SINGLE USE BIOREACTOR
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 62/345,381, filed on June 3, 2016, the contents of which are incorporated herein by reference. U.S. Provisional Application No. 62/354,216, filed June 24, 2016, and the following publications U.S. Patent Publication No. 2011/0312087, U.S. Patent Publication No. 2017/0107476, WO Publication No. WO 2017/072201, are each hereby incorporated by reference in their entirety.
BACKGROUND ART
[0002] Bioreactors, or apparatuses in which biological reactions or processes can be carried out on a laboratory or industrial scale, are used widely within the biopharmaceutical industry. Bioreactors can be used in fed-batch applications, wherein substrates are supplied at certain times to a bioreactor and wherein products remain in the bioreactor until the end of the reaction time, or in perfusion applications, wherein a continuous supply of substrate is supplied to the bioreactor while damaging by-products are continuously removed. Bioreactors can also be used in continuous batch applications,
[0003] Since the late 1990's there has been increasing interest in single use bioprocessing solutions within the biopharmaceutical industry. These solutions reduce the capital costs and validation time for new facilities, improve plant throughput by reducing turnaround time between batches, and reduce the burden of cleaning validation.
[0004] This interest in single use bioprocessing solutions has included the bioreactor unit operation. As a result, single use bioreactors (SUBs) are becoming standard work horses in the biopharmaceutical industry. These SUBs are supplied by vendors as off the shelf designs, limiting the cell culture engineer's ability to match the geometry of the SUB to the geometry of their existing stirred tank reactor (STR) capacity. For example, the first generation of SUBs departed from conventional stirred tank bioreactor (STR) geometry in terms of impeller number and orientation and sparger hole diameter. Moreover, one marked feature of single use bioreactors SUB bioreactors was that they could be operated at lower volumes than conventional STRs, bringing considerable operational flexibility. This practice, however, further negated the principle of geometric similarity.

culture. This is compatible with the controller systems of Figure 36. The single-use bioreactor has an agitator, a sparger, a gas filter inlet ports for sparger, and an exhaust gas outlet filter port with bifurcating line. It also has seven feed addition ports. Ideally, two are subsurface discharging in the impeller region and one discharging above the impeller region. It also has two medium fill ports, one harvest port designed to enable harvest the complete contents of the single-use bioreactor, one sample port, one condenser or equivalent on the gas exit line, and at least six measurement probe ports. These sample and harvest ports have animal derived component free (ADCF) C-flex tubing to enable aseptic connection for addition and removal of liquids. In addition, it has gas filters.
[00340] It is also preferable to have a fill line or lines directed such that the liquid flows down the side of the SUB to avoid splashing and foaming during the fill operation.
Example 2
Reactor Geometry
[00341] This example relates to the effect of changing reactor geometry on scale up of mammalian cell culture processes using multivariate data analysis to compare different geometries and different fill volumes. This approach uncovered a surprising result when working at half volume, which may not have been spotted using conventional data analysis methods.
[00342] Mass transfer studies were performed with two manufacturing scale SUB systems and a miniature SUB system using the gassing-out approach. A scale independent kLaCh model developed according to the geometry described in U.S. Publication No. US 2011/0312087 (referred to herein as "Lonza Geometry") was used to predict kLaO2 in both SUBs. The results have been compared to results generated using the STR geometry described in U.S. Publication No. US 2011/0312087 from 10 to 20,000 L. The vessel geometry has a substantial impact on mass transfer.
[00343] Multivariate analysis of the data showed that there were substantial differences in cell culture performance between different STR-scaled vessels. The results of this testing are presented in Figures 11-35.
[00344] As described herein, Figure 11 shows the results of a comparison of the Van't Reit model built with data from single-use bioreactors that were designed at least partially

according to the geometry described in U.S. Publication No. US 2011/0312087 at six different scales.
[00345] As described herein, Figure 12 shows the results of a comparison of the Van't Reit model built with data from single-use bioreactors that were designed at least partially according to the Lonza Geometry at six different scales as compared to a single-use bioreactor that did not incorporate the Lonza Geometry (red diamonds).
[00346] As described herein, Figure 13 shows the results of a comparison of the Van't Reit model built with data from single-use bioreactors that were designed at least partially according to the Lonza Geometry at six different scales as compared to a single-use bioreactor that did not incorporate the Lonza Geometry (red diamonds), two single-use bioreactors at two different scales built at least partially according to the Lonza Geometry when half full (blue triangles), and two single-use bioreactors at two different scales built at least partially according to the Lonza Geometry when full.
[00347] Cell culture evaluations were also performed with a model cell line in the two single-use bioreactor systems discussed above and one stainless steel/glass. The results were compared to historical data obtained in 10 L STR and 10 L airlift vessels ("ALR"). A total of fifteen measurements were taken for sixteen days in all four of the vessel geometries. The data were analyzed using the principal component analysis which projects high dimensional data sets onto lower dimension space to aid in data interpretation. Principal component analysis (PCA) and the calculation of associated statistics was performed in MATLAB Version 7.11.0.584 (The MathWorks Inc) using the PLS Tool Box Version 6.2 (Eigenvector Research, Inc.). The results are summarized in Figures 14, 15, and 16. These data show that the first four principal components captured 63% of the variance of the dataset, as shown in Figure 15. The cell cultures performed similarly in the ALRs, the STRs, and SUB1 at full volume. However, SUB2, which does not possess Lonza's geometry, performed outside the 95% confidence interval, as shown in Figure 16. Furthermore, ALRs and STRs performed similarly in principal components one, two, and three, as shown in Figure 17.
[00348] The impact of operating at half volume was investigated for one vessel design at two different vessel volumes, as shown in Figures 18-19. Here, the data show that the cultures in SUB1 at two scales, which contains at least partially Lonza's geometry,

performed similarly at full volume with the STR cultures on all principal components. However, when at half volume, that those same SUB at two scales displayed substantial differences in performance on the first three principal components indicates dissimilarity in culture properties.
[00349] Multivariate data analysis showed that there was considerable difference in behavior of the cultures performed at half volume when compared to cultures performed in the conventional scale-down model. For example, in Figures 20-21, cultures in SUB2 were performed at three scales with two bioprocess container materials.
[00350] The experiments conducted in Example 2 highlight the importance of bioreactor design, including the single-use bioreactors that are the object of the present disclosure. For example, loadings for principal component one normally track growth and/or culture progression. Loadings for a model built with STR data alone followed this norm. However, when the tests were expanded to include all four vessels designs of ALR, STR, SUB1, and SUB2, growth and/or culture progression was relegated to principal component two.
[00351] Additionally, Example 2 shows that geometric similarity is indicative of performance. The analysis indicated that there was also a difference in behavior of the half-volume cultures in different size vessels. Specifically, SUB 1 and STR cultures cluster well at full volume but not at half volume. At full volume, SUB 1 has a high degree of geometric similarity to the STR. However, at half volume, just one of these geometric parameters has been altered. Furthermore, culture performance was radically altered. Interestingly, kxaCh performance was not altered. Half-volume SUBl's performance was not consistent across scales as shown by the data where half volume cultures don't form a cluster.
[00352] Furthermore, the selection of bioprocess container material has an impact on SUB 2 culture performance. This is additionally supported by Figures 22-35 where the principal components were assessed over time for the various fills, volumes, and bioprocess container materials.
[00353] This indicated a lack of scalability between half-volume cultures performed in different scale vessels, which was not apparent when the same vessels were run at full volume.

