Continuous Blade Impeller

Abstract

The impeller can be used in a variable diameter bioreactor having multiple vessel sections with continuously increasing and decreasing volumes. The impeller includes an impeller blade extending along an impeller blade axis between first and second axial ends and having opposing impeller blade faces, a leading edge, and a trailing edge. Each of the leading edge and the trailing edge defines a spiral or helix between the axial ends of the impeller blade. In certain devices, the impeller blade is one of at least two impeller blades coupled together along an impeller shaft extending axially along the impeller blade axis, and the spiral or helix has approximately the same pitch for half the length between the first and second axial ends.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 836,922, filed April 22, 2019, the entire contents of which are incorporated by reference herein.

[0002] The present invention relates to a novel impeller for use in a bioreactor. The impeller according to the invention is suitable for use in bioreactors of any type or capacity, including variable diameter bioreactors. [Background technology]

[0003] U.S. Patent Application Publication No. 2017 / 0369828 A1 to Mietzner et al. discloses certain variable diameter bioreactor vessel configurations for the production of biological materials. The bioreactor vessel can have multiple vessel sections with different diameters, and in certain arrangements, an agitator assembly extends into the interior volumes of the different bioreactor vessel sections, with multiple agitators disposed on agitator shafts located within the interior volumes of each vessel section. The entire disclosure of U.S. Patent Application Publication No. 2017 / 0369828 to Mietzner et al. is incorporated by reference herein as non-essential material.

[0004] The bioreactors, bioreactor trains, and methods disclosed in U.S. Patent No. 9,783,771, U.S. Patent No. 9,670,446, and U.S. Patent Application Publication No. 2017 / 0267962 to Khan are also suitable vessels in which the novel impellers of the present invention may be used, and these patents and applications are incorporated by reference herein in their entireties.

[0005] The impeller of the present invention may also be used in disposable bioreactors such as those disclosed in U.S. Patent Application Publication No. 2017 / 0369828, WO 2017 / 223269, U.S. Patent Application Publication No. 2017 / 0349874, and WO 2017 / 207822.

[0006] Conventional bioreactor processing of microbial cultures typically uses multiple impellers mounted on a shaft with an offset distance between the bottom of the vessel and the air-liquid interface and between other impellers. Seed vessels (e.g. N-1, N-2) are used to volumetrically increase the capacity relative to cell density and production scale. Bioreactor processing at full scale (e.g. N) using conventional impellers is subject to limitations that prevent steady medium volume addition during the growth cycle without the use of seed vessels. Bolus addition is used to achieve height above the agitator without excessive foaming at the impeller level. Variable diameter bioreactor (VDB) technology also requires purpose-designed impellers to enable steady volume addition and avoid limitations imposed by conventional impellers. The design requires consideration of optimal continuous mixing conditions (e.g. mixing time and shear), stable and adequate power per unit volume as well as minimal foaming conditions while providing agitation to the dynamically changing reactor volume and eliminating growth delays from conventionally designed agitators. This also allows for optimization of processing at existing downstream scales by varying the bioreactor volume, making the process adaptable for low, medium and high titer production lines.

[0007] In some examples, production scale bioreactor processing is achieved by using a bioreactor where the liquid height is greater than the vessel diameter (aspect ratio >1:1, liquid height to vessel diameter) and a multiple blade impeller arranged on a shaft is used to mix the culture. Other examples use aspect ratios as disclosed in U.S. Pat. No. 9,783,771, U.S. Pat. No. 9,670,446, and U.S. Patent Application Publication No. 2017 / 0267962 to Khan. These bioreactors are sized to increase the volume of the culture from an inoculum in an intermediate seed reactor, the volume of which increases until the production volume is reached. Typically, a bioreactor consists of a vertical vessel with a dish-shaped top and bottom and, optionally, either a tank or a disposable bag in a support structure. Typically, the impeller consists of a shaft-coupled (e.g., mechanical, magnetic) motor with a separate agitator attached (e.g., welded, set screw) in a configuration that mixes solution and gas into the culture to promote efficient growth.

