# Patent

## Title
Continuous blade impeller

## Abstract

An impeller is usable in a variable diameter bioreactor having multiple vessel sections of successively increasing or decreasing volume. The impeller includes an impeller blade extending along an impeller blade axis between first and second axial ends and having opposed impeller blade faces, an impeller blade leading edge, and an impeller blade trailing edge. Each of the leading and trailing edges defines a helix or spiral between the axial ends of the impeller blade. In certain arrangements, the impeller blade is one of at least two impeller blades joined together along an impeller shaft extending axially along the impeller blade axis mentioned, and the helix or spiral has a pitch approximately equal to one half of a length between the first and second axial ends.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to and the benefit of U.S. Provisional Application No. 62/836,922, filed Apr. 22, 2019, the contents of which are incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to novel impellers for use in bioreactors. Impellers according to the present invention are suitable for use in any type or volume of bioreactor, including variable diameter bioreactors.
DESCRIPTION OF RELATED ART
U.S. Patent Application Publication No. 2017/0369828 A1 to Mietzner et al. discloses certain variable diameter bioreactor vessel configurations for production of biologic material. The bioreactor vessels can include multiple vessel sections with different diameters, and, in certain arrangements, an agitator assembly extends into different bioreactor vessel section internal volumes so that multiple agitators disposed on an agitator shaft are located within respective vessel section internal volumes. The entire disclosure of the Mietzner et al., US 2017/0369828 publication is incorporated herein by reference as non-essential material.
The bioreactors, bioreactor trains, and methods disclosed in U.S. Pat. Nos. 9,783,771; 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 can be used. These patents and applications are hereby incorporated by reference in their entireties.
The impeller of the present invention may also be used in single-use bioreactors, such as those disclosed in U.S. Patent Application Publication No. 2017/0369828; International Publication No. WO 2017/223269; U.S. Patent Application Publication No. 2017/0349874; and International Publication No. WO 2017/207822.
Traditional bioreactor processing of microorganism cultures typically use multiple impellers installed on a shaft with offset distances between the vessel bottom, the air liquid interface, and between other impellers. Seed vessels (e.g., N-1, N-2) are used to volumetrically bulk up the cell density and volume to production scale. Bioreactor processing at full scale (e.g., N) using traditional impellers, without the use of seed vessels, experience limitations prohibiting consistent volumetric addition of media throughout the growth cycle. A bolus addition is used to achieve a height over the agitator to avoid excessive foaming at the impeller level. Variable diameter bioreactor (VDB) technology also requires a purpose designed impeller to allow consistent volumetric addition and avoid limitations experienced with traditional impellers. This design needs to consider optimal continuous mixing conditions (e.g., mixing time and shear), consistent and appropriate power per unit volume conditions, as well as minimal foaming conditions while providing agitation to a dynamically changing reactor volume to eliminate growth lag from traditional designed agitators. This also allows flexible processing for low, mid, and high titer production lines via varying bioreactor volume to optimize processing at the existing downstream scale.
In some instances, production scale bioreactor processing is accomplished through the use of bioreactors where liquid height is greater than vessel diameter (aspect ratio>1:1, liquid height to vessel diameter) and an array of multiple blade impellers on a shaft are used to mix the culture. Others use aspect ratios disclosed in U.S. Pat. Nos. 9,783,771; 9,670,446; and U.S. Patent Application Publication No. 2017/0267962 to Khan. These bioreactors are designed and sized to scale up the volume of the culture from inoculum in interim seed reactors increasing in volume until production volume has been reached. Typically bioreactors consist of vertical vessels with dished heads and bottoms, either tanks or disposable bags in support structures as necessary. Typically impellers consist of motors coupled with shafts (e.g., mechanical, magnetic) with discrete agitators attached (e.g., welded, set screws) in configurations to mix solutions and gases with culture to promote efficient growth.
There are certain limitations or drawbacks of the known process, product, and apparatus. Each traditional reactor/impeller system involves a transfer from one reactor to another and introduces the culture to conditions that differ from the end of the previous reactor. The reactor/agitator system design works optimally at a narrow volumetric band. This typically produces a “lag phase” effect where the cell growth stalls for a period before attaining exponential growth again. For large scale, this typical processing requires multiple reactors, increased footprint, and increased preparation activities. This leads to increased draw on utilities (water, steam, waste), more clean-in-place (CIP) systems, more steam-in-place (SIP) steps, and greater risk of contamination. There is a limited flexibility in traditional reactor/impeller systems with volumetric scalability. The traditional agitator design applied to the Variable Diameter Bioreactor has been shown to create detached mixing zones (See FIGS. 2A-2C).
SUMMARY OF THE INVENTION
An impeller according to the present invention is particularly suitable for use in a variable diameter bioreactor having multiple vessel sections of successively increasing or decreasing volume. The impeller includes an impeller blade extending along an impeller blade axis between first and second axial ends and having opposed impeller blade faces, an impeller blade leading edge, and an impeller blade trailing edge. Each of the leading and trailing edges defines a helix or spiral between the axial ends of the impeller blade. In certain arrangements, the impeller blade is one of at least two impeller blades joined together along an impeller shaft extending axially along the impeller blade axis mentioned, and the helix or spiral has a pitch approximately equal to one half of a length between the first and second axial ends.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1D are schematic cross sectional illustrations of 20,000 liter bioreactor vessels, with FIG. 1A showing a conventional bioreactor, FIGS. 1B and 1C showing variable diameter bioreactors (VDBs) with conventional impellers, and FIG. 1D showing a VDB with a continuous agitator or impeller.
FIGS. 2A-2D are schematic representations of fluid velocity vectors at similar values of P/V for 20,000 liter bioreactor vessels, with FIG. 2A showing vectors for a conventional bioreactor, FIGS. 2B and 2C showing vectors for variable diameter bioreactors (VDBs) with conventional impellers, and FIG. 2D showing vectors for a VDB with a continuous agitator or impeller.
FIG. 3 illustrates an initial position of a tracer added to a conventional bioreactor and a VDB for computational fluid dynamics (CFD) simulations of mixing time.
FIG. 4A supplies a comparison of calculated blend times from CFD simulations for certain configurations of the present invention, although the analysis had a geometrical issue that was fixed and adjusted in subsequent updates.
FIG. 4B supplies a comparison that is roughly the same as that supplied by FIG. 4, but which includes an updated optimal design.
FIG. 5 is a VDB modelling update, although, again, the analysis had a geometrical issue that was fixed and adjusted in subsequent updates.
FIG. 6 supplies a comparison of a 20 kl conventional bioreactor and three VDB bioreactor designs designated V6, V7, and V8.
FIG. 7 supplies a comparison of a fluid velocity vectors and fluid pathlines for the VDB bioreactor designs designated V6, V7, and V8 mentioned.
FIG. 8 provides a preliminary graphical comparison of blend times of VDB designs relative to a conventional 20,000 kl bioreactor.
FIG. 9 supplies an example of flow number calculation.
FIG. 10 supplies a comparison of a 20 kl conventional bioreactor and five VDB bioreactor designs designated V1, V2, V3, V4, and V5.
FIG. 11 supplies a comparison of fluid pathlines for the 20 kl conventional bioreactor and 20 kl VDB bioreactor designs designated V1, V2, V3, V4, and V5 mentioned.
FIG. 12 is an illustration of fluid velocity vectors for the 20 kl conventional bioreactor and the 20 kl VDB bioreactor designs designated V1, V2, V3, V4, and V5 mentioned.
FIG. 13 is another preliminary VDB modelling update similar to but more recent than that of FIG. 5.
FIGS. 14A-14B provide schematic representations of axial distribution of mixing energy at an equal power to volume ratio for VDB bioreactor designs designated V9, V16, V17, and V18.
FIG. 15 is a schematic representation of fluid velocity vectors at an equal pressure to volume ratio for VDB bioreactor designs designated V9, V16, V17, V18, V19, and V20.
FIG. 16 supplies a comparison of fluid pathlines for the VDB bioreactor designs designated V9, V16, V17, V18, V19, and V20 mentioned.
FIG. 17 illustrates a spatial analysis simulation of blend time for a VDB bioreactor design designated V20.
FIGS. 18A-18B illustrate a spatial analysis simulation of blend time for a VDB bioreactor design designated V19.
FIG. 19 illustrates a spatial analysis simulation of blend time for a VDB bioreactor design designated V18.
FIGS. 20A-20B illustrate a spatial analysis simulation of blend time for a VDB bioreactor design designated V17.
FIGS. 21A-21B illustrate a spatial analysis simulation of blend time for a VDB bioreactor design designated V16.
FIG. 22 illustrates the configuration of a potential VDB prototype.
FIG. 23 provides views of a conventional 20,000 liter bioreactor, including a geometric representation similar to FIG. 1A, a fluid velocity vector representation similar to FIG. 2A but with different rpm settings, and a fluid pathline representation similar to those of FIGS. 7, 11, and 16 but at with different rpm settings.
FIG. 24 provides a geometric representation of the VDB V1 design included in FIG. 10, a fluid velocity vector illustration similar to the V1 illustration included in FIG. 12, and a fluid pathline illustration similar to the V1 illustration supplied by FIG. 11 but with different rpm settings.
FIG. 25 provides a geometric representation of the VDB V2 design included in FIG. 10 but with different rpm settings, a fluid velocity vector illustration similar to the V2 illustration included in FIG. 12 but with different rpm settings, and a fluid pathline illustration similar to the V2 75 rpm illustration supplied by FIG. 11 but with different rpm settings.
FIG. 26 provides a geometric representation of the VDB V3 with impeller design included in FIG. 10, a fluid velocity vector illustration similar to the V3 illustration included in FIG. 12 but with different rpm settings, and a fluid pathline illustration similar to the V3 illustration supplied by FIG. 11 but with different rpm settings.
FIG. 27 provides a geometric representation of the VDB V4 with impeller design included in FIG. 10, a fluid velocity vector illustration similar to the V4 46 rpm illustration included in FIG. 12, and a fluid pathline illustration similar to the V4 illustration supplied by FIG. 11 but with different rpm settings.
FIG. 28 provides a geometric representation of the VDB V5 with impeller design included in FIG. 10, a fluid velocity vector illustration similar to the V5 illustration included in FIG. 12 but with different rpm settings, and a fluid pathline illustration similar to the V5 illustration supplied by FIG. 11 but with different rpm settings.
FIG. 29 provides a geometric representation of the VDB V6 with impeller design included in FIG. 6, a fluid velocity vector illustration similar to the V6 illustration included in FIG. 7 but with different rpm settings, and a fluid pathline illustration similar to the V6 illustration also supplied by FIG. 7 but with different rpm settings.
FIG. 30 provides a geometric representation of the VDB V7 with impeller design included in FIG. 6, a fluid velocity vector illustration similar to the V7 illustration included in FIG. 7 but with different rpm settings, and a fluid pathline illustration similar to the V7 illustration also supplied by FIG. 7 but with different rpm settings.
FIG. 31 provides a geometric representation of the VDB V8 with impeller design included in FIG. 6, a fluid velocity vector illustration similar to the V8 illustration included in FIG. 7 but with different rpm settings, and a fluid pathline illustration similar to the V8 illustration also supplied by FIG. 7 but with different rpm settings.
FIG. 32 provides a geometric representation of the VDB V9 design included in FIGS. 14A-14B but with different rpm settings, a fluid velocity vector illustration similar to the V9 illustration included in FIG. 15 but with different rpm settings, and a fluid pathline illustration similar to the V9 rpm illustration supplied by FIG. 16 but with different rpm settings.
FIG. 33 provides a geometric representation of a further VDB bioreactor designs, designated VDB Design V10, a fluid velocity vector illustration for the VDB Design V10, and a fluid pathline illustration for the VDB Design V10.
FIG. 34 provides a geometric representation of a further VDB bioreactor designs, designated VDB Design V11, a fluid velocity vector illustration for the VDB Design V11, and a fluid pathline illustration for the VDB Design V11.