[00354] Single-use bioreactor geometry does matter when scaling processes up and should be a key consideration in a quality by design approach to minimizing differences in culture behavior during cell culture process scale up. Moreover, multivariate data analysis can provide useful supplemental insight in bioreactor process performance comparisons.
Example 3
A 1,000 L Bioreactor Set Up
[00355] The single-use bioreactors of the present disclosure are suitable for use in the production processes described in WO 2017/072201 A2, which is incorporated by reference in its entirety herein.
[00356] The bioprocess container shell was a jacketed stainless steel container, which supported the SUB container. The shell incorporated two doors that open outwards for operators to fit the SUB bioprocess container. These were fastened shut by clamps. The shell incorporated a water jacket at the bottom for regulation of temperature. This was connected to the controller of the present disclosure.
[00357] At the bottom of the shell there was a drain port for harvesting and two openings for control probes and sampling. For non-disposable probes the shell had shelving set at 15 degrees from horizontal to support the probes.
[00358] At the top of the bioprocess container holder there was a motor to which the SUB container impeller was connected via a magnetic coupling. The motor attached to the 200 liter shell could be moved, but in the motor attached to the 1000 liter shell was fixed. There was a gas filter holder, pressure sensor and manual pressure relief valve situated on the arm of the motor.
[00359] The SUB bioprocess container incorporated a pressure release valve which actuated if pressure exceeded 100 mbar. Both the pressure transmitter and the relief valve were connected to the SUB container via a 0.22 um filter.
[00360] The controller of the present disclosure contained: two Watson Marlow pumps one for acid and one for base control, rotameters for control of gas flow, a human machine interface (HMI), a thermocirculator and gas mass flow controllers (MFCs) built into the tower. The pH probes, dissolved oxygen tension (DOT) probes, temperature probes,

pressure sensor and vent heater were external to but connected to a controller of the present disclosure.
[00361] Set points were entered into the HMI screen for all control parameters. The controller used these values to regulate culture temperature, gas flow rates and pump speed. The HMI also displayed current values of all measured parameters.
[00362] Temperature measurement was performed using a ptlOO probe inserted into a pocket in the SUB container.
[00363] Inside the SUB container there was: (i) an agitator shaft with a choice of two impeller designs (see Figure 1A and Figure 1B); (ii) disposable optical pH and DOT probes; (iii) a combination sparger (option of micro (0.15 mm) macro (0.8 mm) holes; and (iv) surface and subsurface feed lines.
[00364] On the outside of the SUB bioprocess container there were C flex lines for inoculum, medium and feed additions and OPTA connections for gas filters and feed additions. At the bottom of the SUB bioprocess container there were four connections for non-disposable probes, a sample line, and an insert for a pt100 probe. The harvest line was at the bottom of the SUB bioprocess container.
[00365] Hydrophobic 0.22 urn gas filters came autoclaved separately and were connected to the SUB bioprocess container using OPTA connections. Each SUB bioprocess container had connections for two gas outlet filters, one pressure sensor filter, one filter for headspace aeration and filter each for micro and macro spargers.
[00366] The pressure filter was connected to the pressure sensor and the gas inlet and outlet filters were open before inflation was started.
[00367] The sparger and head space gas filters were connected to the controller of the present disclosure using silicon tubing, which in turn was connected to the main gas supplies via nylon tubing. The main gas supply pressures were set to 1.8 barg for all gases. The MFCs had a turn down ratio of 1:50 and range of up to 100 L/min. As a result an additional calibrated rotatmeter was required supply of the CO2 ballast because this flow rate was too low to control with the MFC.
[00368] For safety reasons it was important to ensure that gas outlet line and pressure sensor line were not kinked during inflation.

[00369] Inflation of the SUB bioprocess container was started slowly with a low gas flow rate. A scientist had to hold the SUB bioprocess container in place until the agitator shaft 8 and motor were magnetically coupled. To prevent damage to the SUB bioprocess container it had to be inflated such that no components inside the bioprocess container (agitator blades or dip tubes) touched the bioprocess container. Inflation had to be stopped once it was possible to couple the agitator and the motor.
[00370] The agitator magnetic coupling was then slowly lifted up to the motor. Once in place the SUB bioprocess container was rotated slowly into position to align the probe ports with the probe holders and to align the seal of the SUB bioprocess container with the middle of where the two doors met. When in final position the agitator shaft 8 was secured in place to the motor using a tri clamp. The filters were fitted into position on the filter holder. A vent heater was placed around the gas outlet filter. The SUB bioprocess container was then fully inflated. A continuous air flow (at the air cap described in the pilot fermentation process description (FPD)) was maintained through the sparger and headspace in order to keep the SUB bioprocess container inflated.
[00371] One standard pH and one standard DOT probe were calibrated prior to starting each batch using the standard calibration procedure used the Slough pilot plant. These probes were fitted into the probe sleeves with connections and autoclaved on a fluid cycle. The probes were fitted into the SUB bioprocess container using the connections and placed onto the probe holder shelf set at a 15° degree angle to the horizontal.
[00372] Once pH and DOT probes were fitted the medium or buffer as appropriate was filtered into the SUB bioprocess container using a pre irradiated 0.1 urn filter welded on onto the dip tube. The Bioprocess container holders tested did not have a load cell, so a floor balance was used to weigh in the medium / buffer. During medium fill / buffer fill a constant air flow (at the air cap described in the pilot FPD) was maintained to avoid liquid going into the gas inlet line.
[00373] Once the required volume was achieved the jacket was filled with DI water and temperature and agitation control was initiated. Following medium fill pH control was initiated based on the reusable probe using C02 to prevent the pH from drifting outside the acceptable range for medium hold. The disposable pH and DOT probes were then

