce mammalian cells comprises a first vessel section having a first diameter and configured to hold liquid medium and biological source material, the first vessel section being conically designed such that a base of the first vessel section is narrower than a top of the first vessel section, the variable diameter bioreactor vessel also comprising a second vessel section, the bottom of the second vessel section having a diameter equal to the diameter of a top of the second vessel section, and the second vessel section being arranged such that the liquid medium and biological source material can be increased from a first volume to a second volume within the vessel.

[0014] In another aspect of the present disclosure, a variable diameter bioreactor system is provided comprising a bioreactor vessel having a first diameter and a second diameter such that the vessel diameter varies with vessel height, at least one agitator disposed within the bioreactor vessel such that desired agitation is provided at a given liquid height in the bioreactor vessel, and a control system operable to expand the bioreactor vessel from a first volume to a second volume.

[0015] In another aspect of the disclosure, a method for producing a fermentation product using a reduced number of reactors in a seed stage train and production reactor is provided that includes inoculating a variable diameter bioreactor with growth medium and an inoculum at a first volume, adding additional growth medium to the variable diameter bioreactor and expanding the volume of the variable diameter bioreactor from the first volume to a second volume after completion of the inoculation stage in the first volume, and adding additional growth medium to the variable diameter bioreactor and expanding the volume of the variable diameter bioreactor from the second volume to a third volume after completion of the seed stage in the second volume.

[0016] In another aspect of the disclosure, a biological production facility is provided that includes an initial inoculum growth reactor and a variable diameter bioreactor configured in fluid communication with the inoculum growth reactor to provide a train of seed stage reactors.

[0017] The variable diameter bioreactors of the invention can be made to be used at any scale, starting at small volumes for an inoculation stage up to production scale, including 20,000 L. However, the variable diameter bioreactors of the invention can also be used as part of a train of bioreactors, as described in U.S. Patent No. 9,670,446, which is incorporated by reference in its entirety.

[0018] The description herein will be more fully understood in light of the following drawings. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a side view of a variable diameter bioreactor (VDB). [Diagram 2] FIG. 1 is a side view of a variable diameter bioreactor (VDB). [Diagram 3] FIG. 1 is a side view of a variable diameter bioreactor (VDB). [Figure 4] FIG. 1 is a schematic diagram of a variable diameter bioreactor (VDB). [Diagram 5] FIG. 1 is a schematic diagram of a variable diameter bioreactor (VDB). [Figure 6] FIG. 1 is a schematic diagram of a typical bioreactor with uniform diameter. [Figure 7] FIG. 1 is a schematic diagram of an example of a variable diameter bioreactor (VDB). [Figure 8] FIG. 1 is a schematic diagram of an example of a variable diameter bioreactor (VDB). [Figure 9] FIG. 1 is a schematic diagram of an example of a variable diameter bioreactor (VDB). [Figure 10] FIG. 1 is a schematic diagram of an example of a variable diameter bioreactor (VDB). [Figure 11] FIG. 1 is a schematic diagram of an example of a variable diameter bioreactor (VDB). [Figure 12] FIG. 1 is a top view of an example of a variable diameter bioreactor (VDB). [Figure 13]FIG. 1 is a top view of an example of a variable diameter bioreactor (VDB). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] As noted above, the present disclosure relates to systems, devices, and methods for culturing cellular biological material in a bioreactor vessel, which will now be described in detail with reference to the accompanying drawings, in which it should be noted that like reference numbers refer to like elements throughout the different embodiments.

[0021] As used herein, the articles "a" and "an" preceding an element or component are not intended to be limiting with respect to the number of instances (i.e., occurrences) of the element or component. Thus, "a" or "an" includes one or at least one, and the singular form of an element or component should also be read to include the plural unless the numeral clearly implies a singular number.

[0022] The term "invention" or "present invention" as used herein is not a term of limitation and is not intended to refer to any single embodiment of a particular invention, but rather encompasses all possible embodiments described in the specification and claims.

[0023] As used herein, the term "about" to modify the quantity of an ingredient, component, or reactant used refers to the variation in quantity that may occur through typical measuring and liquid handling procedures used, for example, to make a concentrate or solution. Additionally, variation may occur from random error in measuring procedures, differences in manufacture, source, or purity of ingredients used to make a component or perform the procedure, and the like. In one embodiment, the term "about" means within 10% of the reported numerical value. In another embodiment, the term "about" means within 5% of the reported numerical value. Additionally, in another embodiment, the term "about" means within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the reported numerical value.

[0024] Bioreactor processing of biological feedstocks (including, but not limited to, microbial and mammalian cultures) in variable diameter bioreactors (VDBs), such as those described herein, is designed to begin with a minimal inoculum, maintain growth conditions, utilize continuous and / or bolus media and / or feed additions to maintain cell growth throughout the growth period, and obtain a culture of sufficient volume to produce the desired product. By achieving cell growth and production in a single VDB, multiple, relatively small volume bioreactors can be eliminated. A single VDB reduces the overall footprint of the bioreactor equipment required to produce the desired product, eliminates the need for multiple seed reactors, multiple CIPs, SIPs, start-up activities, post-operational operations, and minimizes non-logarithmic cell growth or lag phase effects currently seen with the use of multiple seed bioreactors, thereby simplifying overall facility operations and resulting in time and cost savings.

[0025] For example, a single 20,000 L VDB can replace a 200 L N-3 step, a 1000 L N-2 step, and a 5000 L N-1 step seed bioreactor, and it is estimated that replacing three seed bioreactors with a single VDB can eliminate over 300 square feet of clean room space.

[0026] In some embodiments, the use of a conical or smaller diameter cylindrical geometry at the bottom of the bioreactor and a cylindrical design at the top allows for controlled scale-up within a single bioreactor providing important design advantages for mixing and aeration. For example, using a variable diameter conical or smaller diameter cylindrical bottom tank, the aspect ratio (liquid height vs. width of vessel at liquid level) can be maintained at greater than 1:1, accommodating a minimal inoculum with sufficient head for oxygen transfer in the bulk to a larger volume culture. The volume of the culture can then be bulked up by the addition of medium to maintain cell growth. Alternative bottom designs may allow for higher aspect ratios and smaller volume operation compared to typical fixed diameter cylindrical tank bioreactor designs.

[0027] As used herein, "biological material" is understood to mean a particle consisting of living or dead cellular or viral material and / or a product produced and expressed by a cellular or viral culture, in whole or in part. This can include, for example, eukaryotic or prokaryotic cells, such as bacteria, mammalian, plant, fungi, viruses, such as talimogene laherparepvec (T-VEC), or any other desired therapeutic or biochemical product. In some embodiments, "biological material" includes cells produced for cell therapy programs. In some embodiments, "biological material" includes viruses produced for viral gene therapy, viral immunotherapy, or viral therapy, including protozoan viral therapy. In some embodiments, "biological material" includes cell cultures or viral cultures for fermentative production of desired compounds, including, but not limited to, proteins, polypeptides, polymers, DNA, RNA, antigens, monoclonal antibodies, or any other desired compounds. In some embodiments, the biological material can include inert materials, such as substrates or immobilization materials. Additionally, as used herein, "liquid medium" should be understood to mean any liquid typically used in bioreactor processes, such as growth medium, water, inoculum, and biological feedstocks. Liquid media can have solid particles and / or gasses suspended, emulsified, entrained, or otherwise present in the liquid medium.