[0008] Known processes, products, and equipment have certain limitations and drawbacks. Each conventional reactor / impeller system involves transfer from one reactor to another, introducing the culture into a state different from the end of the previous reactor. The design of the reactor / agitator system works best in a narrow capacity band. This typically results in a "lag phase" effect, where cell growth stalls for a period of time before reaching exponential growth again. This typical process requires multiple reactors at large scale, resulting in a large facility footprint and additional preparatory work. This results in increased utility costs (water, steam, waste), increased cleaning in place (CIP) systems, increased sterilization in place (SIP) steps, and increased contamination risks. The versatility of conventional reactor / impeller systems with capacity scalability is limited. A conventional agitator design applied to a variable diameter bioreactor was shown to separate the mixing zones (see Figures 2A-C). Summary of the Invention

[0009] The impeller according to the invention is particularly suitable for use in variable diameter bioreactors having multiple vessel sections with continuously increasing and decreasing volumes. The impeller comprises an impeller blade extending along an impeller blade axis between first and second axial ends and having opposing impeller blade faces, an impeller blade leading edge, and an impeller blade trailing edge. Each of the leading edge and trailing edge defines a spiral or helix between the axial end of the impeller blade. In a particular device, the impeller blade is one of at least two impeller blades coupled together along an impeller shaft extending axially along the impeller blade axis described, and the spiral or helix has approximately the same pitch for half the length between the first and second axial ends. [Brief description of the drawings]