DESCRIPTION OF THE INVENTION
Key concepts of the invention, as well as the purpose and operation of the invention including technical characteristics, will now be identified. Using a conical bottomed tank with continuous agitation, an aspect ratio of greater than 1:1 (liquid height to vessel diameter at liquid level) can be maintained to support minimal inoculation volume with sufficient liquid head for oxygen transfer during bulk up to larger scale. The culture volume is then bulked up through addition of media to sustain logarithmic growth via the continuous agitation design concept.
Initial VDB designs consisted of using multiple impellers of conventional design (i.e. the same types of impellers used in conventional bioreactors), but altering their size, number, and overall placement. CFD simulations of these designs indicated that an unacceptably large number of impellers (five or more) would be required in order to avoid separated fluid mixing zones and achieve adequate overall fluid mixing. Optimization of impeller type and size using four impellers resulted in blend times of approximately 200 to 400 seconds for a 20,000 liter fill VDB compared to approximately 60 seconds for a 20,000 liter conventional bioreactor. VDB designs were shifted to use largely a novel continuous agitator that runs nearly the entire vertical length of the reactor and provide more consistent agitation as a function of height. Numerous design changes of impeller pitch and blade number as well as baffle size resulted in blend times of approximately 68-100 seconds for a 20,000 liter fill VDB compared to approximately 60 seconds for a 20,000 liter conventional bioreactor.
FIG. 1 shows schematically (A) a 20,000 liter conventional bioreactor, (B and C) a 20,000 liter VDB with multiple conventional impellers, and (D) a 20,000 liter VDB with a continuous agitator.
The impellers for the VDB in FIG. 1B are sized and placed according to industry best practices. The impellers in FIG. 1C have been optimized to reduce mixing time at the same P/V value compared to FIG. 1B.
FIGS. 2A-2D show fluid velocity vectors from computational dynamics (CFD) simulations for a vertical cross section for each of the bioreactors operated at a similar average power/volume. FIGS. 2A-2C show four conventional impellers in a VDB result in detached mixing zones rather than single, connected fluid flow seen in the conventional bioreactor. FIG. 2D shows that these detached mixing zones are greatly reduced with the continuous agitator design.
CFD simulations of fluid mixing times were conducted. Mixing time is quantitatively defined as the time required for all points in the bioreactor to be within +/−5% of the final, mean value for an added tracer fluid. For these simulations, tracer fluid was added as a single bolus to the top of each bioreactor as shown in FIG. 3 and then the mixing of that bolus tracked as a function of time until uniformity was reached.
Results of these simulations are shown in FIGS. 4A and 4B, with FIG. 4B including a currently optimal Design V20. Blend time (as defined previously) for a VDB with 4 conventional impellers (Design V1) range from 420 seconds (using impellers sized using industry best practices) to 264 seconds (for impellers optimized to maximize fluid mixing—Design V2). These results suggest that greater than four impellers are likely needed to achieve acceptable mixing relative to a conventional bioreactor. Blend time for a VDB with a continuous agitator (Design V20) shows a blend time of about 100 seconds, indicating a significant improvement in mixing relative to the same VDB bioreactor with conventional impellers.
The particular illustrations supplied by FIGS. 14A-16 show what are presently considered to be optimal designs. Referring by way of example to FIGS. 14A-14B, each of the impeller configurations shown is suitable for use in multiple vessel section VDBs such as those disclosed by the Mietzner et al. '828 A1 publication referred to above. As shown in FIG. 14A, an impeller 100 is usable in a variable diameter bioreactor 102 having multiple vessel sections 104, 106, and 108 of successively increasing or decreasing volume. The impeller 100 illustrated includes an impeller blade 110 extending along an impeller blade axis 112 between first and second axial ends 114, 116 and having opposed impeller blade faces 118, 120, an impeller blade leading edge 122, and an impeller blade trailing edge 124. As illustrated, the leading and trailing edges 122 and 124 each define a helix or spiral between the first and second axial ends 114, 116 of the impeller blade 110.
In each arrangement shown in FIG. 14A-14B, the impeller blade 110 is one of at least two impeller blades 110, 126 joined together along an impeller shaft 118 extending axially along the impeller blade axis 112 mentioned. The helix or spiral has a pitch approximately equal to one half of a length between the first and second axial ends 114, 116; as illustrated in FIG. 14A, the pitch mentioned is one half of the length mentioned. Differences between the versions V9, V16, V17, and V18 shown in FIGS. 14A-14B reside primarily in the combined diameter of shaft 118 and widths of the blades 110 and 126, with the V9 combined diameter and widths being 0.35 m between the first and second axial ends 114, 116, the V16 combined diameter and widths tapering from 0.75 m at the first axial end 114 to 0.35 m at the second axial end 116, the V17 combined diameter and widths tapering from 0.45 m at the first axial end 114 to 0.35 m at the second axial end 116, and the V18 combined diameter and widths tapering from 0.50 m at the first axial end 114 to 0.35 m at the second axial end 116.
Features considered of the invention considered different (i.e. novel) when compared to the state of the art include the use of a continuous impeller alone or in conjunction with another impeller (twin shaft design) within a typical cylindrical bioreactor or in a Variable Diameter Bioreactor.
With respect to envisioned products, processes, or applications, the biggest benefit would be bioreactor systems for contract manufacturing. Ease of configuration for total production volume and minimized footprint are key attributes for this application (flexibility for low, mid, and high titer processes). Ease of modification of existing bioreactors with this agitator technology to eliminate seed train equipment (N-1 and possibly more).
It is clear from the present disclosure that a scaled down version could be fabricated and used for any desired mixing, mass transfer, and cleaning in place attributes. A scaled down version could be used to replace seed train and feed a production bioreactor (traditional or VDB).
The examples below are set out in the Mietzner et al. '828 A1 publication mentioned above, which is incorporated herein by reference, but these examples are explicitly included here as well for the sake of completeness.
Examples
The relationship between the volume, diameter and properties for cell growth of the variable diameter bioreactors of the present invention requires the consideration of many factors. The below equation provides a useful guide when designing bioreactors of the present invention:



 
  
   
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For example, when designing the bioreactor of the present invention to work with volumes up to 20,000 L, the variable diameter bioreactor would have the following proportions:
Total Volume: 20,000 L
Cone Volume: 15,000 L
Diametertop: 7 ft
Diameterbottom: 3 ft
Total Height: 30.2 ft
Cylinder Volume: 5,000 L
Cone Height: 25.6 ft
Cylinder Height: 4.6 ft
As another example, when designing the bioreactor of the present invention to fit into a certain space in a manufacturing facility or the like, where the height is limited to twenty feet, the above equation would yield the following proportions:
Total Volume: 16,458 L
Cone Volume: 9,341 L
Diametertop: 8 ft
Diameterbottom: 2 ft
Total Height: 20 ft
Cylinder Volume: 7,117 L
Cone Height: 15 ft
Cylinder Height: 5 ft
The above example can have four impellers.
As another example, the design of the present invention allows variable diameter bioreactors to be built in excess of 20,000 L, which is new to the art. Specifically, a variable diameter bioreactor could be built with the following proportions:
Total Volume: 25,000 L
Cone Volume: 15.000 L
Diametertop: 7.9 ft
Diameterbottom: 2.5 ft
Total Height: 30 ft
Cylinder Volume: 10,000 L
Cone Height: 22.8 ft
Cylinder Height: 7.2 ft
Unless described otherwise above, the above description may be further understood as follows. The devices, facilities and methods described herein are suitable for use in and with culturing any desired cell line including prokaryotic and/or eukaryotic cell lines. Further, in embodiments, the devices, facilities and methods are suitable for culturing suspension cells or anchorage-dependent (adherent) cells and are suitable for production operations configured for production of pharmaceutical and biopharmaceutical products—such as polypeptide products, nucleic acid products (for example DNA or RNA), or cells and/or viruses such as those used in cellular and/or viral therapies.
In embodiments, 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 cells include, but are not limited to, antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimetics (polypeptide molecules that bind specifically to antigens but that are not structurally related to antibodies such as e.g. 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), cell 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 (such as e.g. siRNA) or DNA (such as e.g. plasmid DNA), antibiotics or amino acids. In embodiments, the devices, facilities and methods can be used for producing biosimilars.
As mentioned, in embodiments, devices, facilities and methods allow for the production of eukaryotic cells, e.g., mammalian cells or lower eukaryotic cells such as for example yeast cells or filamentous fungi cells, or prokaryotic cells such as Gram-positive or Gram-negative cells and/or products of the eukaryotic or prokaryotic cells, e.g., proteins, peptides, antibiotics, amino acids, nucleic acids (such as DNA or RNA), synthesized by the eukaryotic cells in a large-scale manner. Unless stated otherwise herein, the devices, facilities, and methods can include any desired volume or production capacity including but not limited to bench-scale, pilot-scale, and full production scale capacities.
Moreover and unless stated otherwise herein, the devices, facilities, and methods can include any suitable reactor(s) 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 herein, “reactor” can include a fermentor or fermentation unit, or any other reaction vessel and the term “reactor” is used interchangeably with “fermentor.” For example, in some aspects, an example bioreactor unit can perform one or more, or all, of the following: feeding of nutrients and/or carbon sources, injection of suitable gas (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH level, agitation (e.g., stirring), and/or cleaning/sterilizing. Example reactor units, such as a fermentation unit, may contain multiple reactors within the unit, for example the unit can 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 unit and/or a facility may contain multiple units having a single or multiple reactors within the facility. In various embodiments, the bioreactor can be suitable for batch, semi fed-batch, fed-batch, perfusion, and/or a continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor can have a volume between about 100 mL and about 50,000 L. Non-limiting examples include a volume of 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 can be multi-use, single-use, disposable, or non-disposable and can be formed of any suitable material including metal alloys such as stainless steel (e.g., 316 L or any other suitable stainless steel) and Inconel, plastics, and/or glass.
In embodiments and unless stated otherwise herein, the devices, facilities, and methods described herein can also include any suitable unit operation and/or equipment not otherwise mentioned, such as operations and/or equipment for separation, purification, and isolation of such products. Any suitable facility and environment can be used, such as traditional stick-built facilities, modular, mobile and temporary facilities, or any other suitable construction, facility, and/or layout. For example, in some embodiments modular clean-rooms can be used. Additionally and unless otherwise stated, the devices, systems, and methods described herein can be housed and/or performed in a single location or facility or alternatively be housed and/or performed at separate or multiple locations and/or facilities.
By way of non-limiting examples and without limitation, U.S. Patent Application Publication Nos. 2012/0077429; and 2009/0305626; and U.S. Pat. Nos. 9,388,373, 8,771,635, 8,298,054; 7,629,167; and 5,656,491, which are hereby incorporated by reference in their entirety, describe example facilities, equipment, and/or systems that may be suitable.
In embodiments, the cells are eukaryotic cells, e.g., mammalian cells. The mammalian cells can be for example human or rodent or bovine cell lines or cell strains. Examples of such cells, cell lines or cell strains are e.g. mouse myeloma (NSO)-cell lines, Chinese hamster ovary (CHO)-cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cell), VERO, SP2/0, YB2/0, Y0, C127, L cell, COS, e.g., COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLA, EB1, EB2, EB3, oncolytic or hybridoma-cell lines. Preferably the mammalian cells are CHO-cell lines. In one embodiment, the cell is a CHO cell. In one embodiment, 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 knock-out cell, a CHO FUT8 GS knock-out cell, a CHOZN, or a CHO-derived cell. The CHO GS knock-out cell (e.g., GSKO cell) is, for example, a CHO-K1 SV GS knockout cell. The CHO FUT8 knockout cell is, for example, the Potelligent® CHOK1 SV (Lonza Biologics, Inc.). Eukaryotic cells can also be avian cells, cell lines or cell strains, such as for example, EBx® cells, EB14, EB24, EB26, EB66, or EBv13.