[0028] As shown, the variable diameter bioreactor can have multiple configurations that allow efficient scale-up from inoculation to seed and production in a single bioreactor vessel or by reducing the number of reactors from a traditional inoculation to seed and production train. In some embodiments, the variable diameter bioreactor can have a more suitable aspect ratio when the medium volume of the bioreactor is smaller compared to a traditional vertical cylindrical uniform diameter reactor. Also, medium or feed addition from a small inoculation to production volume provides a stable environment for cell growth as waste is diluted and fresh nutrients are continuously introduced and mixed. In some embodiments, the illustrative variable diameter bioreactor can be configured for fermentation processes and can be batch, fed-batch, or continuous, and the production method can be changed within the bioreactor vessel depending on the stage of the culture and the stage of the volume. For example, a batch or fed-batch process can be used for the initial inoculation stage. Then, fed-batch, continuous, or perfusion processes can be used as the cell growth stage reaches maturity and the bioreactor volume is expanded to its desired limit. The variable diameter bioreactors described herein can be made of any suitable material and can be configured for single-use, disposable systems, including but not limited to those described in U.S. Application No. 15 / 613,954, filed June 5, 2017. In some aspects, the reactors can be configured for use in a single system or a multi-product production suite.

[0029] Additionally, the variable diameter bioreactor can be configured to have any desired total volume. As described in more detail below, the VDB can have a total volume of about 20,000 liters (L), although it is possible to design the VDB with a total volume of 1,000 L, or even, for example, with a total volume of 10 L. For example, a VDB with a total volume of 10 L can also be used for process development or scale-down studies, while a VDB with a volume of 1000 L can act as a pilot-scale bioreactor. Figures 1-3 show an example variable diameter bioreactor having a conical lower portion and a cylindrical upper portion, whereby the height of the cylindrical upper portion is varied to achieve various desired volumes.

[0030] 1 shows a variable diameter bioreactor (VDB) 100. The variable diameter bioreactor 100 comprises a first vessel portion 102 having a first diameter and configured to hold a liquid medium or a culture of biological material such as suitable cells, and a second vessel 104 having a second diameter greater than the first diameter and allowing the liquid medium to be expanded from a first volume to a second volume within the vessel 100. The variable diameter bioreactor 100 also comprises at least one inlet 106, such as a manway, and at least one outlet 108.

[0031] Figure 2 shows a variable diameter bioreactor (VDB) 200 with a reduced height of its upper cylindrical portion compared to the height of the upper cylindrical portion of the variable diameter bioreactor shown in Figure 1. The variable diameter bioreactor 200 comprises a first vessel portion 202 having a first diameter and configured to hold a liquid medium, and a second vessel 204 having a second diameter that is greater than the first diameter. The variable diameter bioreactor 200 also comprises at least one inlet 206, such as a manway, and at least one outlet 208.

[0032] Figure 3 shows a variable diameter bioreactor (VDB) 300 with a reduced height of its upper cylindrical portion compared to the height of the upper cylindrical portion of the variable diameter bioreactor shown in Figure 2. The variable diameter bioreactor 300 comprises a first vessel portion 302 having a first diameter and configured to hold a liquid medium, and a second vessel portion 304 having a second diameter that is greater than the first diameter. The variable diameter bioreactor 300 also comprises at least one inlet 306, such as a manway, and at least one outlet 308.

[0033] 4 illustrates a variable diameter bioreactor (VDB) 400. The variable diameter bioreactor 400 comprises a first vessel section 402, a second vessel section 404, and a third vessel section 406. The first vessel section varies in diameter with vessel height, i.e., the diameter of the first vessel section 402 and the diameter of the second vessel section 404 increase towards the top of the bioreactor 400. However, as illustrated, the diameter of the third vessel section 406 remains relatively uniform throughout the vessel section 406.

[0034] 5 shows a variable diameter bioreactor (VDB) 500. The variable diameter bioreactor 500 comprises a first vessel section 502, a second vessel section 504, and a third vessel section 506. The first vessel section has a diameter that varies stepwise along the vessel height, i.e., moving up the vessel, and the third vessel section 506 has a diameter that is greater than the volume of the second vessel section 504, which in turn is greater than the volume of the first vessel section 502. As shown, in this embodiment, the diameter of each stage is uniform throughout the stage, with a step increase in diameter between the first stage 502 and the second stage 504, and another step increase in diameter between the second stage 504 and the third stage 506.

[0035] Figures 6-9 show examples of aspect ratios and capacities of various bioreactor designs. As noted above, aspect ratio is defined as the height to width or diameter of the vessel. As shown, the reactors of Figures 6-9 can have capacities ranging between about 0 and 25,000 liters (L).

[0036] FIG. 6 shows a typical bioreactor 600 having a uniform diameter (i.e., not a variable diameter bioreactor). The typical bioreactor 600 has only a single vessel section 608 and has a bioreactor height 602, volume 604, and aspect ratio 606. The typical bioreactor 600 has the bioreactor height 602 and aspect ratio 606 shown in Table 1. As shown, at small volumes, e.g., 800 L, typical uniform diameter reactors have aspect ratios significantly lower than 0.3. Furthermore, uniform diameter bioreactors must operate at aspect ratios of at least 0.65 or greater, which is shown in FIG. 6 for a volume of about 10,000 L. That is, for optimal operation, a uniform diameter bioreactor requires incremental growth of the culture volume with multiple seed bioreactors to reach the desired culture volume.

[0037] [Table 1]

[0038] Figures 7, 8 and 9 show variable diameter bioreactors in various configurations, all capable of operating at the desired volumes required to eliminate the need for multiple seed bioreactors of 200 L, 1000 L, and 4000 L, respectively.

[0039] 7 illustrates an example variable diameter bioreactor (VDB) 700 having a bioreactor height 702, volume 704, and aspect ratio 706. As shown, the bioreactor 700 includes a first vessel section 708, a second vessel section 710, and a third vessel section 712. The example bioreactor 700 has the bioreactor height 702, aspect ratio 706, and volume 704 shown in Table 2.

[0040] [Table 2]

[0041] 8 illustrates an example variable diameter bioreactor (VDB) 800 having a bioreactor height 802, a volume 804, and an aspect ratio 806. As shown, the bioreactor 800 includes a first vessel section 808, a second vessel section 810, and a third vessel section 812.

[0042] 9 illustrates an example variable diameter bioreactor (VDB) 900 having a bioreactor height 902, volume 904, and aspect ratio 906. As shown, the bioreactor 900 includes a first vessel section 908 and a second vessel section 910. The example reactors 800, 900 have bioreactor heights 802, 902 and aspect ratios 806, 906 as shown in Table 3.

[0043] [Table 3]

[0044] 10 and 11 show example variable diameter bioreactor vessels 1000 and 1100. As shown, the variable diameter bioreactors 1000, 1200 can include various ports, probes, spargers, and other components, such as at least one of an agitator shaft, an agitator such as an impeller, a sparger, a probe port, a fill port, a condenser, a vent filter, a bubble breaker plate, a sample port, a level probe, and a load cell.

[0045] FIG. 10 is a schematic diagram of a VDB 1000 comprising a first vessel portion 1002 and a second vessel portion 1004. In one embodiment, the first vessel portion 1002 has an increasing diameter, such that the first vessel portion 1002 is conical. The second vessel portion 1004 can have a constant diameter, such that the first vessel portion 1002 is cylindrical. As shown, the VDB 1000 can have an overall bioreactor height A. In one embodiment, the overall bioreactor height A can range from about 5 feet to about 50 feet. For example, the overall bioreactor height can be about 20 feet. Additionally, as shown, the top of the bioreactor can have a height B, the bottom can have a height C, and the bioreactor can have a liquid height E. The liquid height E can vary based on what stage of production is desired. In some embodiments, the diameter of the lower portion can vary along height C, and in some embodiments, the diameter of the upper portion can remain constant along height B.