[0010] [Figure 1] 1A-1D show schematic cross-sectional views of a 20,000 L bioreactor vessel, where FIG. 1A shows a conventional bioreactor, FIGS. 1B and 1C show a variable diameter bioreactor (VDB) with a conventional impeller, and FIG. 1D shows a VDB with a continuous agitator or impeller. [Diagram 2] 2A-2D are schematic diagrams of fluid velocity vectors at similar P / V values ​​for a 20,000 L bioreactor vessel, where FIG. 2A shows the vectors for a conventional bioreactor, FIGS. 2B and 2C show the vectors for a variable diameter bioreactor (VDB) with a conventional impeller, and FIG. 2D shows the vectors for a VDB with a continuous agitator or impeller. [Diagram 3] Figure 3 shows the initial positions of tracers added to the conventional bioreactor and the VDB for the computational fluid dynamics (CFD) simulation of mixing times. [Figure 4A] FIG. 4A provides a comparison of compounding times calculated from a CFD simulation for a particular configuration of the present invention, where the analysis had the geometry problem fixed and adjusted in a subsequent update. [Figure 4B] FIG. 4B provides a comparison that is nearly identical to that provided in FIG. 4, but includes an updated optimal format. [Diagram 5] Figure 5 shows the VDB modeling update, but again, the analysis ended up having geometric issues that were fixed and adjusted in subsequent updates. [Figure 6] FIG. 6 provides a comparison between a 20 kl conventional bioreactor and three VDB bioreactor formats designated V6, V7, and V8. [Figure 7] FIG. 7 provides a comparison of fluid velocity vectors and fluid paths for the VDB bioreactor modes designated V6, V7, and V8 described. [Figure 8] FIG. 8 provides a preliminary graphical comparison of blend times for the VDB mode versus a conventional 20,000 kl bioreactor. [Figure 9] FIG. 9 provides an example of the flow number calculation. [Figure 10] FIG. 10 provides a comparison between a 20 kl conventional bioreactor and five VDB bioreactor styles designated V1, V2, V3, V4, and V5. [Figure 11] FIG. 11 provides a comparison of the fluid paths for a 20 kl conventional bioreactor and a 20 kl VDB bioreactor configuration designated V1, V2, V3, V4, and V5 as described. [Figure 12] FIG. 12 is a fluid velocity vector diagram for the 20 kl conventional bioreactor and the 20 kl VDB bioreactor modes designated V1, V2, V3, V4, and V5 as described. [Figure 13] FIG. 13 is another preliminary VDB modeling update similar to, but more recent than, that depicted in FIG. [Figure 14A] 14A and 14B provide schematic diagrams of the axial distribution of mixing energy at the same output volume ratio for VDB bioreactor formats designated V9, V16, V17, and V18. [Figure 14B] Same as above. [Figure 15]FIG. 15 is a schematic diagram of fluid velocity vectors at the same pressure volume ratio for VDB bioreactor configurations designated V9, V16, V17, V18, V19, and V20. [Figure 16] FIG. 16 provides a comparison of fluid pathways for the VDB bioreactor designs designated V9, V16, V17, V18, V19, and V20 described. [Figure 17] FIG. 17 shows a spatial analysis simulation of blend times for the VDB bioreactor mode specified by V20. [Figure 18A] 18A and 18B show spatial analysis simulations of blend times for the VDB bioreactor mode designated by V19. [Figure 18B] Same as above. [Figure 19] FIG. 19 shows a spatial analysis simulation of blend times for the VDB bioreactor mode designated by V18. [Figure 20A] 20A and 20B show spatial analysis simulations of blend times for the VDB bioreactor mode designated by V17. [Figure 20B] Same as above. [Figure 21A] 21A and 21B show spatial analysis simulations of blend times for the VDB bioreactor mode designated by V16. [Figure 21B] Same as above. [Figure 22] FIG. 22 shows the construction of a possible VDB prototype. [Diagram 23] FIG. 23 shows a diagram of a conventional 20,000 L bioreactor including a geometric representation similar to that shown in FIG. 1A, a fluid velocity vector representation similar to that shown in FIG. 2A but with different rpm settings, and a fluid path representation similar to that shown in FIGS. 7, 11, and 16 but with different rpm settings. [Figure 24] FIG. 24 provides a geometric representation of the V1 style of the VDB included in FIG. 10, a fluid velocity vector diagram similar to the V1 diagram included in FIG. 12, and a fluid path diagram similar to the V1 diagram presented in FIG. 11 but with a different rpm setting. [Diagram 25] FIG. 25 provides a geometric representation of the V2 design of the VDB included in FIG. 10 but with a different rpm setting, a fluid velocity vector diagram similar to the V2 diagram included in FIG. 12 but with a different rpm setting, and a fluid path diagram similar to the 75 rpm diagram of the V2 presented in FIG. 11 but with a different rpm setting. [Figure 26] FIG. 26 provides a V3 geometric representation of a VDB with the impeller design included in FIG. 10, a fluid velocity vector diagram similar to the V3 diagram included in FIG. 12 but with a different rpm setting, and a fluid path diagram similar to the V3 diagram presented in FIG. 11 but with a different rpm setting. [Figure 27] FIG. 27 provides a geometric representation of V4 of the VDB with the impeller design contained in FIG. 10, a fluid velocity vector diagram similar to the V4 46 rpm diagram contained in FIG. 12, and a fluid path diagram similar to the V4 diagram presented in FIG. 11 but with a different rpm setting. [Figure 28] FIG. 28 provides a geometric representation of V5 of a VDB with the impeller design included in FIG. 10, a fluid velocity vector diagram similar to the V5 diagram included in FIG. 12 but with a different rpm setting, and a fluid path diagram similar to the V5 diagram presented in FIG. 11 but with a different rpm setting. [Figure 29] FIG. 29 provides a geometric representation of V6 of a VDB with the impeller design included in FIG. 6, a fluid velocity vector diagram similar to the V6 diagram included in FIG. 7 but with a different rpm setting, and a fluid path diagram similar to the V6 diagram presented in FIG. 7 but with a different rpm setting. [Diagram 30] FIG. 30 provides a V7 geometric representation of a VDB with the impeller design included in FIG. 6, a fluid velocity vector diagram similar to the V7 diagram included in FIG. 7 but with a different rpm setting, and a fluid path diagram similar to the V7 diagram presented in FIG. 7 but with a different rpm setting. [Diagram 31]FIG. 31 provides a V8 geometric representation of a VDB with the impeller design included in FIG. 6, a fluid velocity vector diagram similar to the V8 diagram included in FIG. 7 but with different rpm settings, and a fluid path diagram similar to the V8 diagram presented in FIG. 7 but with different rpm settings. [Diagram 32] FIG. 32 provides a geometric representation of the V9 design of the VDB included in FIGS. 14A and 14B but with different rpm settings, a fluid velocity vector diagram similar to the V9 diagram included in FIG. 15 but with different rpm settings, and a fluid path diagram similar to the rpm diagram of the V9 presented in FIG. 16 but with different rpm settings. [Diagram 33] FIG. 33 provides additional VDB bioreactor formats, a geometric representation of the designated VDB format V10, a fluid velocity vector diagram for the VDB format V10, and a fluid path diagram for the VDB format V10. [Diagram 34] FIG. 34 provides additional VDB bioreactor formats, a geometric representation of the designated VDB format V11, a fluid velocity vector diagram for the VDB format V11, and a fluid path diagram for the VDB format V11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The main concepts of the present invention will now be identified, as well as the purpose and operation of the present invention, including technical characteristics. A continuously stirred conical bottom tank is used to support a minimum inoculum size with sufficient head for oxygen transfer while being able to maintain an aspect ratio (liquid height to vessel diameter at liquid level) of greater than 1:1, while scaling up to larger scales. The volume of the culture is then bulked up by the addition of medium to support logarithmic growth, due to the continuous stirring design concept.