activated. The pH and DOT probes were left to equilibrate overnight in the medium or buffer.
[00374] Sample bioprocess containers were welded onto the sample line situated next to the disposable pH and DOT probes in order to ensure the sample was representative of the environment experience by the probes. Samples were removed the day after the vessel was filled and analyzed for pH and pCO2. The results from these measurements were used to perform single point calibrations on the DOT and pH probes.
[00375] For inoculation an S200 cell bioprocess container was connected to the SUB bioprocess container using sterile c flex tubing attached to the dip tube line. The required volume of inoculum was pumped to the SUB bioprocess container using a calibrated Watson Marlow 600 series pump.
[00376] The feeds, alkali and antifoam were all welded onto the SUB bioprocess container using c flex tubing, each had dedicated lines. Alkali addition was via the Watson Marlow 100 series alkali pump built into the control tower. Alkali was added as required to control the pH. Antifoam was added manually using the second Watson Marlow 100 series pump built into the control tower.
[00377] Feeds were added using Watson Marlow 500 series pumps. Flow rates and addition volumes were determined using appropriately sized balances correcting for the density of the feeds. The flow rate of the continuous feeds SF70 and 400 g/L D glucose were adjusted on a daily basis according to the viable cell concentration (VCC) and glucose concentration of the culture. Shot feeds SF71, SF72 and SF73 were added according to the FPD.
[00378] Each day samples were taken as part of daily monitoring of the bioreactors to check cell concentrations, viabilities, metabolites and dissolved gases using sample bioprocess containers attached to the sample line.
[00379] One point adjustments for online pH probes were performed when necessary according to UKSL 182 using results from a calibrated offline pH probe (Mettle Toledo offline 405 DPAS SC K8S/120 with pHM220 meter).
EXAMPLE 4
Use of a Single Use Bioreactor in a Production System

[00380] In another example, this single-use bioreactor can also be used in the systems and methods disclosed in WO 2017/072201 A2, the entirety of which is incorporated by reference.
[00381] In WO 2017/072201 A2, bioreactors are used during both the inoculum expansion and production process steps. The single-use bioreactors of the present disclosure provide advantages to this system because they can be made ready for different runs more quickly and efficiently, thereby reducing bioreactor "down time" needed for cleaning and sterilizing.
[00382] This will allow the systems of WO 2017/072201 A2 to produce high quality, safe, and cost effective active pharmaceutical ingredients (APIs) and biopharmaceutical products in a more timely and cost-effective manner. For instance, there would be greater flexibility in vessel architecture and components used when designing processes to manufacture proteins and cells, significantly reduced operating costs (e.g., labor, utility, and maintenance), improved facility throughput as batch turnaround times are condensed, clean in place and steam in place operations.
[00383] As part of the process disclosed in WO 2017/072201 A2, there are purification steps. During the purification processes, numerous resins can be used during purification, including but not limited to, MabSelect SuRe / MabSelect SuRe LX / MabSelect SuRe pcc (GE Healthcare), Amsphere A and Amsphere A3 (JSR micro), Praesto AP and Praesto AC (Purolite), KanCapA (Pall), Toyopearl AF-rProtein A HC (Tosoh), Poros MabCapture A (Thermo-Fisher), and the like. Other purification material would be known to a person of ordinary skill in the art and this is by no means an exhaustive list.
[00384] It should be recognized that the one or more examples in the disclosure are non-limiting examples and that the present disclosure is intended to encompass variations and equivalents of these examples.
cool the cell culture within the bioprocess container so as to avoid the formation and/or perpetuation of hot and cold spots during single use bioreactor operation. The bioreactor jacket may partially or completely surround the shell and/or bioprocess container. In at least one embodiment, the thermal jacket at least partially, preferably fully, covers the lower portion of the bioreactor vessel above the probe shelving. In some embodiments, the thermal jacket may be in fluid communication with at least one of a heated or a chilled fluid. The bioreactor jacket may, in one embodiment, comprise a water jacket.
[00208] Exemplary embodiments of the bioreactor jacket 280 are shown in FIG. 6 and FIG. 10. As shown in FIG. 6 and FIG. 10, in some embodiments, the bioreactor jacket 280 covers the bottom half of the shell 110. The bioreactor jacket 280 may be configured such that at least one open space or hole is provided, such as for placement of a probe belt. As shown in FIG. 10, in one embodiment, the bioreactor jacket 280 may cover the bottom half of the shell that is above the probe belt(s) 290. This configuration may promote efficient heat transfer. The probe shelving 260 may be located on the bioreactor jacket 280.
[00209] In one embodiment, the at least one door 270 of the shell 110 may have a thermal jacket that may be separate from or connected to the thermal jacket of the bottom half of the shell. In a further embodiment, the top jacket may be connected to the bottom jacket via flexible tubing or the like in order to ensure that the at least one door can be opened.
[00210] In one embodiment, the temperature sensor senses the temperature of the cell culture medium during operation of the single use bioreactor. In embodiments wherein the temperature sensor is in communication with the controller, the controller may be configured to receive information from the temperature sensor and, based on that information, control the flow of a fluid into the bioreactor thermal jacket for increasing or decreasing the temperature of a culture media that is contained within the bioprocess container. As such, in some embodiments, the culture media is maintained within preset temperature limits.
[00211] The temperature sensor, in one embodiment, comprises a resistance temperature detector. In operation, in some embodiments, temperature set points can be provided as a temperature band such that temperature corrective action is taken when the measured temperature is outside the temperature band. In at least one embodiment, the temperature

control system is configured to maintain temperature at +/- 0.2 °C over a range of 10 to 40 °C. In at least one embodiment, the temperature control system is configured to maintain temperature at +/- 1.0 °C over the range of 5 to 20 °C. In at least one embodiment, the temperature control system is configured such that temperature overshoot and undershoot does not exceed +0.8 °C for transitions between any set points in the range 10 to 40 °C. In at least one embodiment, the temperature control system is configured to control temperature constantly at +/- 0.1 °C during fermentation over a range of 10 to 40 °C. In at least one embodiment, the temperature control system is configured such that the temperature control system is unable to heat above a certain temperature, such as 40 °C, to avoid damage to any disposable component parts.
[00212] In one embodiment, the medium is brought to operating temperature by process control. In one embodiment, this is achieved by "gentle" heating or cooling of the jacket. For example, in one embodiment, very high or very low temperatures are avoided at the vessel wall. In at least one embodiment, the temperature control system is configured such that the thermal jacket warms 1000 L of cell culture medium from ambient to 34 to 40 °C in less than 6 hours. In at least one embodiment, the temperature control system is configured such that the thermal jacket chills 1000 L of cell culture medium from 34 to 40 °C to 10 °C in less than 6 hours. In one embodiment, the temperature control range during operation is 36 to 38° C. with an accuracy of ±0.2° C. at set point.
[00213] In one particular embodiment, the bioreactor jacket area may be specified with the following considerations in mind: (i) warming up of medium from 10° C. to 40° C; (ii) all points within the bioreactor must reach ±0.2° C. of set point, typically e.g. 37° C, as measured by thermocouples, and (iii) chilling of medium from 40° C. to 10° C.
Probes 28/Probe Belt 290:
[00214] Referring to Figure 7, in at least one embodiment, one or more of the various probes and/or sensors described herein are disposed in at least one probe belt 290 configured to position the various probes and/or sensors appropriately with respect to the bioprocess container. The at least one probe and at least one probe belt may be configured in any suitable location in or on the shell. For example, in one particular embodiment, the sample line is to be situated next to the pH probes to ensure close proximately when taking offline pH samples. In at least one embodiment, two or more probe belts are provided.