[0046] As described herein, the diameter of the VDB bioreactor can vary as one moves through the overall bioreactor height A or the lower height C. As shown, the first vessel section 1002 can have an increasing diameter as the lower height C moves up the reactor height A. The diameters increase, for example, to a second diameter D2, a third diameter D3, and a fourth diameter D4. In one non-limiting embodiment, for example, D1 can be from about 1 foot to about 3 feet, D2 can be from about 1 foot to about 5 feet, D3 can be from about 2 feet to about 10 feet, and D4 can be from about 3 feet to about 20 feet. As one non-limiting example, the height A of a VDB bioreactor may be about 20 feet with a bottom height C (cone height) of about 15 feet, a top diameter (D4) of about 10 feet, a bottom diameter (D1) of about 2 feet, D2 of about 3.25 feet, and D3 of about 4.8 feet, resulting in a total volume of about 24,909 liters (L), a bottom (cone) volume of 13,789 L, and a top (cylindrical) volume of 11,120 L. Note that in one embodiment as shown in FIG. 10, the top can have a uniform diameter and D4 is equal to D5. Additionally, as shown, the bottom can have a cone shape with angle θ, which can be any angle suitable to provide the bottom with the desired diameter and volume. It is understood that the volume can have a dished bottom 1016. It should also be understood that the angled apex 1018 is shown for illustrative purposes only and need not be present in the reactor.

[0047] Additionally, the VDB 1000 includes a number of agitators 1010a, 1010b, 1010c, and 1010d. The agitators can be configured to provide agitation configured for the particular vessel portion 1002, 1004 in which the particular agitator 1010a, 1010b, 1010c, and 1010d is disposed. As shown, the agitator 1010d can be disposed at a height H within the bioreactor, the agitator 1010c can be disposed at a height I within the bioreactor, the agitator 1010b can be disposed at a height J within the bioreactor, and the agitator 1010a can be disposed at a height K. For example, the heights H, I, J, and K can be within a range of approximately 1 foot to 20 feet. In some embodiments, the agitators can have a single drive (not shown) disposed along a midpoint 1011 of the VDB 1000. In one embodiment, the VDB 1000 can include bulkheads 1012 throughout the bioreactor 1000. As shown, the bulkheads 1012 can extend along height G or F of the bioreactor. In one embodiment, the VDB 1000 can include multiple ports 1014. The ports 1014 can be configured to be inlets, outlets, probes for pH, temperature, oxygen, etc., or any other desired probe or sensor. The VDB 1000 can also include a single agitator, such as a single impeller.

[0048] FIG. 11 is a schematic diagram of an example of a VDB bioreactor 1100. The VDB bioreactor 1100 includes an inlet port 1102 and a bottom outlet valve 1104 configured to add and remove media from the bioreactor. The VDB bioreactor 1100 can include a first vessel section 1102, a second vessel section 1104, and a third vessel section 1106. The bioreactor includes an agitator 1108 including a lower agitator 1110, a middle agitator 1112, an upper agitator 1114, and an agitator motor and drive 1116. Additionally, the bioreactor can include at least one sparger 1118 configured to allow air or other nutrients to bubble through the liquid media of the bioreactor. Additionally, the bioreactor can include at least one probe or addition port 1120. The bioreactor can also include at least one CIP port 1122. As shown, the bioreactor can be configured with spargers 1118, probe and addition ports 1120, and CIP ports 1122 in each of the vessel sections 1102, 1104, 1106. The bioreactor can include any suitable control system for controlling the bioreactor system, including monitoring and controlling air bubble injection, liquid medium addition and removal, cell growth and production, oxygen levels, volume, temperature, pH, and any other desired components. In certain embodiments, the control system is configured to expand the capacity of the bioreactor in either a continuous or batch mode approach. Additionally, the bioreactor can include at least one bulkhead 1124 disposed therein and configured to provide suitable mixing conditions without causing undue stress to the bioreactor inoculum that may result in cell death. Additionally, the bioreactor can include a heat transfer shell 1126, which can include external insulation. The VDB 1000 can also include a single agitator, such as a single impeller.

[0049] The variable diameter bioreactors described herein can have any cross-sectional shape. In some embodiments, the variable diameter bioreactors can have a non-circular cross-sectional shape. In the case of a non-circular cross-section, "diameter" should be understood to mean the cross-sectional area of ​​each stage. In some embodiments, the variable diameter bioreactors can have a cross-sectional shape that is any geometric shape, including but not limited to circular, square, rectangular, triangular, pentagonal, hexagonal, octagonal, heptagonal, decagonal, and any other shape.

[0050] The variable diameter bioreactor of the invention can also include an agitator, for example, which can include: (i) an orbital rocking or vibration that creates ripples on the surface, allowing the surface layer to mix with the bulk of the liquid, (ii) agitators placed non-centrosymmetrically on the agitator shaft or mounted off-center on the bottom of a conical vessel, allowing axial mixing by swirling the liquid around the area of ​​the agitator, (iii) a centrally mounted agitator in a septum-less vessel with a composite base / base plate design that allows the bulk of the radially flowing liquid to be axially deflected (agitator cluster), and (iv) a non-circular vessel (cube) where the vessel is agitated to overcome the lack of axial flow caused by the lack of a septum.

[0051] In some embodiments, an agitator with at least one blade element is used as the aeration device. The blade element can be folded toward a rotatable shaft. In some embodiments, the rotatable shaft is coupled with a first agitator and a second agitator, both of which can be equipped with at least one foldable blade element. Again, there may be retaining ring positions and agitator disengagement positions for holding the agitators in a vertical position during mixing or in a folded, folded position, respectively.

[0052] In one embodiment, the rotatable shaft comprises a metal reinforcing rod surrounded by a sheath. The metal reinforcing rod may be made of stainless steel and may be made of multiple pieces bonded together. The top of the reinforcing rod may comprise a magnetic member for magnetically engaging the motor. The sheath may be made of a polymeric material. The agitator on the shaft may also be made of a polymeric material, such as a hydrophilic polymer. For example, the sheath and the agitator may comprise a polyethylene polymer modified by being subjected to irradiation, light or plasma induction, or oxidation. A single agitator mounted off-center, as opposed to multiple agitators arranged non-centrosymmetrically, offers the considerable advantage of being able to continuously change the working volume during a fed-batch process without having to consider the effect of the liquid surface being cut by a rotating agitator that is not submerged in the liquid.

[0053] According to the present disclosure, the rotatable shaft can be coupled to a top impeller and a bottom impeller. Both the top impeller and the bottom impeller can be made of a polymeric material. For example, in one embodiment, the impellers can be made with a 3D printer. Both the top impeller and the bottom impeller can define a hydrophilic surface. For example, the polymeric material used to form the impellers can include a hydrophilic polymer or can include a polymer that has been surface-modified to make the surface hydrophilic.

[0054] In some embodiments, for example, the top and bottom impellers are made of a polyolefin polymer, such as polyethylene or polypropylene. In one embodiment, low density polyethylene can be used. The low density polyethylene may be modified by irradiation, light or plasma induction, or by oxidation to form a hydrophilic surface.