[0012] Early VDB configurations consisted of using multiple impellers of the conventional configuration (i.e., the same type of impellers used in conventional bioreactors) but changing the size, number, and overall arrangement. CFD simulations of these configurations indicated that an unacceptably large number of impellers (five or more) could be required to avoid mixing regions of the separated fluids and achieve adequate overall fluid mixing. Using four impellers, optimizing the impeller type and size resulted in blending times of about 200-400 seconds for a 20,000 L packed VDB, compared to about 60 seconds for a 20,000 L conventional bioreactor. The VDB configuration transitioned to mostly using a novel continuous agitator that covered almost the entire vertical length of the reactor and provided more consistent mixing as a function of height. Numerous modifications of impeller pitch and blade count, as well as baffle size, resulted in blending times of approximately 68-100 seconds for a 20,000 L packed VDB, compared to approximately 60 seconds for a 20,000 L conventional bioreactor.

[0013] FIG. 1 shows schematics of (A) a 20,000 L conventional bioreactor, (B and C) a 20,000 L VDB equipped with multiple conventional impellers, and (D) a 20,000 L VDB equipped with a continuous agitator.

[0014] The impeller for the VDB of Figure 1B is sized and positioned according to best practices in the art. The impeller of Figure 1C has been optimized to reduce mixing time at the same P / V value compared to Figure 1B.

[0015] Figures 2A-D show fluid velocity vectors from computational dynamic (CFD) simulations for vertical cross sections for each of the bioreactors operated at similar average power / volume. Figures 2A-C show that the four conventional impellers in the VDB result in separate mixing regions rather than the single, connected fluid stream found in conventional bioreactors. Figure 2D shows that these separate mixing regions are greatly reduced in the continuous agitator format.

[0016] CFD simulations were performed of the mixing time of the fluids. Mixing time is quantitatively defined as the time required for all points in the bioreactor to be within + / - 5% of the final average value for the tracer fluid added. For these simulations, the tracer fluid was added as a single bolus to the top of each bioreactor, as shown in Figure 3, and then mixing of that bolus was tracked as a function of time until homogeneity was achieved.

[0017] The results of these simulations are shown in Figures 4A and 4B, with Figure 4B including the currently optimized mode V20. Blending times (as previously defined) for a VDB with four conventional impellers (mode V1) range from 420 seconds (using impellers sized using best practices in the industry) to 264 seconds (for impellers optimized to maximize fluid mixing, mode V2). These results suggest that more than four impellers may be required to achieve acceptable mixing for conventional bioreactors. Blending times for a VDB with a continuous stirrer (mode V20) show blending times of approximately 100 seconds, indicating a significant improvement in mixing over the same VDB bioreactor with conventional impellers.

[0018] The particular illustrations provided by Figures 14A-16 show a currently optimal format. By way of example, and referring to Figures 14A and 14B, each of the impeller configurations shown are suitable for use in a multi-vessel VDB such as that disclosed by the above-referenced Mietzner et al. '828 A1 publication. As shown in Figure 14A, the impeller 100 can be used in a variable diameter bioreactor 102 having multiple vessel sections 104, 106, and 108 that have successively increasing and decreasing volumes. The illustrated impeller 100 includes an impeller blade 110 extending along an impeller blade axis 112 between first and second axial ends 114, 116 and having opposing impeller blade faces 118, 120, an impeller blade leading edge 122, and an impeller blade trailing edge 124. As shown, the leading and trailing edges 122 , 124 each define a spiral or helix between the first and second axial ends 114 , 116 of the impeller blade 110 .

[0019] 14A and 14B, the impeller blade 110 is one of at least two impeller blades 110, 126 coupled together along an axially extending impeller shaft 118 along a depicted impeller blade axis 112. The spiral or helix has approximately the same pitch halfway along its length between the first and second shaft ends 114, 116, and as shown in FIG 14A, the depicted pitch is half the depicted length. The differences between the styles V9, V16, V17, and V18 shown in Figures 14A and 14B are primarily in the combined diameter of the shaft 118 and the width of the blades 110 and 126, with V9 having a combined diameter and width of 0.35m between the first and second shaft ends 114, 116, V16 having a combined diameter and width that tapers from 0.75m at the first shaft end 114 to 0.35m at the second shaft end 116, V17 having a combined diameter and width that tapers from 0.45m at the first shaft end 114 to 0.35m at the second shaft end 116, and V18 having a combined diameter and width that tapers from 0.50m at the first shaft end 114 to 0.35m at the second shaft end 116.