In one embodiment, the eukaryotic cells are stem cells. The stem cells can be, for example, pluripotent stem cells, including embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue specific stem cells (e.g., hematopoietic stem cells) and mesenchymal stem cells (MSCs).
In one embodiment, the cell is a differentiated form of any of the cells described herein. In one embodiment, the cell is a cell derived from any primary cell in culture.
In embodiments, the cell is a hepatocyte such as a human hepatocyte, animal hepatocyte, or a non-parenchymal cell. For example, the cell can be a plateable metabolism qualified human hepatocyte, a plateable induction qualified human hepatocyte, plateable Qualyst Transporter CertifiedTM human hepatocyte, suspension qualified human hepatocyte (including 10-donor and 20-donor pooled hepatocytes), human hepatic kupffer cells, human hepatic stellate cells, dog hepatocytes (including single and pooled Beagle hepatocytes), mouse hepatocytes (including CD-1 and C57BI/6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, 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). Example hepatocytes are commercially available from Triangle Research Labs, LLC, 6 Davis Drive Research Triangle Park, N.C., USA 27709.
In one embodiment, the eukaryotic cell is a lower eukaryotic cell such as e.g. a yeast cell (e.g., Pichia genus (e.g. Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta), Komagataella genus (e.g. Komagataella pastoris, Komagataella pseudopastoris or Komagataella phaffii), Saccharomyces genus (e.g. Saccharomyces cerevisae, cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum), Kluyveromyces genus (e.g. Kluyveromyces lactis, Kluyveromyces marxianus), the Candida genus (e.g. Candida utilis, Candida cacaoi, Candida boidinii), the Geotrichum genus (e.g. Geotrichum fermentans), Hansenula polymorpha, Yarrowia lipolytica, or Schizosaccharomyces pombe. Preferred is the species Pichia pastoris. Examples for Pichia pastoris strains are X33, GS115, KM71, KM71H; and CBS7435.
In one embodiment, the eukaryotic cell is a fungal cell (e.g. Aspergillus (such as A. niger, A. fumigatus, A. orzyae, A. nidula), Acremonium (such as A. thermophilum), Chaetomium (such as C. thermophilum), Chrysosporium (such as C. thermophile), Cordyceps (such as C. militaris), Corynascus, Ctenomyces, Fusarium (such as F. oxysporum), Glomerella (such as G. graminicola), Hypocrea (such as H. jecorina), Magnaporthe (such as M. orzyae), Myceliophthora (such as M. thermophile), Nectria (such as N. heamatococca), Neurospora (such as N. crassa), Penicillium, Sporotrichum (such as S. thermophile), Thielavia (such as T. terrestris, T. heterothallica), Trichoderma (such as T. reesei), or Verticillium (such as V. dahlia)).
In one embodiment, the eukaryotic cell is an insect cell (e.g., Sf9, MimicTM Sf9, Sf21, High FiveTM (BT1-TN-5B1-4), or BT1-Ea88 cells), an algae cell (e.g., of the genus Amphora, Bacillariophyceae, Dunaliella, Chlorella, Chlamydomonas, Cyanophyta (cyanobacteria), Nannochloropsis, Spirulina, or Ochromonas), or a plant cell (e.g., cells from monocotyledonous plants (e.g., maize, rice, wheat, or Setaria), or from a dicotyledonous plants (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens or Arabidopsis).
In one embodiment, the cell is a bacterial or prokaryotic cell.
In embodiments, the prokaryotic cell is a Gram-positive cells such as Bacillus, Streptomyces Streptococcus, Staphylococcus or Lactobacillus. Bacillus that can be used is, e.g. the B. subtilis, B. amyloliquefaciens, B. licheniformis, B. natto, or B. megaterium. In embodiments, the cell is B. subtilis, such as B. subtilis 3NA and B. subtilis 168. Bacillus is obtainable from, e.g., the Bacillus Genetic Stock Center, Biological Sciences 556, 484 West 12th Avenue, Columbus, Ohio 43210-1214.
In one embodiment, the prokaryotic cell is a Gram-negative cell, such as Salmonella spp. or Escherichia coli, such as e.g., TG1, TG2, W3110, DH1, DHB4, DH5a, HMS 174, HMS174 (DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue and Origami, as well as those derived from E. coli B-strains, such as for example BL-21 or BL21 (DE3), all of which are commercially available.
Suitable host cells are commercially available, for example, from culture collections such as the DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).
In embodiments, the cultured cells are used to produce proteins e.g., antibodies, e.g., 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 a molecular weight of about 4000 daltons to greater than about 140,000 daltons can be produced. In embodiments, these molecules can have a range of complexity and can include posttranslational modifications including glycosylation.
In embodiments, the protein is, e.g., BOTOX, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxins), alglucosidase alpha, daptomycin, YH-16, choriogonadotropin alpha, filgrastim, cetrorelix, interleukin-2, aldesleukin, teceleulin, denileukin diftitox, interferon alpha-n3 (injection), interferon alpha-n1, DL-8234, interferon, Suntory (gamma-1a), interferon gamma, thymosin alpha 1, tasonermin, DigiFab, ViperaTAb, EchiTAb, CroFab, nesiritide, abatacept, alefacept, Rebif, eptoterminalfa, teriparatide (osteoporosis), calcitonin injectable (bone disease), calcitonin (nasal, osteoporosis), etanercept, hemoglobin glutamer 250 (bovine), drotrecogin alpha, collagenase, carperitide, recombinant human epidermal growth factor (topical gel, wound healing), DWP401, darbepoetin alpha, epoetin omega, epoetin beta, epoetin alpha, desirudin, lepirudin, bivalirudin, nonacog alpha, Mononine, eptacog alpha (activated), recombinant Factor VIII+VWF, Recombinate, recombinant Factor VIII, Factor VIII (recombinant), Alphnmate, octocog alpha, Factor VIII, palifermin, Indikinase, tenecteplase, alteplase, pamiteplase, reteplase, nateplase, monteplase, follitropin alpha, rFSH, hpFSH, micafungin, pegfilgrastim, lenograstim, nartograstim, sermorelin, glucagon, exenatide, pramlintide, iniglucerase, galsulfase, Leucotropin, molgramostim, triptorelin