[0055] In another embodiment, the variable diameter bioreactor of the present invention can be designed to maintain its size and characteristics as discussed in U.S. Patent No. 9,670,466, which is incorporated by reference in its entirety. For example, the variable diameter bioreactor of the present invention can include two agitators. The top impeller can include a hydrofoil impeller, while the bottom impeller can include a four pitch-blade high stiffness impeller. The impeller-to-tank diameter ratio can be from about 0.35 to about 0.55, such as from about 0.44 to about 0.46. The top impeller and bottom impeller can be configured with a power number (N) of from about 0.1 to about 0.9. p ) and a flow number (N q ).

[0056] Non-limiting examples of impellers suitable for use in the agitation system of the present disclosure include hydrofoil impellers, high stiffness pitch-blade impellers, high stiffness hydrofoil impellers, Rushton-type impellers, pitch-blade impellers, gentle marine impellers, Cerigen Celllift impellers, A320 impellers, HE3 impellers, and the like. Spin filters can also be used, such as when the device is operated in perfusion mode. In the multiple impeller embodiments of the single-use bioreactor of the present disclosure, the impellers can include the same or different materials, designs, and manufacturing methods. For example, in one embodiment, the top impeller can be a hydrofoil impeller or one of similar designs, such as one made using a 3D printer.

[0057] 12 is a top view of an example bioreactor vessel 1200 having various non-circular cross sections. That is, the bioreactor 1200 has a square cross-sectional shape. Specifically, the vessel 1200 includes a bottom 1208, a first vessel portion 1202 having a first diameter 1228 and configured to hold liquid medium and biological raw materials, and a second vessel portion 1204 having a second diameter 1230 greater than the first diameter 1228 and configured to hold liquid medium and biological raw materials such that the liquid medium and biological raw materials can be increased from a first volume to a second volume within the vessel 1200, and the vessel 1200 is configured to hold liquid medium and biological raw materials. One of ordinary skill in the art will appreciate that such shape configurations may be present in any or all of the embodiments shown in FIGS. 1-11 as well as other embodiments disclosed herein when viewed from the side. For example, an increase in volumetric size can be achieved by changing the diameter of one of four sides, two of four sides, three of four sides, or all four sides. The increase does not have to be contiguous on any or all sides.

[0058] In one embodiment, impellers suitable for use herein include those manufactured by 3D printing that look like any impeller known in the art, even if the dimensions of the impeller vary.

[0059] FIG. 13 is a top view of an example bioreactor vessel 1300 having various non-circular cross sections. That is, the bioreactor 1300 has a triangular cross-sectional shape. Specifically, the vessel 1300 includes a first vessel portion 1302 having a first diameter 1328 and configured to hold liquid medium and biological raw materials, and a second vessel portion 1304 having a second diameter 1330 greater than the first diameter 1328 and configured to hold liquid medium and biological raw materials such that the liquid medium and biological raw materials can be increased from a first volume to a second volume within the vessel 1300, and the vessel 1300 is configured to hold liquid medium and biological raw materials. One of ordinary skill in the art will appreciate that such shape configurations may be included in any or all of the embodiments shown in FIGS. 1-11 as well as other embodiments disclosed herein when viewed from the side. For example, an increase in volumetric dimension may be achieved by changing the diameter of one of the three sides, two of the three sides, or all three sides. The increase need not be continuous on any or all sides.

[0060] Those of skill in the art will appreciate that other non-circular bioreactors are disclosed herein, such as oval, hexagonal, octagonal, etc.

[0061] For non-circular vessel geometries, such as the cubic geometry shown in Figure 12 and the triangular geometry shown in Figure 13, the radial flow created by the agitator can be deflected as it strikes each of the four sides of the vessel. Such a design offers the advantage of mounting to a steel frame in that each corner of a horizontally packed bioprocess container can be easily aligned with a corner of the steel frame during mounting.

[0062] In use, the variable diameter bioreactors described herein can be used to culture living cells and produce biological feedstocks, allowing efficient use of floor space by limiting the number of reactors required in a train to a single bioreactor. In particular, production of a biological feedstock, such as the production of a fermentation product, can be accomplished in a single VDB bioreactor by inoculating the bioreactor with growth medium and inoculum at a first volume, and adding additional growth medium to the bioreactor after completion of the inoculation stage and expanding the volume of the bioreactor to a second volume. In one aspect, using the bioreactor can include adding additional growth medium to the bioreactor after completion of the growth stage and expanding the volume of the bioreactor to a third volume.

[0063] That is, by condensing the inoculum bioreactor and all necessary subsequent propagation or seed reactors into a single bioreactor vessel, the footprint of a particular facility is minimized. For example, for a desired production scale of 20,000 liters (L), only one 20,000 L bioreactor can be used, consisting of a first vessel section (i.e., inoculum vessel section), a second seed or propagation section, and a third seed or propagation vessel section. For example, the first vessel section (inoculum vessel section) can have a first diameter corresponding to a volume of about 100 L to about 200 L, and a desired aspect ratio between about 0.3:1 and about 2:1. The second and third seed vessel sections can then expand the volume of the bioreactor to the desired volume of 20,000 L while maintaining the desired aspect ratio range. For example, the aspect ratio can remain between about 0.3:1 and about 3:1. The 20,000 L bioreactor unit may perform one or more or all of the following steps: supplying nutrients and / or carbon sources, injecting a suitable gas (e.g., oxygen), flowing fermentation or cell culture media in and out, separating gas and liquid phases, maintaining growth temperature, maintaining pH levels, agitation (e.g., stirring), and / or cleaning / sanitizing.

[0064] For example, the illustrative 20,000 L bioreactor may, in one embodiment, be inoculated with an inoculum, such as growth medium and mammalian cells, in a first volume. In this inoculation step, the reactor may be inoculated with a first volume, and this volume of the reactor is suitable for the initial growth of the inoculum. After a suitable period of time to allow for the desired cell growth, the bioreactor may be scaled up to a second reactor volume to achieve a second growth stage of the inoculum. That is, after completion of the inoculation step, additional growth medium and any other desired components required for growth may be added to the bioreactor to expand the volume of the bioreactor to a second volume. This second volume may be any desired volume suitable for the desired continued growth conditions required for the inoculum. Further cell growth and spreading may be achieved in this second volume. In one embodiment, a third, fourth, or any number of additional volume growth stages may be utilized to continue expanding the reactor volume to a desired volume.

[0065] Variable diameter bioreactors as described herein can be used in many types of manufacturing equipment, including but not limited to those disclosed in U.S. Patent Application No. 15 / 455,836, filed March 10, 2017, and Publication No. WO / 2017 / 072201 A2, both of which are incorporated by reference in their entirety, in which one or more of the bioreactors discussed in the above applications or other similar equipment can be replaced with a variable diameter bioreactor of the present invention.

[0066] The variable diameter bioreactors of the present invention can also be governed by control systems known to those of skill in the art, including, but not limited to, those disclosed in U.S. Application No. 15 / 613,954, filed June 5, 2017, U.S. Application No. 15 / 612,769, filed June 2, 2017, U.S. Provisional Application No. 62 / 451,470, filed January 27, 2017, and others. EXAMPLES

[0067] The relationship between the volume, diameter, and cell growth characteristics of a variable diameter bioreactor of the invention requires consideration of many factors. The following equations provide useful guidance when designing a bioreactor of the invention.