[0020] The features contemplated in this invention that are found to be different (i.e., novel) compared to the state of the art include the use of a sequential impeller alone or in conjunction with another impeller (two-shaft design) inside a typical cylindrical bioreactor or in a variable diameter bioreactor.

[0021] For the envisioned product, process, or application, the greatest advantage would be the bioreactor system for contract manufacturing. Easy configuration for total production volume and minimized facility footprint are key attributes for this application (adaptability for low, medium, and high titer processes). The ease of configuring existing bioreactors with this agitator technology eliminates the need for seed stage train equipment (N-1 and potentially more).

[0022] As will be apparent from this disclosure, the scaled-down format can be constructed and used for any desired mixing, mass transfer, and clean-in-place attributes. The scaled-down format can be used to feed a production bioreactor (conventional or VDB) in place of a seed stage train.

[0023] The following examples are described in the Mietzner et al. '828 A1 publication referenced above and are incorporated herein by reference, and are expressly incorporated herein as if for completeness. EXAMPLES

[0024] The relationship between the volume, diameter and cell growth characteristics of the variable diameter bioreactor of the present invention requires consideration of many factors. The following equations provide useful guidance in designing the bioreactor of the present invention.

number

[0025] For example, in designing a bioreactor of the invention to operate with a capacity of up to 20,000 L, the variable diameter bioreactor would have the following dimensions:

[0026] Total capacity: 20,000L

[0027] Cone capacity: 15,000L

[0028] Top diameter: 7 feet

[0029] Base diameter: 3 feet

[0030] Overall Height: 30.2 ft

[0031] Cylindrical capacity: 5,000L

[0032] Cone Height: 25.6 ft

[0033] Barrel Height: 4.6 ft

[0034] As another example, in 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 formula would have the following dimensions:

[0035] Total volume: 16,458L

[0036] Cone volume: 9,341L

[0037] Top diameter: 8 feet

[0038] Base diameter: 2 feet

[0039] Overall height: 20 feet

[0040] Cylindrical capacity: 7,117L

[0041] Cone Height: 15 feet

[0042] Cylindrical height: 5 feet

[0043] The above embodiment may have four impellers.

[0044] As another example, the design of the present invention allows variable diameter bioreactors to be made in excess of 20,000 L, which is new to the industry. In particular, variable diameter bioreactors can be made in the following dimensions:

[0045] Total capacity: 25,000L

[0046] Cone volume: 15,000L

[0047] Top diameter: 7.9 feet

[0048] Base diameter: 2.5 feet

[0049] Overall height: 30 feet

[0050] Cylindrical capacity: 10,000L

[0051] Cone Height: 22.8 ft

[0052] Barrel Height: 7.2 ft

[0053] Unless otherwise stated above, the above description may be further understood as follows: The devices, equipment and methods described herein are suitable for use in culturing and culturing any desired cell line, including prokaryotic and / or eukaryotic cell lines. Furthermore, in embodiments, the devices, equipment and methods are suitable for culturing suspension cells or anchorage-dependent (adherent) cells, and are suitable for production processes configured for the manufacture of pharmaceuticals and biopharmaceuticals, such as polypeptide products, nucleic acid products (e.g., DNA or RNA), or cells and / or viruses, such as for use in cell therapy and / or virus therapy.

[0054] In embodiments, the cells express or make a product, such as a recombinant therapeutic or diagnostic product. As described in more detail below, examples of products made 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 antigens but are not structurally related to antibodies, such as 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) or DNA (e.g., plasmid DNA), antibiotics, or amino acids. In embodiments, the devices, equipment, and methods can be used to generate biosimilars.

[0055] As described, in embodiments, the devices, equipment, and methods allow for the production of eukaryotic or prokaryotic products, such as proteins, peptides, antibiotics, amino acids, nucleic acids (such as DNA or RNA), synthesized in a large-scale manner by 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 cells. Unless otherwise stated herein, the devices, equipment, and methods can include any desired capacity or production capability, including, but not limited to, laboratory scale, pilot production scale, and full production scale capabilities.