Each probe belts may be capable of operationally housing at least one, such as at least two, such as at least three, such as at least four, such as at least five, such as at least six probes and/or sensors each. In one embodiment, the probe belts include two pH sensors and two DO sensors. In some embodiments, at least seven, such as at least eight, such as at least nine, such as at least ten additional probes may be accommodated. The probes may be opposite each other. In one particular embodiment, the probe shelving is configured to operationally support two probe belts, each capable of housing six probes opposite each other. In one embodiment, a probe belt, such as a probe belt containing spectroscopic probes, may be configured in order to protect the belt and/or the probes from light or other environmental conditions.
[00215] In one embodiment, the probes can rest on shelving. Referring to FIG. 10, in at least one embodiment, the lower portion of the shell 110 and/or the bioreactor jacket 280 includes probe shelving 260 configured to operationally support at least one probe belt 290 having various probes thereon. The shelving may be permanently or removably fixed to the bioreactor shell. The shelving may be oriented at a certain angle, such as greater than 1°, such as greater than 5°, such as greater than 10°, such as greater than 15°, such as greater than 30°, such as greater than 45°, such as greater than 60° degrees, with respect to the shell. In one embodiment, the probe shelving is oriented at an acute angle with respect to the shell exterior surface. In one particular embodiment, the probe shelving is oriented at a 30° angle with respect to the shell exterior surface.
[00216] In one embodiment, the various probes are in wired and/or wireless communication with the controller and/or their respective systems, and are configured to transmit respective data thereto. In one non-limiting embodiment, spectroscopic probes are either the RAMAN or NIR type. In some embodiments, the spectroscopic probes can receive and monitor characteristics including but not-limited to viable cell concentration, culture viability, glucose concentration, amino acid concentrations, lactic acid concentration, and ammonium concentration. In some embodiments, further measurement and analysis using additional tools may be necessary for product characterization. In one embodiment, the controller is preferably configured to control the various system set points (e.g., pH, Temperature, DOT, agitation, nitrogen flow rate, air cap) and pump flow rates (all integral pumps and external pumps) based on the output of the spectroscopic probe.

Methods
[00217] In a preferred embodiment of the present disclosure, the method according to the disclosure takes place in at least one single-use bioreactor of the present disclosure. In one embodiment, the present disclosure includes a method for comparing the performance of a bioreactor vessel across scale and vessel size. In another embodiment, the present disclosure includes a method for validation bioprocess container performance beyond the intended operating ranges, such as for scaling up or down. In a further embodiment, the present disclosure includes a method for theoretically or experimentally determining the number and size of holes in at least one sparger during scaling of the bioprocess container.
[00218] The disclosure also includes a method for cultivating and propagating cells and/or cell products, wherein at least one cell is cultivated under suitable conditions and in a suitable culture medium in a first bioreactor with a first volume, the medium containing the cells obtained by propagation from the at least one mammalian cells is transferred into a second bioreactor having a second volume, wherein the second volume is greater than the first volume, the transferred cells are cultivated in the second bioreactor, the medium containing the cells from the second bioreactor is transferred into a third bioreactor having a third volume, wherein the third volume is greater than the second volume, and the transferred cells are cultivated in the third bioreactor.
[00219] In one particular embodiment, the disclosure also includes a method for cultivating and propagating cells and/or cell products, characterized in that a) at least one mammalian cell is cultivated under suitable conditions and in a suitable culture medium in a first single use bioreactor with a volume of at least 10 L, such as at least 500 L, such as at least 1000 L, b) the medium containing the cells obtained by propagation from the at least one mammalian cell is transferred into a second single use bioreactor with a volume of at least 1000 L, such as at least 2000 L, such as at least 4000 L, c) the transferred cells are cultivated in the second single use bioreactor, d) the medium containing the cells obtained in step c) is transferred into a third single use bioreactor with a volume of at least 10,000 L, such as at least 20,000 L, and e) the transferred cells are cultivated in the third single use bioreactor. In one embodiment, the system may include a plurality of single use bioreactors in fluid communication with each other. The bioreactors can be controlled by a single controller or by multiple controllers. Each single use bioreactor in the system can, in

one embodiment, have the same size. The volume of each single use bioreactor, for instance, can be e.g. 1000 L, 2000 L, 4000 L, 10,000 L, 20,000 L, etc.
[00220] In one embodiment of the disclosure, the method is characterized in that at least one of the bioreactors used is a bioreactor according to the disclosure. In a further embodiment, all bioreactors used are bioreactors according to the disclosure.
[00221] Bioreactors according to the disclosure are in this context all bioreactors described in this description, in the examples and in the claims.
[00222] In one embodiment, the bioreactor of step e) is operated in batch or fed batch mode. In one embodiment, the cells are cultivated in step e) preferably for 6 to 20 days.
[00223] Step a) is also called stage N-3 and/or N-2. Step c) is also called stage N-l. Step e) is also called stage N.
[00224] In one embodiment, the cultivation conditions in the bioreactors of steps a), c) and e) are the same. In one embodiment, the cultivation conditions in the bioreactors of steps a), c) and e) have a homogenous environment with respect to process parameters such as pH, dissolved oxygen tension and temperature. In one embodiment, pH, dissolved oxygen tension, and temperature in the bioreactors of steps a), c) and e) are the same.
[00225] In one embodiment of the disclosure, the seeding ratio after the transfer steps b) and/or d) is at least 10% v/v, such as at least 11% v/v (1 in 9 dilution) and at most 30% v/v, such as at most 20% v/v (1 in 5 dilution).
Train
[00226] The single-use bioreactor system according to the disclosure can also be used in a bioreactor train or device.
[00227] The bioreactor train, in one embodiment, can comprise different bioreactors, which are also called stages. For example, a bioreactor with a volume of at least 500 L, such as at least 1000 L may correspond to stage N-3 and/or N-2. The bioreactor with a volume of at least 2000 L, such as at least 4000 L may correspond to stage N-l. The bioreactor with a volume of at least 10,000 L, such as at least 20,000 L may correspond to stage N. In one embodiment of the disclosure there is a further bioreactor, such as a 50 L bioreactor, corresponding to stage N-4. In one embodiment of the disclosure, the N-4