acetate, histrelin (subcutaneous implant, Hydron), deslorelin, histrelin, nafarelin, leuprolide sustained release depot (ATRIGEL), leuprolide implant (DUROS), goserelin, Eutropin, KP-102 program, somatropin, mecasermin (growth failure), enlfavirtide, Org-33408, insulin glargine, insulin glulisine, insulin (inhaled), insulin lispro, insulin deternir, insulin (buccal, RapidMist), mecasermin rinfabate, anakinra, celmoleukin, 99 mTc-apcitide injection, myelopid, Betaseron, glatiramer acetate, Gepon, sargramostim, oprelvekin, human leukocyte-derived alpha interferons, Bilive, insulin (recombinant), recombinant human insulin, insulin aspart, mecasenin, Roferon-A, interferon-alpha 2, Alfaferone, interferon alfacon-1, interferon alpha, Avonex′ recombinant human luteinizing hormone, dornase alpha, trafermin, ziconotide, taltirelin, diboterminalfa, atosiban, becaplermin, eptifibatide, Zemaira, CTC-111, Shanvac-B, HPV vaccine (quadrivalent), octreotide, lanreotide, ancestirn, agalsidase beta, agalsidase alpha, laronidase, prezatide copper acetate (topical gel), rasburicase, ranibizumab, Actimmune, PEG-Intron, Tricomin, recombinant house dust mite allergy desensitization injection, recombinant human parathyroid hormone (PTH) 1-84 (sc, osteoporosis), epoetin delta, transgenic antithrombin III, Granditropin, Vitrase, recombinant insulin, interferon-alpha (oral lozenge), GEM-21S, vapreotide, idursulfase, omnapatrilat, 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 (inhaled, pulmonary disease), icatibant, ecallantide, omiganan, Aurograb, pexigananacetate, ADI-PEG-20, LDI-200, degarelix, cintredelinbesudotox, Favld, MDX-1379, ISAtx-247, liraglutide, teriparatide (osteoporosis), tifacogin, AA4500, T4N5 liposome lotion, catumaxomab, DWP413, ART-123, Chrysalin, desmoteplase, amediplase, corifollitropinalpha, 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 (hepatitis C viral infection (HCV)), interferon alpha-n3 (oral), belatacept, transdermal insulin patches, AMG-531, MBP-8298, Xerecept, opebacan, AIDSVAX, GV-1001, LymphoScan, ranpirnase, Lipoxysan, lusupultide, MP52 (beta-tricalciumphosphate carrier, bone regeneration), melanoma vaccine, sipuleucel-T, CTP-37, Insegia, vitespen, human thrombin (frozen, 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, 99 mTc-Hynic-Annexin V, kahalalide F, CTCE-9908, teverelix (extended release), ozarelix, rornidepsin, BAY-504798, interleukin4, PRX-321, Pepscan, iboctadekin, rhlactoferrin, TRU-015, IL-21, ATN-161, cilengitide, Albuferon, Biphasix, IRX-2, omega interferon, PCK-3145, CAP-232, pasireotide, huN901-DMI, ovarian cancer immunotherapeutic 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), pegsunercept, thymosinbeta4, plitidepsin, GTP-200, ramoplanin, GRASPA, OBI-1, AC-100, salmon calcitonin (oral, eligen), calcitonin (oral, osteoporosis), examorelin, capromorelin, Cardeva, velafermin, 131I-TM-601, KK-220, T-10, ularitide, depelestat, hematide, Chrysalin (topical), rNAPc2, recombinant Factor V111 (PEGylated liposomal), bFGF, PEGylated recombinant staphylokinase variant, V-10153, SonoLysis Prolyse, NeuroVax, CZEN-002, islet cell neogenesis therapy, rGLP-1, BIM-51077, LY-548806, exenatide (controlled release, Medisorb), AVE-0010, GA-GCB, avorelin, ACM-9604, linaclotid eacetate, CETi-1, Hemospan, VAL (injectable), fast-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 diseases/inflammation), talactoferrin (topical), rEV-131 (ophthalmic), rEV-131 (respiratory disease), oral recombinant human insulin (diabetes), RPI-78M, oprelvekin (oral), CYT-99007 CTLA4-Ig, DTY-001, valategrast, interferon alpha-n3 (topical), IRX-3, RDP-58, Tauferon, bile salt stimulated lipase, Merispase, alaline 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 (virosomes, PeviPRO), ALTU-135, parvovirus B19 vaccine, influenza vaccine (recombinant neuraminidase), malaria/HBV vaccine, anthrax vaccine, Vacc-5q, Vacc-4x, HIV vaccine (oral), HPV vaccine, Tat Toxoid, YSPSL, CHS-13340, PTH(1-34) liposomal cream (Novasome), Ostabolin-C, PTH analog (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-targeting vaccine (dust mite allergy), PR1 peptide antigen (leukemia), mutant ras vaccine, HPV-16 E7 lipopeptide vaccine, labyrinthin vaccine (adenocarcinoma), CIVIL vaccine, WT1-peptide vaccine (cancer), IDD-5, CDX-110, Pentrys, Norelin, CytoFab, P-9808, VT-111, icrocaptide, telbermin (dermatological, diabetic foot ulcer), rupintrivir, reticulose, rGRF, HA, alpha-galactosidase A, ACE-011, ALTU-140, CGX-1160, 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 peptides, 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 radioimmunotherapeutics (cancer), Re-188-P-2045, AMG-386, DC/1540/KLH vaccine (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-1 vaccine (peptides), NA17.A2 peptides, melanoma vaccine (pulsed antigen therapeutic), 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 (nasal, osteoporosis), F-18-CCR1, AT-1100 (celiac disease/diabetes), JPD-003, PTH(7-34) liposomal cream (Novasome), duramycin (ophthalmic, dry eye), CAB-2, CTCE-0214, GlycoPEGylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, Factor XIII, aminocandin, PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, teverelix (immediate release), EP-51216, hGH (controlled release, Biosphere), OGP-I, sifuvirtide, TV4710, ALG-889, Org-41259, rhCC10, F-991, thymopentin (pulmonary diseases), r(m)CRP, hepatoselective insulin, subalin, L19-IL-2 fusion protein, elafin, NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist (thrombocytopenic disorders), 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 pneumoniae pediatric vaccine, malaria vaccine, Neisseria meningitidis Group B vaccine, neonatal group B streptococcal vaccine, anthrax vaccine, HCV vaccine (gpE1+gpE2+MF-59), otitis media therapy, HCV vaccine (core antigen+ISCOMATRIX), hPTH(1-34) (transdermal, ViaDerm), 768974, SYN-101, PGN-0052, aviscumnine, BIM-23190, tuberculosis vaccine, multi-epitope tyrosinase peptide, cancer vaccine, enkastim, APC-8024, GI-5005, ACC-001, TTS-CD3, vascular-targeted TNF (solid tumors), desmopressin (buccal controlled-release), onercept, and TP-9201.
In some embodiments, the polypeptide is adalimumab (HUMIRA), infliximab (REMICADETM), rituximab (RITUXANTM/MAB THERATM) etanercept (ENBRELTM) bevacizumab (AVASTINTM), trastuzumab (HERCEPTINTM), pegrilgrastim (NEULASTATM), or any other suitable polypeptide including biosimilars and biobetters.
Other suitable polypeptides are those listed below and in Table A of US 2016/0097074:








TABLE A





Protein Product
Reference Listed Drug







interferon gamma-1b
Actimmune ®


alteplase; tissue plasminogen activator
Activase ®/Cathflo ®


Recombinant antihemophilic factor
Advate


human albumin
Albutein ®


Laronidase
Aldurazyme ®


Interferon alfa-N3, human 
Alferon N ®


leukocyte derived



human antihemophilic factor
Alphanate ®


virus-filtered human coagulation factor IX
AlphaNine ® SD


Alefacept; recombinant, dimeric 
Amevive ®


fusion protein LFA3-Ig



Bivalirudin
Angiomax ®


darbepoetin alfa
Aranesp TM


Bevacizumab
Avastin TM


interferon beta-1a; recombinant
Avonex ®


coagulation factor IX
BeneFix TM


Interferon beta-1b
Betaseron ®


Tositumomab
BEXXAR  ®


antihemophilic factor
Bioclate TM


human growth hormone
BioTropin TM


botulinum toxin type A
BOTOX ®


Alemtuzumab
Campath ®


acritumomab; technetium-99 labeled
CEA-Scan ®


alglucerase; modified form of 
Ceredase ®


beta-glucocerebrosidase



imiglucerase; recombinant form of beta-
Cerezyme ®


glucocerebrosidase



crotalidae polyvalent immune Fab, ovine
CroFab TM


digoxin immune fab [ovine]
DigiFab TM


Rasburicase
Elitek ®


Etanercept
ENBREL ®


epoietin alfa
Epogen ®


Cetuximab
Erbitux TM


algasidase beta
Fabrazyme ®


Urofollitropin
Fertinex TM


follitropin beta
Follistim TM


Teriparatide
FORTEO ®


human somatropin
GenoTropin ®


Glucagon
GlucaGen ®


follitropin alfa
Gonal-F ®


antihemophilic factor
Helixate ®


Antihemophilic Factor; Factor XIII
HEMOFIL


adefovir dipivoxil
Hepsera TM


Trastuzumab
Herceptin ®


Insulin
Humalog ®


antihemophilic factor/von Willebrand 
Humate-P ®


factor complex-human



Somatotropin
Humatrope ®


Adalimumab
HUMIRA TM


human insulin
Humulin ®


recombinant human hyaluronidase
Hylenex TM


interferon alfacon-1
Infergen ®


Eptifibatide
Integrilin TM


alpha-interferon
Intron A ®


Palifermin
Kepivance


Anakinra
Kineret TM


antihemophilic factor
Kogenate ® FS


insulin glargine
Lantus ®


granulocyte macrophage 
Leukine ®/


colony-stimulating factor
Leukine ® Liquid


lutropin alfa for injection
Luveris


OspA lipoprotein
LYMErix TM


Ranibizumab
LUCENTIS ®


gemtuzumab ozogamicin
Mylotarg TM


Galsulfase
Naglazyme TM


Nesiritide
Natrecor ®


Pegfilgrastim
Neulasta TM


Oprelvekin
Neumega ®


Filgrastim
Neupogen ®


Fanolesomab
NeutroSpec TM 



(formerly



LeuTech ®)


somatropin [rDNA]
Norditropin ®/



Norditropin



Nordiflex ®


Mitoxantrone
Novantrone ®


insulin; zinc suspension;
Novolin L ®


insulin; isophane suspension
Novolin N ®


insulin, regular;
Novolin R ®


Insulin
Novolin ®


coagulation factor VIIa
NovoSeven ®


Somatropin
Nutropin ®


immunoglobulin intravenous
Octagam ®


PEG-L-asparaginase
Oncaspar ®


abatacept, fully human soluable 
Orencia TM


fusion protein



muromomab-CD3
Orthoclone OKT3 ®


high-molecular weight hyaluronan
Orthovisc ®


human chorionic gonadotropin
Ovidrel ®


live attenuated Bacillus Calmette-Guerin
Pacis ®


peginterferon alfa-2a
Pegasys ®


pegylated version of interferon alfa-2b
PEG-Intron TM


Abarelix (injectable suspension); 
Plenaxis TM


gonadotropin-releasing hormone



Antagonist



epoietin alfa
Procrit ®


Aldesleukin
Proleukin, IL-2 ®


Somatrem
Protropin ®


dornase alfa
Pulmozyme ®


Efalizumab; selective, reversible 
RAPTIVA TM


T-cell blocker



combination of ribavirin and 
Rebetron TM


alpha interferon



Interferon beta 1a
Rebif ®


antihemophilic factor
Recombinate ® rAHF/


antihemophilic factor
ReFacto ®


Lepirudin
Refludan ®


Infliximab
REMICADE ®


Abciximab
ReoPro TM


Reteplase
Retavase TM


Rituxima
Rituxan TM


interferon alfa-2a
Roferon-A ®


Somatropin
Saizen ®


synthetic porcine secretin
SecreFlo TM


Basiliximab
Simulect ®


Eculizumab
SOLIRIS (R)


Pegvisomant
SOMAVERT ®


Palivizumab; recombinantly 
Synagis TM


produced, humanized mAb



thyrotropin alfa
Thyrogen ®


Tenecteplase
TNKase TM


Natalizumab
TYSABRI ®


human immune globulin intravenous 
Venoglobulin-S ®


5% and 10% solutions



interferon alfa-n1, lymphoblastoid
Wellferon ®


drotrecogin alfa
Xigris TM


Omalizumab; recombinant 
Xolair ®


DNA-derived humanized



monoclonal



antibody targeting immunoglobulin-E



Daclizumab
Zenapax ®


ibritumomab tiuxetan
Zevalin TM


Somatotropin
ZorbtiveTM 



(Serostim ®)









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







TABLE B







Exemplary Products









Therapeutic




Product type
Product
Trade Name





Hormone
Erythropoietin, Epoein-α
Epogen, Procrit



Darbepoetin-α
Aranesp



Growth hormone (GH),
Genotropin , Humatrope, 



somatotropin
Norditropin,




NovIVitropin, Nutropin, 




Omnitrope,




Protropin, Siazen, 




Serostim, Valtropin



Human follicle-stimulating
Gonal-F, Follistim



hormone (FSH)