[0056] 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, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. As used in the variants, "reactor" may include a fermenter or fermentation apparatus, or any other reaction vessel, and the term "reactor" is used interchangeably with "fermenter." For example, in some embodiments, a typical bioreactor apparatus may perform one or more or all of the following: feeding nutrients and / or carbon sources, injecting a suitable gas (e.g., oxygen), inflow and outflow of fermentation or cell culture medium, separating gas and liquid phases, maintaining temperature, maintaining oxygen and carbon dioxide concentrations, maintaining pH levels, agitation (e.g., stirring), and / or cleaning / sanitization. A typical reactor apparatus, such as a fermenter, may include multiple reactors within the apparatus, for example, an apparatus may have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors in each apparatus, and / or a facility may include multiple apparatuses with single or multiple reactors within the facility. In various embodiments, the bioreactor may be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter may be used. In embodiments, the bioreactor may have a capacity of about 100 mL to about 50,000 L. Non-limiting examples include 100mL, 250mL, 500mL, 750mL, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 15L, 20L, 25L, 30 L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, ​​200L, 250L, 300L, 350L, 400L, 450L, 500L, 550L, 600 L, 650L, 700L, 750L, 800L, 850L, 900L, 950L, 1000L, 1500L, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 6000L, 7000L, 8000L, 9000L, 10,000L, 15,000L, 20,000L, and / or 50,000L volumes.Additionally, suitable reactors may be multi-use, single-use, disposable, or non-disposable, and may be formed from any suitable material, including metal alloys such as stainless steels (e.g., 316L or any other suitable stainless steel) and Inconel, plastic, and / or glass.

[0057] In embodiments, unless otherwise noted herein, the apparatus, systems, and methods described herein can also include any suitable apparatus operations and / or equipment not otherwise mentioned, such as operations and / or equipment for separation, purification, and isolation of such products. Any suitable equipment and environment can be used, such as conventional field-assembled facilities, modularly assembled mobile or temporary facilities, or any other suitable structure, equipment, and / or configuration. For example, in some embodiments, modularly assembled clean rooms can be used. Additionally, unless otherwise noted herein, the apparatus, systems, and methods described herein can be contained and / or performed in a single location or facility, or alternatively, can be contained and / or performed in separate or multiple locations and / or facilities.

[0058] By way of non-limiting example, and without limitation, U.S. Patent Application Publication Nos. 2012 / 0077429 and 2009 / 0305626, and U.S. Patent Nos. 9,388,373, 8,771,635, 8,298,054, 7,629,167, and 5,656,491, the contents of which are incorporated herein by reference in their entireties, describe exemplary equipment, devices, and / or systems that may be suitable.

[0059] In an embodiment, the cell is a eukaryotic cell, such as 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 such as COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLA, EBl, EB2, EB3, oncolytic cells or hybridoma cell lines. Preferably, the mammalian cell is a CHO cell line. In one embodiment, the cell is a CHO cell. In one embodiment, the cell is a CHO-K1 cell, a CHO-K1SV 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 can also be an avian cell, cell line, or cell line, such as an EBx® cell, EB14, EB24, EB26, EB66, or EBvl3.

[0060] 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).

[0061] In one embodiment, the cell is a differentiated form of any of the cells described herein. In one embodiment, the cell is derived from any primary cell in culture.

[0062] In an embodiment, the cell is a hepatocyte, such as a human hepatocyte, an animal hepatocyte, or a non-parenchymal cell. For example, the cell can be an adherent metabolically adapted human hepatocyte, an adherent induction adapted human hepatocyte, an adherent Qualyst Transporter™ adapted human hepatocyte, a suspension adapted human hepatocyte (including 10 donor and 20 donor pooled hepatocytes), a human hepatic Kupffer cell, a human hepatic stellate cell, a dog hepatocyte (including a single pooled Beagle dog hepatocyte), a mouse hepatocyte (including CD-1 hepatocyte and C57BI / 6 hepatocyte), a rat hepatocyte (including Sprague-Dawley hepatocyte, Wistar Han hepatocyte, and Wistar hepatocyte), a monkey hepatocyte (including Cynomolgus or Rhesus monkey hepatocyte), a cat hepatocyte (including Domestic Shorthair hepatocyte), and a rabbit hepatocyte (including New Zealand White hepatocyte). Exemplary hepatocytes are commercially available from Triangle Research Labs, LLC, 6 Davis Drive, Research Triangle Park, North Carolina, USA 27709.

[0063] 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.

[0064] 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), Hypocrea (H.jecorina, etc.), Magnaporthe (M.orzyae, etc.), Myceliophthora (M.thermophile, etc.), Nectria (N.heamatococca, etc.), Neurospora (N.crassa, etc.), Penici llium, Sporotrichum (S.thermophile, etc.), Thielavia (T.terrestris, T.heterothallica, etc.), Trichoderma (T.reesei, etc.), or Verticillium (V.dahlia, etc.)).