bioreactor is a S-200 seed rocking bioreactor or a 100 L stirred tank reactor. In a one embodiment of the disclosure, the aspect ratio HL/T is at least 0.17 and at most 1.96.
[00228] In one embodiment, the bioreactor train may include a plurality of single use bioreactors in fluid communication with each other. The plurality of single use bioreactors can be controlled by a single controller or by multiple controllers. In one particular embodiment, the single use bioreactors can have the same volume, such as any of the volumes described above.
[00229] In one embodiment, the design of the bioreactor train is based on the need to ensure a homogenous environment with respect to process parameters such as pH, dissolved oxygen tension (DOT) and temperature, maintaining a well-mixed cell suspension and blending nutrient feeds within the bioreactor. In some embodiments, the bioreactors of the bioreactor train show geometric similarity. This can allow a scale-down model to develop, for example at 5 L laboratory scales or 500 1 pilot scales. In some embodiments, the bioreactors of the stages N-3, N-2 and N-l are used as seed-bioreactors, while the bioreactor of stage N is used as a production-bioreactor. The design of the seed-and production-bioreactors can be based on the same principles. However, in certain embodiments, some departures can be required to allow for flexibility in processing.
[00230] In another embodiment, the single-use bioreactors of the present disclosure can be used in series for fed batch or perfusion with a single controller, as described below. In one embodiment, a single controller as described below could control perfusion in series as much as fed batch in series. Yet another aspect of the present disclosure allows inoculation perfusion to be automated once the cells entered an inoculum/production vessel. In certain embodiments, this would enable support of development scales as well as smaller scale facility to increase output of production.
[00231] In one embodiment, the single-use bioreactor of the present disclosure may be used in perfusion applications. For instance, referring to FIG. 38, one embodiment of a bioreactor system 400 for carrying out a perfusion process is shown. The bioreactor system 400 includes a bioprocess container 402 made in accordance with the present disclosure. The bioprocess container 402, for instance, can be made from a flexible film and can be inserted into a rigid metallic shell. The bioprocess system 400 can include a mixing device which includes a rotatable shaft 408 coupled to a first impeller 407 and a second impeller

406. As shown, the first impeller 407 is spaced from the second impeller 406. The first impeller 407 is located in a middle section of the bioprocess container 402, while the second impeller 406 is located in a bottom section of the bioprocess container 402.
[00232] A feed tube 417 is included for feeding fresh feed medium applied at a desired flow rate. The feed tube 417 can terminate with a one-way valve to prevent fluids from flowing into the feed tube 417.
[00233] The bioreactor system 400 can also include at least one sparger. For instance, in the embodiment illustrated in FIG. 38, the bioreactor system includes a first sparger 405. The first sparger 405 is a subsurface sparger located below the impeller 406. The sparger 405 can be used to feed air, oxygen, nitrogen, carbon dioxide and other gases into a culture media contained within the bioprocess container 402.
[00234] The bioreactor system 400 includes a second sparger 420. The second sparger 420 can be a supersurface sparger that feeds gases into the head space of the bioreactor container 402. The sparger 420, for instance, can feed overlay gases such as air, oxygen, nitrogen and carbon dioxide into the bioprocess container.
[00235] The bioreactor system 400 can further include a vent 422 in order to release gases from the system.
[00236] As shown, the bioprocess container 402 is in fluid communication with a recirculation line 424. The recirculation line 424 is in fluid communication with a cell retention chamber 426. A pressure gauge 428 can be used to monitor the pressure within the cell retention chamber 426.
[00237] The cell retention chamber 426 can be in fluid communication with a filtrate outlet 430. The filtrate outlet 430 is placed in association with a biofilter. The filtrate outlet 430 is configured to remove liquids from the cell retention chamber 426, such as spent liquids. The biofilter, however, is permeable to liquids but impermeable to biological materials, such as cells. Thus, filtrate can be removed from the cell retention chamber 426 without loss of biomaterial. The position of the recirculation line 424 can vary. The recirculation line 424 can be positioned at the top section, at the middle section or at the bottom section of the bioprocess container 402.

[00238] The bioreactor system 400 can further include a flow regulator 432. The flow regulator 432, for instance, may comprise an alternating tangential flow regulator. In the embodiment illustrated, the flow regulator 432 is in communication with a vacuum source 434 and a pressurized gas source 436 which may be an air pressure source. Upstream from the vacuum source and the gas pressure source, the flow regulator 432 is in fluid communication with a reciprocating diaphragm 438. The flow regulator 432 is configured to alternatively apply a vacuum or a gas pressure to a fluid contained in the cell retention chamber 426 by using, for instance, the reciprocating diaphragm 438. The reciprocating diaphragm 438, for instance, can alternate between applying pressure and applying a suction force to fluid contained in the cell retention chamber 426. In this manner, fluids such as a culture media can be recycled back and forth between the bioprocess container and the cell retention chamber for carrying out a perfusion process.
System
[00239] The present disclosure also relates to the use of single-use bioreactors in systems. The required system settings are covered in the single-use bioreactor control system described herein.
[00240] Forming single-use bioreactors of the present disclosure can, in one embodiment, be accomplished by fitting and/or inflation of a single use product contact bioprocess container to be inserted into a stainless steel shell and inflated. In another aspect of the present disclosure, filters may be fitted to the shell after inflation. In yet another aspect of the present disclosure, probes and sampling system may be fitted to the SUB after inflation.
[00241] In one embodiment, production may be commenced when the growth medium is filtered into the single-use bioreactor via gamma-irradiated sterilizing grade filters. In some embodiments, these filters can be welded onto the additional lines prior to or after gamma irradiation, but do not need to be. In some embodiments, the culture medium and gas inlet filters may be provided in the bioprocess container prior to gamma irradiation. Next, in some embodiments, the medium would be allowed to equilibrate in the single-use bioreactor (temperature, pH and dissolved oxygen) under agitation prior to inoculation. During the production process additional substrates, pH controlling solutions and antifoam