[0068]

number

[0069] For example, when designing a bioreactor of the present invention to operate with a volume of up to 20,000 L, the variable diameter bioreactor will have the following dimensions: Total capacity: 20,000L Cone capacity: 15,000L Top Diameter top ):2.13m(7ft) Bottom Diameter bottom ):0.91m(3ft) Overall height: 9.20 meters (30.2 feet) Cylindrical capacity: 5,000L Cone height: 7.80 meters (25.6 feet) Barrel height: 1.40 meters (4.6 feet)

[0070] As another example, when designing a bioreactor of the present invention to fit into a space such as in a manufacturing facility where the height is limited to 20 feet, the above equations yield the following dimensions: Total volume: 16,458L Cone volume: 9,341L Top Diameter top ):2.44m(8ft) Bottom Diameter bottom ):0.61m(2ft) Overall height: 6.10 meters (20 feet) Cylindrical capacity: 7,117L Cone height: 4.57 meters (15 feet) Cylindrical height: 1.52 meters (5 feet)

[0071] The example may include four impellers, such as that shown in FIG.

[0072] As another example, the design of the present invention allows for the creation of variable diameter bioreactors in excess of 20,000 L, which are new to the industry. Specifically, the variable diameter bioreactors can be made in the following sizes: Total capacity: 25,000L Cone capacity: 15,000L Top Diameter top ):2.41m(7.9ft) Bottom Diameter bottom ): 0.76m (2.5ft) Overall height: 9.14 meters (30 feet) Cylindrical capacity: 10,000L Cone height: 6.95 meters (22.8 feet) Barrel height: 2.19 meters (7.2 feet)

[0073] If not otherwise specified above, the above description may be further understood as follows: The devices, equipment and methods described herein are suitable for use in and with any desired cell line culture, including prokaryotic and / or eukaryotic cell lines. Furthermore, in embodiments, the devices, equipment and methods are suitable for culturing suspension or anchorage-dependent (adherent) cells, and are suitable for production processes adapted to produce pharmaceuticals and biologics, such as polypeptide products, nucleic acid products (e.g. DNA or RNA), or cells and / or viruses such as those used in cell and / or virus therapy.

[0074] In an embodiment, the cells express or produce a product, such as a recombinant therapeutic or diagnostic product. As described in more detail below, examples of products produced by the cells include, but are not limited to, antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimetics (polypeptide molecules that specifically bind to an antigen but are not structurally related to antibodies, such as, for example, DARPins, affibodies, adnectins, or IgNARs), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), viral therapeutics (e.g., anti-cancer oncolytic viruses, viral vectors for gene therapy and viral immunotherapy), cellular therapeutics (e.g., pluripotent stem cells, mesenchymal stem cells, and adult stem cells), vaccines or lipid-encapsulated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA, etc.) or DNA (e.g., plasmid DNA, etc.), antibiotics, or amino acids. In an embodiment, the devices, equipment, and methods can be used to generate biosimilars.

[0075] As described, in embodiments, the devices, equipment, and methods allow for the production of eukaryotic cells, e.g., mammalian cells, or lower eukaryotic cells, e.g., yeast cells or filamentous fungal cells, or prokaryotic cells, e.g., gram-positive or gram-negative cells, and / or eukaryotic or prokaryotic products synthesized by eukaryotic cells in a large-scale manner, e.g., proteins, peptides, antibiotics, amino acids, nucleic acids (e.g., DNA or RNA).Unless otherwise described herein, the devices, equipment, and methods can include any desired volume or production capacity, including, but not limited to, laboratory scale, pilot production scale, and full production scale capacities.

[0076] Additionally, unless otherwise stated herein, the devices, equipment, and methods may include any suitable reactor, including, but not limited to, stirred tank, airlift, fiber, microfiber, hollow fiber, magnetic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. As used herein, "reactor" may include a fermenter or fermentation unit, or any other reaction vessel, and the term "reactor" is used interchangeably with "fermenter." For example, in certain embodiments, an example bioreactor unit may perform one or more or all of the following: supplying nutrients and / or carbon sources, injecting a suitable gas (e.g., oxygen), inflowing and outflowing fermentation or cell culture medium, separating gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitating (e.g., stirring), and / or cleaning / sanitizing. An illustrative reactor unit, such as a fermentation unit, can include multiple reactors within the unit, e.g., a unit can house 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors within each unit, and / or a facility can include multiple units with single or multiple reactors within the facility. In various embodiments, the bioreactors can be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactors can have a volume between about 100 mL and about 50,000 L.Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, , 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. Additionally, suitable reactors may be multi-use, single-use, disposable, or non-disposable, and may be formed of any suitable material, including stainless steel (e.g., 316L or any other suitable stainless steel) and alloys such as Inconel, plastic, and / or glass.

[0077] In embodiments, unless otherwise noted herein, the apparatus, equipment, and methods described herein may also include any suitable unit operations and / or equipment not specifically mentioned, such as operations and / or equipment for separation, purification, and isolation of such products. Any suitable equipment and environment may be used, such as conventional wooden equipment, modular equipment, mobile temporary equipment, or any other suitable structure, equipment, and / or layout. For example, in an embodiment, a modular clean room may be used. In addition, unless otherwise noted herein, the apparatus, systems, and methods described herein may be contained and / or performed in a single location or facility, or alternatively, may be contained and / or performed in separate or multiple locations and / or facilities.

[0078] As non-limiting examples, and without limitation, U.S. Publication Nos. 2012 / 0077429 and 2009 / 0305626, as well as U.S. Patent Nos. 9,388,373, 8,771,635, 8,298,054, 7,629,167, and 5,656,491, which are incorporated by reference herein in their entireties, describe example equipment, devices, and / or systems that may be suitable.

[0079] In an embodiment, the cell is a eukaryotic cell, for example a mammalian cell. The mammalian cell may be, for example, a human or rodent or bovine cell line or cell strain. Examples of such cells, cell lines or cell strains are, for example, mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat cells, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS, for example COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLA, EB1, EB2, EB3, oncolytic cells or hybridoma cell lines. The mammalian cell is preferably a CHO cell line. In one embodiment, the cell is a CHO cell. In one example, the cell is a CHO-K1 cell, a CHO-K1 SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS, a CHO GS knockout cell, a CHO FUT8 GS knockout cell, a CHOZN, or a CHO derived cell. A CHO GS knockout cell (e.g., a GSKO cell) is, for example, a CHO-K1 SV GS knockout cell. A CHO FUT8 knockout cell is, for example, Potelligent® CHOK1 SV (Lonza Biologics, Inc.). The eukaryotic cell may also be an avian cell, cell line, or cell strain, such as, for example, an EBx® cell, EB14, EB24, EB26, EB66, or EBvl3.

[0080] In one embodiment, the eukaryotic cell is a stem cell. The stem cell may be a pluripotent stem cell, including, for example, an embryonic stem cell (ESC), an adult stem cell, an induced pluripotent stem cell (iPSC), a tissue-specific stem cell (e.g., a hematopoietic stem cell), and a mesenchymal stem cell (MSC).

[0081] In one embodiment, the cell is a differentiated form of any of the cells described herein, hi one embodiment, the cell is derived from any primary cell in culture.