[0065] In one embodiment, 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)).

[0066] In one embodiment, the cell is a bacterial cell or a prokaryotic cell.

[0067] 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 is available, for example, from the Bacillus Genetic Stock Center, Biological Sciences 556, 484 West 12th Avenue, Columbus OH 43210-1214.

[0068] In one embodiment, the prokaryotic cells are gram-negative cells such as Salmonella spp. or Escherichia coli, e.g., TG1, TG2, W3110, DH1, DHB4, DH5a, HMS174, HMS174(DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue, and Origami, and cells from E. coli B strains, e.g., BL-21 or BL21(DE3), all of which are commercially available.

[0069] 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).

[0070] In embodiments, the cultured cells are used to produce proteins, e.g., antibodies, such as monoclonal antibodies, and / or recombinant proteins for therapeutic use. In embodiments, the cultured cells produce peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites. For example, in embodiments, molecules having molecular weights from about 4,000 daltons to greater than about 140,000 daltons can be produced. In embodiments, these molecules can have a range of complexity and can include post-translational modifications, including glycosylation.

[0071] In embodiments, the protein may be, for example, BOTOX, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxins), 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), rFactor VIII + VWF, Recombinate, rFactor VIII, Factor VIII (recombinant), Alphnmate, Octocog alfa, Factor VIII, Palifermin, Indikinase, Tenecteplase, Alteplase, Pamitoplase, Reteplase, Nateplase, Monteplase, Follitropin alfa, rFSH, hpFSH, 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 rinfabate, anakinra, celmoleukin, 99 mTc-apisitide injection, Myelopid, Betaseron, Glatirama acetate, Gepon, Sargramostim, Oprelvekin, Human leukocyte-derived alpha interferon, Bilive, Insulin (recombinant), Recombinant human insulin, Insulin aspart, Mechasenin, Roferon-A, Interferon-alpha 2, Alphaferon, Interferon alfacon-1, Interferon alpha, Avonex' Recombinant Human Luteinizing Hormone, Dornase alfa, Trafermin, Ziconotide, Taltirelin, Divotermin, Atosiban, Becaplermin, Eptifibatide, Zemaira, CTC-111, Shanvac-B, HPV vaccine (4-valent), Octreotide, Lanreotide, Ancestim, Agalsidase beta, Agalsici alpha, laronidase, copper acetate prezatide (topical gel), rasburicase, ranibizumab, Actimmune, PEG-Intron, Tricomin, recombinant house dust mite allergy desensitization injection, recombinant human parathyroid hormone (PTH) 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, Biojector 2000), 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 (extended release injection), Recombinant G-CSF, Insulin (inhaled, AIR), Insulin (inhaled, Technosphere), Insulin (inhaled, AERx), RGN-303, DiaPep277, Interferon beta (Hepatitis C virus infection (HCV)), Interferon alfa-n3 (oral), Belatacept, Transdermal insulin patch, AMG-531, MBP-8298, Xe recept, opevacan, AIDSVAX, GV-1001, LymphoScan, ranpirnase, Lipoxysan, ruspultide, MP52 (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 (inhalation, cystic fibrosis), SCV-07, OPI-45, endostatin, angiostatin, ABT-510, Bowman Birk Inhibitor Concentrate, XMP-629, 99 mTc-Hynic-Annexin V, Kahalalide F, CTCE-9908, Toberelix (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, Elygen ), calcitonin (oral, osteoporosis), examorelin, capromorelin, Cardeva, verafermin, 131I-TM-601, KK-220, T-10, ularitide, 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, Meripase, 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,LABGHRH, 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-11 60, 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-058, 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, S UN-E7001, 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 Agonists (thrombocytopenia), AL-108, AL-208, Nerve growth factor antagonists (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-8024, GI-5005, ACC-001, TTS-CD3, vascular targeted TNF (solid tumors), desmopressin (buccal controlled release), onercept, and TP-9201.

[0072] In some embodiments, the polypeptide is adalimumab (HUMIRA), infliximab (REMICADE™), rituximab (RITUXAN™ / MABTHERA™), etanercept (ENBREL™), bevacizumab (AVASTIN™), trastuzumab (HERCEPTIN™), pegfilgrastim (NEULASTA™), or any other suitable polypeptide, including biosimilars and biobetters.