Human chorionic
Ovidrel



gonadotropin
Luveris



Lutropin-α
GlcaGen



Glucagon
Geref



Growth hormone releasing
ChiRhoStim (human 



hormone (GHRH)
peptide), SecreFlo



Secretin
(porcine peptide)



Thyroid stimulating
Thyrogen



hormone (TSH),




thyrotropin



Blood
Factor VIIa
NovoSeven


Clotting/ 
Factor VIII
Bioclate, Helixate, 


Coagulation

Kogenate,


Factors

Recombinate, ReFacto



Factor IX




Antithrombin III (AT-III)
Benefix



Protein C concentrate
Thrombate III




Ceprotin


Cytokine/Growth
Type I alpha-interferon
Infergen


factor
Interferon-αn3 (IFNαn3)
Alferon N



Interferon-β1a (rIFN-β) 
Avonex, Rebif



Interferon-β1b (rIFN-β) 
Betaseron



Interferon-γ1b (IFN γ)
Actimmune



Aldesleukin (interleukin
Proleukin



2(IL2), epidermal




theymocyte activating




factor; ETAF
Kepivance



Palifermin (keratinocyte
Regranex



growth factor; KGF)




Becaplemin (platelet-
Anril, Kineret



derived growth factor;




PDGF)




Anakinra (recombinant




IL1 antagonist)



Antibody 
Bevacizumab (VEGFA
Avastin


molecules
mAb)
Erbitux



Cetuximab (EGFR mAb)
Vectibix



Panitumumab (EGFR
Campath



mAb)
Rituxan



Alemtuzumab (CD52
Herceptin



mAb)
Orencia



Rituximab (CD20
Humira



chimeric Ab)
Enbrel



Trastuzumab (HER2/Neu
Remicade



mAb)
Amevive



Abatacept (CTLA Ab/Fc
Raptiva



fusion)
Tysabri



Adalimumab (TNFα mAb)
Soliris



Etanercept (TNF
Orthoclone, OKT3



receptor/Fc fusion)




Infliximab (TNFα




chimeric mAb)




Alefacept (CD2 fusion




protein)




Efalizumab (CD11a mAb)




Natalizumab (integrin α4




subunit mAb)




Eculizumab (C5mAb)




Muromonab-CD3



Other:
Insulin
Humulin, Novolin


Fusion
Hepatitis B surface antigen
Engerix, Recombivax HB


proteins/Protein
(HBsAg)



vaccines/Peptides
HPV vaccine
Gardasil



OspA
LYMErix



Anti-Rhesus(Rh)
Rhophylac



immunoglobulin G 
Fuzeon



Enfuvirtide




Spider silk, e.g., fibrion
QMONOS









In embodiments, the protein is multispecific protein, e.g., a bispecific antibody as shown in Table C.







TABLE C







Bispecific Formats












Name (other







names,


Proposed

Diseases (or


sponsoring
BsAb

mechanisms of 
Development
healthy


organizations)
format
Targets
action
stages
volunteers)





Catumaxomab
BsIgG:
CD3,
Retargeting of T
Approved in
Malignant


(Removab ®,
Triomab
EpCAM
cells to tumor, Fc
EU
ascites in


Fresenius


mediated

EpCAM positive


Biotech, Trion


effector

tumors


Pharma,


functions




Neopharm)







Ertumaxomab
BsIgG:
CD3, HER2
Retargeting of T
Phase I/II
Advanced solid


(Neovii Biotech,
Triomab

cells to tumor

tumors


Fresenius







Biotech)







Blinatumomab
BiTE
CD3, CD19
Retargeting of T
Approved in
Precursor B-cell


(Blincyto ®, AMG


cells to tumor
USA
ALL


103, MT 103,



Phase II and 
ALL


MEDI 538,



III
DLBCL


Amgen)



Phase II
NHL






Phase I



REGN1979
BsAb
CD3, CD20





(Regeneron)







Solitomab (AMG
BiTE
CD3,
Retargeting of T
Phase I
Solid tumors


110, MT110,

EpCAM
cells to tumor




Amgen)







MEDI 565 (AMG
BiTE
CD3, CEA
Retargeting of T
Phase I
Gastrointestinal


211,


cells to tumor

adenocancinoma


MedImmune,







Amgen)







RO6958688
BsAb
CD3, CEA





(Roche)







BAY2010112
BiTE
CD3, PSMA
Retargeting of T
Phase I
Prostate cancer


(AMG 212,


cells to tumor




Bayer; Amgen)







MGD006
DART
CD3, CD123
Retargeting of T
Phase I
AML


(Macrogenics)


cells to tumor




MGD007
DART
CD3, gpA33
Retargeting of T
Phase I
Colorectal


(Macrogenics)


cells to tumor

cancer


MGD011
DART
CD19, CD3





(Macrogenics)







SCORPION
BsAb
CD3, CD19
Retargeting of T




(Emergent


cells to tumor




Biosolutions,







Trubion)







AFM11 (Affimed
TandAb
CD3, CD19
Retargeting of T
Phase I
NHL and ALL


Therapeutics)


cells to tumor




AFM12 (Affimed 
TandAb
CD19, CD16
Retargeting of




Therapeutics)


NK cells to







tumor cells




AFM13 (Affimed
TandAb
CD30,
Retargeting of
Phase II
Hodgkin's


Therapeutics)

CD16A
NK cells to

Lymphoma





tumor cells




GD2 (Barbara
T cells
CD3, GD2
Retargeting of T
Phase I/II
Neuroblastoma


Ann Karmanos
preloaded

cells to tumor

and


Cancer Institute)
with BsAb



osteosarcoma


pGD2 (Barbara
T cells
CD3, Her2
Retargeting of T
Phase II
Metastatic breast


Ann Karmanos
preloaded

cells to tumor

cancer


Cancer Institute)
with BsAb






EGFRBi-armed
T cells
CD3, EGFR
Autologous
Phase I
Lung and other


autologous 
preloaded

activated T cells

solid tumors


activated T cells
with BsAb

to EGFR-




(Roger Williams


positive tumor




Medical Center)







Anti-EGFR-
T-cells 
CD3, EGFR
Autologous
Phase I
Colon and


armed activated
preloaded

activated T cells

pancreatic


T cells (Barbara 
with BsAb

to EGFR-

cancers


Ann Karmanos


positive tumor




Cancer Institute)







rM28 (University 
Tandem
CD28,
Retargeting of T
Phase II
Metastatic


Hospital
scFv
MAPG
cells to tumor

melanoma


Tübingen)







IMCgp100
ImmTAC
CD3, peptide
Retargeting of T
Phase I/II
Metastatic


(Immunocore)

MHC
cells to tumor

melanoma


DT2219ARL
2 scFv
CD19, CD22
Targeting of
Phase I
B cell leukemia


(NCI, University
linked to

protein toxin to

or lymphoma


of Minnesota)
diphtheria

tumor





toxin






XmAb5871
BsAb
CD19,





(Xencor)

CD32b





NI-1701
BsAb
CD47, CD19





(NovImmune)







MM-111
BsAb
ErbB2,





(Merrimack)

ErbB3





MM-141
BsAb
IGF-1R,





(Merrimack)