may be added. The single-use bioreactor can be continuously monitored throughout this process.
[00242] In one embodiment, the cell culture can be harvested via disposable depth filter system to remove the cells and cell debris, prior to filtration and subsequent purification.
SUB Control System
[00243] In accordance with one or more aspects of the disclosure, a control system for controlling the single use bioreactor and its functionalities are provided and will now be described below. By way of example, the control system may include one or more controllers, one or more thermocirculators, one or more scales (e.g., industrial scale), one or more control pumps (e.g., automatic control peristaltic pump), and other suitable types of system components that may be controlled by the controller(s).
[00244] In one embodiment, the controller may control and/or monitor, such as via a sensor, at least the following parameters of the SUB: (1) pH, (2) dissolved oxygen tension (DOT), (3) dissolved CO2 (pCO2), (4) air, O2, CO2, N2, (5) temperature, (6) agitation, (7) alkali, (8) nutrient continuous feed, (9) nutrient shot feed, (10) pressure, (11) foam, (12) level and other suitable types of parameters, all of which will be further described in detail below. The controller may be in communication with at least one sensor, and, based on the information provided by the sensor, may be able to control a material or fluid supply, such as by varying a flow rate of a fluid from the fluid supply into the hollow enclosure of the bioprocess container. As such, in some embodiments, the controller may assist in maintaining within present limits at least one parameter of a culture media contained within the hollow enclosure of the bioprocess container. In another embodiment, the thermocirculator may enable temperature control for fermentation heating (e.g., bioreactor set point of from 34°C to 40°C) and for cooling (e.g., bioreactor set point of 10 °C). In yet another embodiment, the scales may be required (per bioreactor unit) for feed addition control and monitoring; for instance, one scale may be dedicated to alkali addition linked to pH control or to process shot feed additions. In a further embodiment, the automatic control pumps may be required (per bioreactor unit) for further feed additional control and monitoring.
[00245] In one embodiment, the controllers provide increased flexibility, reliability and ease of use in their operation for both research and custom process manufacturing and

development projects. Therefore, in some embodiments, the system must be able to be operated in a GMP environment as well as in a development laboratory. In certain embodiments, the SUB system can be operated as either inoculum reactor or as a production unit. As such, some of the control functions, for example DO control, required may be different from the ones described in paragraph above. In one embodiment, when operating in inoculum mode, pH or DO will not be controlled. In one embodiment, the control system should be flexible to accommodate either mode of operation. In some embodiments, it is likely that more than one disposable bioreactor unit will be operating in manufacturing, with either different or same volumes. All vessels may require the same control functions and each unit may require its own control system. Moreover, in some embodiments, the control package shall comply with current standards for equipment in accordance with cGMP practices, together with European and American regulatory requirements for pharmaceutical industries.
[00246] In embodiments with more than one controller, the controllers can be components of a smart communication system, wherein the controllers may communicate with each other and with a central control system during the culturing process or portions thereof to enable process integration. In various embodiments, the smart communication system may utilize central decision making with a central controller or distributed decision making between unit operators in continuous integrated processes.
Controller
[00247] The controller may be any type of processing hardware, such as a processor or a computing device, configured to control and execute various instructions for one or more components and/or related equipment associated with the single-use bioreactor described herein. In one embodiment, the controller may comprise one or more microprocessors. It may be understood that more than one controller may be used to perform control and the various components of the control system may be connected via a system network.
[00248] Figure 36 illustrates an example system in accordance with one or more aspects of the disclosure. The system may include one or more computing devices, e.g., computer 100, server computer 130, mobile computer 140, smartphone device 150, tablet computer 160, and storage device 170 connected to a network 190. For example, the computer 100 may be a desktop computer, which is intended for use by one or more users. The computer

100 includes various components associated with a desktop computer, such as one or more processors 102, memory 104, e.g., permanent or flash memory (which includes instructions 105 and data 106), one or more interfaces 108, and a display 110. In a further example, similar to the computer 100, the server computer 130 may include at least one processor, memory which also includes instructions and data, one or more interfaces, and/or a display (not shown). Moreover, the mobile computing device 140 may be a laptop (or any type of computer that is portable or mobile, such as an Ultrabook) and also include components similar to the computer 100 and/or server computer 130. The computer 100 may be configured to communicate with the server computer 130, the mobile computer 140, the smartphone device 150, the tablet computer 160 and/or the storage device 170 via the network 190.
[00249] The computer 100 can include a processor 102 (e.g., the controller), which instructs the various components of computer 100 to perform tasks based on the processing of certain information, such as instructions 105 and/or data 106 stored in the memory 104. For example, the processor 102 may be hardware that can be configured to perform one or more operations, e.g., adding, subtracting, multiplying, comparing, jumping from one program to another program, operating input and output, etc., and may be any standard processor, such as a central processing unit (CPU), or may be a dedicated processor, such as an application-specific integrated circuit (ASIC) or a field programmable gate array (FPGA) or an industrial process controller.
[00250] Memory 104, whether permanent or flash, may be any type of hardware configured to store information accessible by the processor 102, such as instructions 105 and data 106, which can be executed, retrieved, manipulated, and/or stored by the processor 102. It may be physically contained in the computer 100 or coupled to the computer 100. For example, memory 104 may be ROM, RAM, CD-ROM, hard drive, write-capable, read-only, etc. Moreover, the instructions 105 stored in memory 104 may include any set of instructions that can be executed directly or indirectly by the processor 102. For example, the instructions 105 may be one or more "steps" associated with software that can be executed by the processor 102 to control various aspects of the SUB control system. According to one aspect of the disclosure, the instructions 105 may include at least a set of executable instructions to read various values and/or parameters associated with the SUB. According to another aspect of the disclosure, the data 106 may include data