[0073] Other suitable polypeptides are those listed below and in Table A of US Publication No. 2016 / 0097074.

[0074] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0075] In embodiments, the polypeptide is a hormone, a clot formation / blood clotting factor, a cytokine / growth factor, an antibody molecule, a fusion protein, a protein vaccine, or a peptide, as shown in Table B. [Table 6] [Table 7] [Table 8]

[0076] In an embodiment, the protein is a multispecific protein, such as a bispecific antibody, as shown in Table C. [Table 9] [Table 10] [Table 11] [Table 12]

[0077] The description of various embodiments of the present invention is provided 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, practical applications, or technical improvements over the art found in the marketplace of the present embodiments, or to enable other skilled in the art to understand the embodiments disclosed herein.

Claims

1. 1. An impeller usable in a variable diameter bioreactor having multiple vessel sections with successively increasing and decreasing volumes, comprising: an impeller blade having an edge extending along an impeller blade axis between first and second axial ends, the impeller blade having opposing impeller blade faces, an impeller blade leading edge, and an impeller blade trailing edge, the leading edge and the trailing edge each comprise an impeller blade defining a spiral or helix between the first and second axial ends of the impeller blade; the impeller blade being one of at least two impeller blades coupled together along an axially extending impeller shaft; Impeller.

2. 2. The impeller of claim 1, wherein the spiral or helix has approximately the same pitch halfway along its length between the first and second axial ends.

3. The impeller of claim 1 , wherein the spiral or helix has a pitch that is greater than half the length between the first and second axial ends.

4. 2. The impeller of claim 1, wherein the spiral or helix has a pitch that is less than half the length between the first and second axial ends.

5. 1. An impeller usable in a bioreactor, comprising: an impeller blade having an edge extending along an impeller blade axis between first and second axial ends, the impeller blade having opposing impeller blade faces, an impeller blade leading edge, and an impeller blade trailing edge, the leading edge and the trailing edge each comprise an impeller blade defining a spiral or helix between the first and second axial ends of the impeller blade; the impeller blade being one of at least two impeller blades coupled together along an axially extending impeller shaft; Impeller.

6. 6. The impeller of claim 5, wherein the spiral or helix has substantially the same pitch halfway along its length between the first and second axial ends.

7. 6. The impeller of claim 5, wherein the spiral or helix has a pitch that is greater than half the length between the first and second axial ends.

8. 6. The impeller of claim 5, wherein the spiral or helix has a pitch that is less than half the length between the first and second axial ends.

9. 1. A variable diameter bioreactor apparatus comprising: A variable diameter bioreactor having multiple vessel sections whose volumes are continuously increased and decreased; an impeller having an impeller shaft and impeller blades, the impeller blades having edges extending along the impeller shaft between first and second axial ends of the shaft, the impeller blades having opposing impeller blade faces, an impeller blade leading edge, and an impeller blade trailing edge; Equipped with the leading edge and the trailing edge each define a spiral or helix between the first and second axial ends of the impeller blade; the impeller blade is one of at least two impeller blades coupled together along the impeller shaft; Variable diameter bioreactor device.

10. 10. The variable diameter bioreactor apparatus of claim 9, wherein the spiral or helix has approximately the same pitch halfway along its length between the first and second axial ends.

11. 10. The variable diameter bioreactor apparatus of claim 9, wherein the spiral or helix has a pitch that is greater than half the length between the first and second axial ends.

12. 10. The variable diameter bioreactor apparatus of claim 9, wherein the spiral or helix has a pitch that is less than half the length between the first and second axial ends.

13. 1. A method for treating a microbial culture comprising: placing the fluid containing the microbial culture in a variable diameter bioreactor apparatus comprising: a variable diameter bioreactor having a plurality of vessel portions for successively increasing and decreasing volumes; and an impeller having an impeller shaft and impeller blades having edges extending along the impeller shaft between first and second axial ends of the shaft, the impeller blades including opposing impeller blade faces, an impeller blade leading edge, and an impeller blade trailing edge, each of the leading edge and trailing edge defining a spiral or helix between the first and second axial ends of the impeller blade; rotating the impeller within the fluid containing the microbial culture; the impeller blade is one of at least two impeller blades coupled together along the impeller shaft; method.

14. The method of claim 13, wherein the impeller is rotated at a speed of 54 to 75 rpm.

15. 14. The method of claim 13, wherein the variable diameter reactor has a fluid capacity of 20,000 L.

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