[0082] In embodiments, the cells are hepatocytes, such as human hepatocytes, animal hepatocytes, or non-parenchymal cells. For example, the cells may be adherent metabolically adapted human hepatocytes, adherent induction adapted human hepatocytes, adherent Qualyst Transporter Certified™ human hepatocytes, suspension adapted human hepatocytes (including 10 donor and 20 donor pooled hepatocytes), human hepatic Kupffer cells, human hepatic stellate cells, dog hepatocytes (including single pooled Beagle hepatocytes), mouse hepatocytes (including CD-1 hepatocytes and C57BI / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley hepatocytes, Wistar Han hepatocytes, and Wistar hepatocytes), monkey hepatocytes (including Cynomolgus or Rhesus monkey hepatocytes), cat hepatocytes (including Domestic Shorthair hepatocytes), and rabbit hepatocytes (including New Zealand White hepatocytes). Exemplary hepatocytes are commercially available from Triangle Research Labs, LLC, 6 Davis Drive Research Triangle Park, North Carolina, USA 27709.

[0083] In one embodiment, the eukaryotic cell is, for example, a yeast cell (e.g., a species of the genus Pichia (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta), the genus Komagataella (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii), the genus Saccharomyces (e.g., Saccharomyces cerevisae, Saccharomyces kluyveri, Saccharomyces uvarum), the genus Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), the genus Candida (e.g., Candida utilis, Candida cacaoi, Candida boidinii), the genus Geotrichum (e.g., Geotrichum fermentans), Hansenula polymorpha, Yarrowia lipolytica, or Schizosaccharomyces pombe. Preferably, the species is Pichia pastoris. Examples of Pichia pastoris strains are X33, GS115, KM71, KM71H, and CBS7435.

[0084] In one embodiment, the eukaryotic cell is a fungal cell (e.g., Aspergillus (e.g., A. niger, A. fumigatus, A. orzyae, A. nidula, etc.), Acremonium (e.g., A. thermophilum), Chaetomium (e.g., C. thermophilum), Chrysosporium (e.g., C. thermophile), Cordyceps (e.g., C. militaris), Corynascus, Ctenomyces, Fusarium (e.g., F. oxysporum), Glomerella (e.g., G. graminicola), Hypocalcaea, ... pocrea (H. jecorina, etc.), Magnaporthe (M. orzyae, etc.), Myceliophthora (M. thermophile, etc.), Nectria (N. heamatococca, etc.), Neurospora (N. crassa, etc.), Penicillium, Sporotrichum (S. thermophile, etc.), Thielavia (T. terrestris, T. heterothallica, etc.), Trichoderma (T. reesei, etc.), or Verticillium (V. dahlia, etc.).

[0085] In one example, the eukaryotic cell is an insect cell (e.g., an Sf9, Mimic™ Sf9, Sf21, High Five™ (BT1-TN-5B1-4), or BT1-Ea88 cell), an algae cell (e.g., an algae cell of the genus Amphora, Bacillariophyceae, Dunaliella, Chlorella, Chlamydomonas, Cyanophyta (blue-green algae), Nannochloropsis, Spirulina, or Ochromonas), or a plant cell (e.g., a cell from a monocotyledonous plant (e.g., maize, rice, wheat, or Setaria), or a cell from a dicotyledonous plant (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens, or Arabidopsis)).

[0086] In one embodiment, the cell is a bacterial cell or a prokaryotic cell.

[0087] In an embodiment, the prokaryotic cell is a gram-positive cell, such as Bacillus, Streptomyces, Streptococcus, Staphylococcus, or Lactobacillus. Bacillus that can be used is, for example, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. natto, or B. megaterium. In an embodiment, the cell is B. subtilis, such as B. subtilis 3NA and B. subtilis 168. Bacillus can be, for example, as described in Biological Sciences 556, 484 West 12, 1999. th These are available from the Bacillus Genetic Stock Center, 1214 University Avenue, Columbus, OH 43210-1214.

[0088] In one example, the prokaryotic cells are gram-negative cells such as Salmonella spp. or Escherichia coli, e.g., TG1, TG2, W3110, DH1, DHB4, DH5a, HMS 174, HMS174(DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue, and Origami, and cells derived from E. coli B strains, e.g., BL-21 or BL21(DE3), all of which are commercially available.

[0089] Suitable host cells are commercially available, for example from culture collections such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0090] In examples, the cultured cells are used to produce proteins, such as antibodies, e.g., monoclonal antibodies, and / or recombinant proteins for therapeutic use. In examples, the cultured cells produce peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites. For example, in examples, molecules having molecular weights from about 4000 Daltons to greater than about 140,000 Daltons can be produced. In examples, the molecules can have a range of complexity and can include post-translational modifications, including glycosylation.