that may be used by the control module, such as sensor readings, data collected by sensors, predetermined parameters, readings associated with valves, pumps, agitators, scales, switches, temperature measurements, pressure measurements, level measurements, dissolved oxygen measurements, etc.
[00251] Interface 108 may be a particular device (such as a field-mounted instrument, processor-to-processor communication, keyboard, mouse, touch sensitive screen, camera, microphone, etc.), a connection or port that allows the reception of information and data, such as interactions from a user or information/data from various components via network 190. Alternatively, the interface 108 may be a graphical user interface (GUI) that is displayed to the user/operator on the display 110. By way of example only, the GUI may be an operator interface (OI) that displays processing units and data to a user or operator. Moreover, the display 110 may be any suitable type of device capable of communicating data to a user. For example, the display 110 may be a liquid-crystal display (LCD) screen, a light emitting diode (LED) screen, a plasma screen, etc.
[00252] The network 190 may be any suitable type of network, wired or wireless, configured to facilitate the transmission of data, instructions, etc. between one or more components of the network. For example, the network 190 may be a local area network (LAN) (e.g., Ethernet or other IEEE 802.03 LAN technologies), Wi-Fi (e.g., IEEE 802.11 standards), wide area network (WAN), virtual private network (VPN), global area network (GAN), or any combinations thereof. In this regard, the computer 100, server computer 130, mobile computer 140, smartphone device 150, and/or tablet computer 160 may connect to and communicate with one another via the network 190.
[00253] While the computer 100 may be a desktop computer in the above-described examples, computer 100 is not limited to just desktop computers, and any of the computers illustrated in Figure 36 may be any device capable of processing data and/or instructions and transmitting and/or receiving data. Moreover, it should be understood that those components may actually include multiple processors, memories, instructions, data or displays that may or may not be stored within the same physical housing.
pH Control
[00254] In accordance with one embodiment of the disclosure, one or more controllers of the SUB control system, such as the one or more processors of computer 100 in Figure

36, may be used to measure and receive pH values of the biomaterial in the bioprocess container via at least one sensor, and in some embodiments at least two sensors, such as electrochemical sensors. During control procedures, for example, only one pH sensor may be used or two or more pH sensors may be used. Each pH sensor used may be in communication with the controller. When two sensors are used, the controller may select between the two sensors, either manually or automatically, depending on whether there are detected errors in the measured pH values. Based on the pH readings, the controller may regulate pH levels by adding requisite amounts of acid or alkali.
[00255] In another example, the controller may use a CO2 gas supply to decrease pH and a pumped liquid alkali to increase pH in order to control to a set point. The CO2 gas supply and/or the liquid alkali supply may be in fluid communication with the bioprocess container. In one embodiment, the ability to operate a "dual" pH set point may be implemented. For instance, a high and low pH set point can be user configurable. Between the high and low set points, no control action (CO2 or alkali) may be required and pH may drift within this band. When pH is less than the low pH set point, alkali may be required, and when the pH is above the high set point, CO2 may be required. In certain embodiments, the controller should not have to "fight" between the addition of CO2 and alkali such that they counteract each other resulting in overdoses of each.
[00256] As such, for example, the controller may set and control pH set points between two different and/or opposing outputs, the first of which may be the CO2 mass flow controlled gas addition and the second of which may be proportional control pumped addition of an alkali solution. Moreover, the controller may be configured to perform temperature compensation based on measure pH values, where temperature values may be selected from the one or more pH sensors.
[00257] In yet another embodiment, the controller may allow a user or operator to enter a separate value and define an upper and lower zone between which there may be no particular control or control action, e.g., no CO2 addition or alkali additions based on the pH measurements and subsequent control. This may be referred to as "deadband" functionality. When using the deadband function, which may be between +/-0.01 to +/-0.30 pH units relative to the process setpoint, the process control of pH and the corresponding CO2 additions, if/when applicable, may have minimal oscillation. In other

examples, the controller may be configured to receive two pH set points (e.g., one at either end of the deadband). It may be understood that when operating with a pH deadband (for example, +/-0.01 pH relative to the minimum set point), there must be no control discrepancies and/or inconsistencies between CO2 and alkali additions
[00258] In at least that regard, one of the numerous advantages of the controller controlling pH is that the system can exhibit responsiveness and adherence to the set point(s) with stable additions of CO2 and/or alkali (e.g., minimal oscillations).
[00259] In a further example, the controller may alert the user or operator by way of an alarm system any deviations, such as a drift between controlling and any non-controlling pH sensors. The range of deviation may be configured by the user/operator using an interface, such as interface 108 of computer 100 in Figure 1. In yet further aspects of the disclosure, single-point calibration may be used to adjust to an off-line pH measured value.
DOT Control
[00260] In accordance with another embodiment of the disclosure, the one or more controllers of the SUB control system, for example the processor(s) of the computer 100, may be used to measure and control dissolved oxygen levels, such as DOT, using at least one sensor, and in some instances at least two sensors, such as electrochemical sensors. Similar to pH control as described herein, during DOT control procedures, only one sensor may be used or two or more sensors may be used. If two sensors are used, the controller may select between the two sensors (manually or automatically) depending on whether there may be detected errors in the DOT measurements.
[00261] In one embodiment, a DOT set point may be controlled based on respective output(s) corresponding to additions of compressed air and compressed oxygen mass flow controlled gas, which may be operated in a cascaded format. Thus, in one embodiment, when using air and oxygen control, DOT levels can be maintained with only air until a configurable airflow point is reached. Moreover, oxygen may meet DOT demand while also maintaining a constant air flow. But, for instance, when there is insufficient demand for oxygen (e.g., at a configurable setpoint), control via the controller may be returned to air in an automatic manner.

[00262] In another example, similar to the pH sensors described herein, the controller may be configured to perform automatic temperature compensation based on measured DOT value and the temperature value may be selected from the one or more DOT sensors.
[00263] As such, an advantage of the controller performing control of the DOT is that the system will exhibit responsiveness and adherence to the set point(s) with stable additions of air and/or O2 (e.g., minimal oscillation). The controller, in examples, may alert the operator via an alarm system when the controller detects a deviation or drift between controlling and any non-controlling DOT and/or pH sensor. In some embodiments, the range of the deviation is configurable by the user. In a further aspect, single-point calibration may be used to adjust to an off-line DOT measured value.
[00264] In a further example, there may be at least two types of DOT control that may be supplied: the capped air method and the gas mixed method. In the capped air method, a user-definable continuous flow of nitrogen introduced through a single mass flow controller (MFC) may be implemented. The DOT control is achieved by increasing air flow-rate via a mass flow controller to match oxygen demand from cells, with the ability to start oxygen supply (via a mass flow controller) when the air flow rate reaches a user defined limit. Under these circumstances the air will be capped at a fixed flow-rate and oxygen added (under PID control) to supplement the demand. When the oxygen is no longer required, control will return to air flow. In the gas mix method, for instance, DOT and pH can be controlled by full 3 plus 1 gas mix system. DOT may be controlled by varying the mix of air/nitrogen and oxygen at a pre-determined, user selectable total gas flow rate. pH control can be performed by the addition of CO2, without increasing the total gas flow rate.
pCO2 Control
[00265] In accordance with yet another embodiment of the disclosure, the one or more controllers of the SUB control system may monitor and control dissolved CO2 (PCO2). For example, pCO2 may be measured using a sensor and the measurement transmitted by a transmitter. The transmitter, in some examples, may physically be mounted within the housing of the controller, but control may be performed externally, e.g., on an interface of the controller, for the user to execute single point and/or two-point calibration via the interface. In further examples, the pCO2 may be linked to an independent air flow via, for