ErbB3





NA (Merus)
BsAb
HER2,







HER3





NA (Merus)
BsAb
CD3,







CLEC12A





NA (Merus)
BsAb
EGFR,







HER3





NA (Merus)
BsAb
PD1,







undisclosed





NA (Merus)
BsAb
CD3,







undisclosed





Duligotuzumab
DAF
EGFR,
Blockade of 2
Phase I and II
Head and neck


(MEHD7945A,

HER3
receptors, ADCC
Phase II
cancer


Genentech,




Colorectal


Roche)




cancer


LY3164530 (Eli 
Not
EGFR, MET
Blockade of 2
Phase I
Advanced or


Lily)
disclosed

receptors

metastatic







cancer


MM-111
HSA body
HER2,
Blockade of 2
Phase II
Gastric and


(Merrimack

HER3
receptors
Phase I
esophageal


Pharmaceuticals)




cancers







Breast cancer


MM-141,
IgG-scFv
IGF-1R,
Blockade of 2
Phase I
Advanced solid


(Merrimack

HER3
receptors

tumors


Pharmaceuticals)







RG7221
CrossMab
Ang2, VEGF
Blockade of 2
Phase I
Solid tumors


(RO5520985,

A
proangiogenics




Roche)







RG7716 (Roche) 
CrossMab
Ang2, VEGF
Blockade of 2
Phase I
Wet AMD




A
proangiogenics




OMP-305B83
BsAb
DLL4/VEGF





(OncoMed)







TF2
Dock and
CEA, HSG
Pretargeting
Phase II
Colorectal,


(Immunomedics)
lock

tumor for PET or

breast and lung





radioimaging

cancers


ABT-981
DVD-Ig
IL-1α, IL-1β
Blockade of 2
Phase II
Osteoarthritis


(AbbVie)


proinflammatory 







cytokines




ABT-122
DVD-Ig
TNF, IL-
Blockade of 2
Phase II
Rheumatoid


(AbbVie)

17A
proinflammatory

arthritis





cytokines




COVA322
IgG-
TNF, IL17A 
Blockade of 2
Phase I/II
Plaque psoriasis



fynomer

proinflammatory







cytokines




SAR156597
Tetravalent
IL-13, IL-4
Blockade of 2
Phase I
Idiopathic


(Sanofi)
bispecific 

proinflammatory 

pulmonary



tandem

cytokines

fibrosis



IgG






GSK2434735
Dual-
IL-13, IL-4
Blockade of 2
Phase I
(Healthy


(GSK)
targeting

proinflammatory

volunteers)



domain

cytokines




Ozoralizumab
Nanobody
TNF, HSA
Blockade of
Phase II
Rheumatoid


(ATN103,


proinflammatory

arthritis


Ablynx)


cytokine, binds 







to HSA to







increase half-life




ALX-0761
Nanobody
IL-7A/F,
Blockade of 2
Phase I
(Healthy


(Merck Serono,

HSA
proinflammatory

volunteers)


Ablynx)


cytokines, binds







to HSA to







increase half-life




ALX-0061
Nanobody
IL-6R, HSA
Blockade of
Phase I/II
Rheumatoid


(AbbVie, Ablynx;


proinflammatory

arthritis





cytokine, binds







increase HSA to







increase half-life




ALX-0141
Nanobody
RANKL,
Blockade of
Phase I
Postmenopausal


(Ablynx,

HSA
bone resorption, 

bone loss


Eddingpharm)


binds to HSA to







increase half-life




RG6013/ACE910
ART-Ig
Factor IXa,
Plasma
Phase II
Hemophilia


(Chugai, Roche)

factor X
coagulation









The descriptions of the various embodiments of the present invention have been presented for purposes of illustration, but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

## Claims

1. An impeller usable in a variable diameter bioreactor having multiple vessel sections of successively increasing or decreasing volume, the impeller comprising:
an impeller blade having an edge extending along an impeller blade axis between first and second axial ends and having opposed impeller blade faces, an impeller blade leading edge, and an impeller blade trailing edge;
wherein the leading and trailing edges each define a helix or spiral between the first and second axial ends of the impeller blade;
wherein the impeller blade is one of at least two impeller blades that are each joined together along an axially extending impeller shaft; and
wherein the at least two impeller blades are attached to the impeller shaft.

2. The impeller according to claim 1, wherein the helix or spiral has a pitch approximately equal to one half of a length between the first and second axial ends.

3. The impeller according to claim 1, wherein the helix or spiral has a pitch higher than one half of a length between the first and second axial ends.

4. The impeller according to claim 1, wherein the helix or spiral has a pitch lower than one half of a length between the first and second axial ends.

5. An impeller usable in a bioreactor, the impeller comprising:
an impeller blade having an edge extending along an impeller blade axis between first and second axial ends and having opposed impeller blade faces, an impeller blade leading edge, and an impeller blade trialing edge, wherein
the leading and trailing edges each define a helix or spiral between the first and second axial ends of the impeller blade,
wherein the impeller blade is one of at least two impeller blades that are each joined together along an axially extending impeller shaft; and


wherein the at least two impeller blades are attached to the impeller shaft.

6. The impeller according to claim 5, wherein the helix or spiral has a pitch approximately equal to one half of a length between the first and second axial ends.

7. The impeller according to claim 5, wherein the helix or spiral has a pitch higher than one half of a length between the first and second axial ends.

8. The impeller according to claim 5, wherein the helix or spiral has a pitch lower than one half of a length between the first and second axial ends.

9. A variable diameter bioreactor comprising:
a variable diameter bioreactor having multiple vessel sections of successively increasing or decreasing volume; and
an impeller having an impeller shaft and an impeller blade, the impeller blade having an edge extending along the impeller shaft between the first and second axial ends of the shaft and having opposed impeller blade faces, an impeller blade leading edge, and an impeller blade trailing edge, wherein
the leading and trailing edges each define a helix or spiral between the first and second axial ends of the impeller blade,
wherein the impeller blade is one of at least two impeller blades that are each joined together along an axially extending impeller shaft; and
wherein the at least two impeller blades are attached to the impeller shaft.

10. The variable diameter bioreactor arrangement according to claim 9, wherein the helix or spiral has a pitch approximately equal to one half of a length between the first and second axial ends.

11. The variable diameter bioreactor arrangement according to claim 9, wherein the helix or spiral has a pitch higher than one half of a length between the first and second axial ends.

12. The variable diameter bioreactor arrangement according to claim 9, wherein the helix or spiral has a pitch lower than one half of a length between the first and second axial ends.

13. A process for treating a microorganism culture comprising:
placing a fluid containing the microorganism culture in a variable diameter bioreactor arrangement, including a variable diameter bioreactor having multiple vessel sections of successively increasing or decreasing volume and an impeller having an impeller shaft, an impeller blade having an edge extending along the impeller shaft between first and second axial ends of the shaft and including opposed impeller blade faces, an impeller blade leading edge, and an impeller blade trailing edge, with the leading and trailing edges each defining a helix or spiral between the first and second axial ends of the impeller blade; and
rotating the impeller in the fluid containing the microorganism culture;
wherein the impeller blade is one of at least two impeller blades that are each joined together along an axially extending impeller shaft; and
wherein the at least two impeller blades are attached to the impeller shaft.

14. The process according to claim 13, wherein the impeller is rotated at a rate of 54-75 rpm.

15. The process according to claim 13, wherein the variable diameter reactor has a 20,000 liter fluid capacity.