[0091] In an embodiment, the protein may be, for example, BOTOX, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxin), alglucosidase alfa, daptomycin, YH-16, chorionic gonadotropin alfa, filgrastim, cetrorelix, interleukin-2, aldesleukin, teceleukin, denileukin diftitox, interferon alfa-n3 (injection), interferon alfa-n1, DL-8234, interferon, Suntory (gamma-1a), interferon alfa-n2, interferon alfa-n1 ... Lon gamma, Thymosin alpha 1, Tasonermin, DigiFab, ViperaTAb, EchiTAb, CroFab, Nesiritide, Abatacept, Alefacept, Rebif, Eptothermin alfa, Teriparatide (osteoporosis), Injectable calcitonin (bone disease), Calcitonin (nasal bone, osteoporosis), Etanercept, Hemoglobin glutamer 250 (bovine), Drotrecogin alfa, Collagenase, Carperitide, Recombinant human epidermal growth factor (topical gel, wound treatment), DWP401, Darbepoetin alfa, Epoetin ·Omega, Epoetin beta, Epoetin alfa, Desirudin, Lepirudin, Bivalirudin, Nonacog alfa, Mononine, Eptacog alfa (activated), rFVIII + VWF, Recombinate, rFVIII, FVIII (recombinant), Alphnmate, Octocog alfa, FVIII, Palifermin, Indikinase, Tenecteplase, Alteplase, Pamitoplase, Reteplase, Nateplase, Monteplase, Follitropin alfa, rFSH, h pFSH, micafungin, pegfilgrastim, lenograstim, nartograstim, sermorelin, glucagon, exenatide, pramlintide, imiglucerase, galsulfase, leucotropin, molgramostim, triptorelin acetate, histrelin (subcutaneous implant, Hydron), deslorelin, histrelin, nafarelin, leuprolide sustained release formulation (ATRIGEL), leuprolide implant (DUROS), goserelin, eutropin, KP-102 program, somatropin, mecasermin (growth failure),Enfuvirtide, Org-33408, Insulin glargine, Insulin glulisine, Insulin (inhaled), Insulin lispro, Insulin detemir, Insulin (buccal, RapidMist), Mecasermin linfabate, Anakinra, Celmoleukin, 99mTc-apisitide injection, Myelopid, Betaseron, Glatirama acetate, Gepon, Sargramostim, Oprelvekin, Human leukocyte-derived alpha interferon, Bilive, Insulin (recombinant), Recombinant human insulin, Insulin aspartate luteo, mechasenin, Roferon-A, interferon-alpha 2, alphaferon, interferon alfacon-1, interferon alpha, Avonex' recombinant human luteinizing hormone, dornase alfa, trafermin, ziconotide, taltirelin, divotarifer, atosiban, becaplermin, eptifibatide, Zemaira, CTC-111, Shanvac-B, HPV vaccine (4-valent), octreotide, lanreotide, ancestim, agalsidase beta, agalsidase alfa, laronidase, copper acetate presatide (topical gel), rasburicase, ranibizumab, Actimmune, PEG-Intron, Tricomin, recombinant house dust mite allergy desensitization injection, recombinant human parathyroid hormone (PTH: parathyroid hormone hormone)1-84 (subcutaneous, osteoporosis), epoetin delta, transgenic antithrombin III, Granditropin, Vitrase, recombinant insulin, interferon-alpha (oral cough drops), GEM-21S, vapreotide, idursulfase, omapatrilat, recombinant serum albumin, certolizumab pegol, glucarpidase, human recombinant C1 esterase inhibitor (angioedema), lanoteplase, recombinant human growth hormone, enfuvirtide (needle-free injection, Biojector2000), VGV-1, interferon (alpha), lucinactant, aviptadil (inhalation, pulmonary disease), icatibant, ecallantide, omiganan, aurograb, pexiganan acetate, ADI-PEG-20, LDI-200, degarelix, syntredekin besudotox, Favld,MDX-1379, ISAtx-247, liraglutide, teriparatide (osteoporosis), tifacogin, AA4500, T4N5 liposome lotion, catumaxomab, DWP413, ART-123, Chrysalin, desmoteplase, amidiprase, corifollitropin alfa, TH-9507, Teduglutide, Diamyd, DWP-412, Growth hormone (sustained release injection), recombinant G-CSF, Insulin (inhaled, AIR), Insulin (inhaled, Technosphere), Insulin (inhaled, AERx), RGN-303, DiaPep277, Interferon beta (for hepatitis C virus infection (HCV)), Interferon alpha-n3 (oral), Belatacept, Transdermal insulin patch, AMG-531, MBP-8298, Xerecept, Opevacan, AIDSVAX, GV-1001, LymphoScan, Ranpirnase, Lipoxysan, Rusprutide, MP 52 (beta-tricalcium phosphate carrier, bone regeneration), melanoma vaccine, sipuleucel-T, CTP-37, Insegia, Vitespen, human thrombin (freezing, surgical bleeding), thrombin, TransMID, alfimeprase, Puricase, terlipressin (intravenous, hepatorenal syndrome), EUR-1008M, recombinant FGF-I (injectable, vascular disease), BDM-E, rotigaptide, ETC-216, P-113, MBI-594AN, duramycin (inhaled, cystic fibrosis), SCV-07, OPI-45, endostatin, angiostatin, ABT-510, Bowman Birk Inhibitor Concentrate, XMP-629, 99mTc-Hynic-Annexin V, Kahalalide F, CTCE-9908, Toverelix (extended release), Ozarelix, Lornidepsin, BAY-504798, Interleukin 4, PRX-321, Pepscan, Ibuoctadequine, Lulactoferrin, TRU-015, IL-21, ATN-161, Cilengitide, Albuferon, Biphasix, IRX-2, Omega Interferon, PCK-3145, CAP-232, Pasireotide, huN901-DMI, Ovarian Cancer Immunotherapy Vaccine, SB-249553, Oncovax-CL, OncoVax-P,BLP-25, CerVax-16, Multi-epitope peptide melanoma vaccine (MART-1, gp100, tyrosinase), Nemifitide, rAAT (inhaled), rAAT (dermatological), CGRP (inhaled, asthma), Pegsnercept, Thymosin beta 4, Plitidepsin, GTP-200, Ramoplanin, GRASPA, OBI-1, AC-100, Salmon calcitonin (oral , Erigen), Calcitonin (oral, osteoporosis), Examorelin, Capromorelin, Cardeva, Verafermin, 131I-TM-601, KK-220, T-10, Uralitide, Depelstat, Hematide, Chrysalin (topical), rNAPc2, Recombinant V111 Factor (pegylated liposomal), bFGF, Pegylated Recombinant Staphylokinase Mutant, V-10153, SonoLysis Prolyse, NeuroVax, CZEN-002, Islet Cell Neoblastic Therapy, rGLP-1, BIM-51077, LY-548806, Exenatide (Controlled Release, Medisorb), AVE-0010, GA-GCB, Avorelin, ACM-9604, Linaclotide Acetate, CETi-1, Hemospan, VAL (Injectable), Rapid Acting Insulin (Injectable, Viadel), Intranasal Insulin, Insulin (Inhaled), Insulin (Oral, Eligen), Recombinant Methionyl human leptin, Pitrakinra (subcutaneous injection, eczema), Pitrakinra (inhaled dry powder, asthma), Multikine, RG-1068, MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10 (autoimmune disease / inflammation), Talactoferrin (topical), rEV-131 (ophthalmology), rEV-131 (respiratory disease), Oral recombinant human insulin (diabetes), RPI-78M, Oprelvekin (oral), CYT-99007 CTLA4-Ig, DTY-001, Balategrast, Interferon alfa-n3 (topical) IRX-3, RDP-58, Tauferon, Bile salt-stimulated lipase, Merispase, Alkaline phosphatase, EP-2104R, Melanotan-II, Bremelanotide, ATL-104, Recombinant human microplasmin, AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, Dynorphin A, SI-6603,LAB GHRH, AER-002, BGC-728, Malaria vaccine (virosome, PeviPRO), ALTU-135, Parvovirus B19 vaccine, Influenza vaccine (recombinant neuraminidase), Malaria / HBV vaccine, Hepatitis B vaccine, Vacc-5q, Vacc-4x, HIV vaccine (oral), HPV vaccine, Tat Toxoid, YSPSL, CHS-13340, PTH(1-34), liposomal cream (Novasome), Ostabolin-C, PTH analogues (topical, psoriasis), MBRI-93.02, MTB72F vaccine (tuberculosis), MVA-Ag85A vaccine (tuberculosis), FARA04, BA-210, recombinant plague FIV vaccine, AG-702, OxSODrol, rBetV1, Der-p1 / Der-p2 / Der-p7 allergen targeted vaccine (dust mite allergy), PR1 peptide antigen (leukemia), mutant RAS vaccine, HPV-16 E7 lipopeptide vaccine, Labyrinthine vaccine (adenocarcinoma), CML vaccine, WT1-peptide vaccine (cancer), IDD-5, CDX-110, Pentrys, Norelin, CytoFab, P-9808, VT-111, Icrocaptide, Telbermin (dermatology, diabetic foot ulcer), Rupintrivir, Reticulose, rGRF, HA, alpha-galactosidase A, ACE-011, ALTU-140, CGX-1 160, Angiotensin Therapeutic Vaccine, D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral, tuberculosis), DRF-7295, ABT-828, ErbB2-specific immunotoxin (anticancer), DT3SSIL-3, TST-10088, PRO-1762, Combotox, Cholecystokinin-B / gastrin receptor binding peptide, 111In-hEGF, AE-37, Trasnizumab-DM1, Antagonist G, IL-12 (recombinant), PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647 (topical), L-19-based radioimmunotherapy (cancer), Re-188-P-2045, AMG-386, DC / 1540 / KLH vaccine (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-1 vaccine (peptide), NA17.A2 peptide,Melanoma vaccine (pulsed antigen therapy), prostate cancer vaccine, CBP-501, recombinant human lactoferrin (dry eye), FX-06, AP-214, WAP-8294A (injectable), ACP-HIP, SUN-11031, peptide YY[3-36] (obesity, intranasal), FGLL, atacicept, BR3-Fc, BN-003, BA-05, 8, Human parathyroid hormone 1-34 (nose, osteoporosis), F-18-CCR1, AT-1100 (celiac disease / diabetes), JPD-003, PTH(7-34) liposome cream (Novasome), Duramycin (ophthalmology, dry eye), CAB-2, CTCE-0214, GlycoPEGylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, Factor XIII, Aminocandin, PN-951, 716155, SUN-E700 1, TH-0318, BAY-73-7977, teverelix (immediate release), EP-51216, hGH (controlled release, Biosphere), OGP-I, sifvirtide, TV4710, ALG-889, Org-41259, rhCC10, F-991, thymopentin (lung disease), r(m)CRP, hepatoselective insulin, subarin, L19-IL-2 fusion protein, elafin, NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist (thrombocytopenia), AL-108, AL-208, nerve growth factor antagonist (pain), SLV-317, CGX-1007, INNO-105, oral teriparatide (Eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 fusion vaccine (Therapore), EP-1043, S pneumonia pediatric vaccine, malaria vaccine, meningococcal group B vaccine, neonatal group B streptococcal vaccine, anthrax vaccine, HCV vaccine (gpE1+gpE2+MF-59), otitis media Treatment, HCV vaccine (core antigen + ISCOMATRIX), hPTH(1-34) (transdermal, ViaDerm), 768974, SYN-101, PGN-0052, aviscamin, BIM-23190, tuberculosis vaccine, multi-epitope tyrosinase peptide, cancer vaccine, encastim, APC-8204, GI-5005, ACC-001, TTS-CD3, vascular targeted TNF (solid tumors), desmopressin (buccal controlled release), onercept, and TP-9201.

[0092] In some embodiments, the polypeptide is adalimumab (HUMIRA), infliximab (REMICADE™), rituximab (RITUXAN™ / MAB THERA™), etanercept (ENBREL™), bevacizumab (AVASTIN™), trastuzumab (HERCEPTIN™), pegfilgrastim (NEULASTA™), or any other suitable polypeptide, including biosimilars and biobetters.

[0093] Other suitable polypeptides are those listed below and in Table A of US Publication No. 2016 / 0097074.

[0094] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0095] In embodiments, the polypeptide is a hormone, a clotting / blood clotting factor, a cytokine / growth factor, an antibody molecule, a fusion protein, a protein vaccine, or a peptide, as shown in Table B.

[0096] [Table 5-1] [Table 5-2]

[0097] In an embodiment, the protein is a multispecific protein, such as a bispecific antibody, as shown in Table C.

[0098] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0099] The description of various embodiments of the present invention has been given for illustrative purposes and is not intended to be exhaustive or limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terms used in this specification are selected to best explain the principles of the embodiments, practical applications, or technical improvements across the art found in the marketplace, or to allow other skilled in the art to understand the embodiments disclosed herein.

Claims

1. A variable-diameter bioreactor container configured to culture living cells, biological raw materials consisting of cells, or mammalian cells, A container configured to hold a liquid culture medium and biological raw materials, At least one stirrer and Equipped with, The container comprises a first container section located at the bottom of the container, a second container section, and a third container section located at the top of the container. The first container portion has a diameter that changes at a first rate along the height of the first container portion. The second container portion has a diameter that changes along the height of the second container portion at a second rate greater than the first rate, The container is a variable-diameter bioreactor container configured to increase the volume of the liquid culture medium and biological raw materials within the container from a first volume to a second volume, and further from the second volume to a third volume.

2. The variable-diameter bioreactor container according to claim 1, wherein the container has a non-circular cross-sectional shape.

3. The variable-diameter bioreactor container according to claim 1, wherein the container has a square cross-sectional shape.

4. The variable-diameter bioreactor container according to claim 1, wherein the first container section is an initial inoculation stage container section configured to allow the inoculant to be directly added to the first container section.

5. The variable-diameter bioreactor container according to claim 1, wherein the second container section is configured as a growth-stage bioreactor.

6. The variable-diameter bioreactor container according to claim 1, wherein at least one container section of the variable-diameter bioreactor is configured as a production-stage bioreactor.

7. A variable-diameter bioreactor vessel according to claim 1, further comprising at least one of a stirrer shaft, a sparger, a probe port, a filling port, a condenser, a vent filter, a bubble breaker plate, a sample port, a level probe, and a load cell.

8. The variable-diameter bioreactor container according to claim 1, wherein the agitator is manufactured using a three-dimensional printer.

9. The variable-diameter bioreactor container according to claim 1, wherein the at least one agitator comprises two agitators.

10. The variable-diameter bioreactor container according to claim 9, wherein one of the two agitators is a hydrofoil impeller.

11. The variable-diameter bioreactor container according to claim 1, wherein the variable-diameter bioreactor container is a single-use bioreactor.

12. The variable-diameter bioreactor container according to claim 1, wherein the variable-diameter bioreactor container is configured for microcarrier culture.

13. The variable-diameter bioreactor container according to claim 1, wherein the variable-diameter bioreactor container is configured for adhesive culture.

14. The variable-diameter bioreactor container according to claim 1, wherein the third container portion has a uniform diameter throughout its entirety.

15. The variable-diameter bioreactor container according to claim 1, wherein the variable-diameter bioreactor container has the minimum height-to-width aspect ratio at each height of at least one of the second container portion and / or the third container portion.

16. The variable-diameter bioreactor container according to claim 1, wherein the maximum diameter of the third container portion is greater than the maximum diameter of the second container portion, and the maximum diameter of the second container portion is greater than the diameter of the first container portion.

17. A variable diameter bioreactor system, A diameter-variable bioreactor container according to any one of claims 1 to 14, A variable-diameter bioreactor system comprising a control system capable of operating the variable-diameter bioreactor container to expand from a first capacity to a second capacity, and further from the second capacity to a third capacity.

18. The variable-diameter bioreactor system according to claim 17, wherein the control system is configured to scale up the variable-diameter bioreactor container in a batch manner.

19. The variable diameter bioreactor system according to claim 17, further comprising a disposable bag configured to be placed inside the variable diameter bioreactor container so that the variable diameter bioreactor system becomes a single-use bioreactor system.

20. A method for producing fermentation products in a seed stage train, The steps include inoculating the first volume of the variable-diameter bioreactor container according to any one of claims 1 to 14 with a growth medium and an inoculant, After the completion of the inoculation step in the first volume, the additional growth medium is added to the variable-diameter bioreactor container to expand the volume of the variable-diameter bioreactor container from the first volume to the second volume. A method comprising the step of adding additional growth medium to the variable-diameter bioreactor container to expand the capacity of the variable-diameter bioreactor container from the second capacity to the third capacity, after the completion of the seeding stage in the second capacity.

21. The method according to claim 20, wherein the inoculant is obtained from an initial seed reactor.

22. The method according to claim 20, wherein the variable-diameter bioreactor container is the last reactor in a seed stage train and is also used as a production reactor.

23. The method according to claim 20, wherein the inoculant is a mammalian cell culture.

24. A biological production facility, Initial inoculant growth reactor, A biological production facility comprising a variable-diameter bioreactor container according to any one of claims 1 to 14, wherein the variable-diameter bioreactor container is in fluid communication with the initial inoculant growth reactor, and is configured such that the variable-diameter bioreactor container becomes a train for the seed stage reactor.

25. The biological production apparatus according to claim 24, wherein the variable-diameter bioreactor container is further configured as a production reactor.

26. The biological production apparatus according to claim 24, further comprising a plurality of variable-diameter bioreactor containers.

27. The biological production apparatus according to claim 24, wherein the variable-diameter bioreactor container is in fluid communication with a downstream processing component.

28. The biological production facility according to claim 24, wherein the variable-diameter bioreactor vessel is controlled by a controller system.

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