example, mass flow control (MFC) to a sparge and also set a minimum CO2 flow output via MFC.
[00266] In one embodiment, the pCO2 measurement values enable control on the airflow to a sparger (which, in some examples, may join with another sparge prior to bioreactor entry and/or sterile filtration) and also the CO2 MFC valve. By way of example, control may be performed to prevent conditions of excessively high or low pCO2 while maintaining suitable set point control of pH and DOT values. The control process for doing so may include the steps of automatically adding a fixed rate of airflow to one sparge, which may be triggered by activation of a first pCO2 alarm value (e.g., "hihi" value). In some instances, the airflow via an open pipe may act to strip out CO2 and thus reduce PCO2. Thereafter, a fixed rate of CO2 to another sparge may automatically be added and the fixed rate of CO2 may be triggered by activation of a second pCO2 alarm value (e.g., "lolo" value). For example, the lolo alarm may trigger the CO2 mass flow control valve to remain open at, for instance, 2 percent of full span (which may be a value set by the operator), regardless of its current state for active pH control.
Redox
[00267] In accordance with a further embodiment of the disclosure, the one or more controllers of the SUB control system may monitor reduction-oxidation (redox) measurements, which may be taken using one or more sensors. In examples, a transmitter for transmitting the redox measurements may be implemented.
Gases
[00268] In accordance with another embodiment of the disclosure, the one or more controllers may be used to control the flow of gas, such as air, oxygen, CO2, N2, which may be related to the control of pH and DOT described herein. Gasses may be introduced into the bioreactor using a single sparger, e.g., located at the base of the bioreactor. Alternatively, two sparger outlets and one outlet to headspace may also be used. In examples, gasses may be introduced to the bioreactor at the same time via the spargers and headspace under maximum level operating conditions in the following full span bioreactor ranges.

[00269] By way of example, the controller may be configured to activate the flow control of gasses via manual activation (e.g., performed by operator) and/or automatic activation (e.g., linked to an in-line pCO2 measurement).
[00270] In another example, the gas overlay (e.g., air) may be controlled through a mass flow control valve. The controller may allow for manual variable set point change during cell culture run. The ranges required are as follows: SUB 50L: 0 to 0.5L; SUB 250L: 0 to 1L; SUB 1000L: 0- 2L. It may be understood that these values may be refined as further operational data is obtained.
[00271] Moreover, the gas overlay flow value may be displayed on an interface, such as a touch screen (or other human machine interface (HMI)). Display screen can show actual value and set point. An alarm may sound if gas overlay set point value falls outside the alarm limits. A message may appear on the alarm screen and be electronically logged. And the ability to switch off gas overlay automatically may be required if it reaches hihi alarm. This is to avoid any pressure build up inside the bioprocess container, as it is not rated as a pressure vessel. A message should appear on the screen flashing to warn that gas overlay has been switched off. This message may also be logged. The restart of the gas flow overlay may then be done manually on the touch screen once operator has acknowledged the alarm and checked that system can cope with the gas flow.
Temperature Control
[00272] In accordance with an embodiment of the disclosure, the one or more controllers may control the temperature of the SUB using a thermal jacket system that is preferably a water jacketed system, as described herein. Moreover, at least one thermocirculator, and in some examples at least two, are used for heating and chilling.
[00273] According to an example, the temperature of the SUB may be controlled based on temperature measurement(s) of the bioreactor vessel contents using a temperature sensor. For instance, an in-line bioreactor temperature sensor may be used for each bioreactor. Alternatively and/or in addition, a depth sensor may be used.
[00274] According to another example, the controller(s) may also be configured to alert the user of any type of deviation via an alarm system, which is capable of detecting a drift between controlling and any non-controlling temperature sensor. The range of this

deviation may be configurable by the user via an interface of the controller, e.g., interface 108 of computer 100.
[00275] As discussed above, circulation and temperature control of vessel contents can be designed to avoid hot and cold spots during bioreactor operation. In one aspect, temperature control can be maintained at ±0.2 degrees Celsius over the range of 10 to 40 degrees Celsius. In another aspect, temperature control can be maintained at ±1.0 degrees Celsius over the range of 10.0 to 20 degrees Celsius and 36 to 40 degrees Celsius. In yet another aspect, the over-shooting and under-shooting of the temperature should not exceed +0.8 degrees Celsius for transitions between any set points in the range 10 to 40 degrees Celsius. In other embodiments, temperature may be controlled constantly at +/- 0.1 degrees Celsius during fermentation over a range of 10 to 40 degrees Celsius. In certain embodiments, heaters are not to be used above 40 degrees Celsius to avoid damage to any of the disposable component parts.
[00276] Moreover, signals can be provided for temperature measurement and control, data logging and alarms, and temperature compensation for the pH sensor unit. A continuous digital display of the temperature value to one decimal place may be provided. Display of the temperature reading must be on the mimic touch screen (or other HMI) should be for both actual reading and desired set point.
[00277] For the heating mechanism, for example, the controller may supply an output for an electrical jacket attached to the SUB reactor. Plugs and sockets may have a positive lock to prevent accidental removal of lead.
[00278] Algorithms may be used for temperature control to the heater actuators. The temperature values used by the controller must be available for logging. There may be user definable set-points with "high high, high" and "low low low" alarm limits. There must be the ability to auto-tune various terms.
Agitation
[00279] In accordance with another embodiment of the disclosure, the one or more controllers may control the mechanical circulation of the liquids in the bioreactor vessel (e.g. 400L vessel) via an impeller, e.g., a dual impeller system.

[00280] For example, the controller may measure and control the agitation speed based at least in part on inputs from a calibrated tachometer that may be mounted next to the top of the motor.
Feeds Addition during Fermentation
[00281] In accordance with yet another embodiment of the disclosure, the one or more controllers of the SUB control system may allow peristaltic addition pumps to be run in automatic or manual modes. For example, the addition pumps may be used for alkali addition, which may be monitored via a dedicated scale and/or a dedicated pump totalizer. Moreover, there may be multiple continuous feed additions at variable rates as well as multiple shot feed additions (which may be monitored via a dedicated scale and/or a dedicated pump totalizer). As will be further described below, automation software may be executed by the controller for running, for instance, shot addition sequence.
[00282] Additions feeds can be operated via the control system and can allow for gradual feed addition or single shot based on quantity over a period of time.
[00283] In one example, three industrial scales per bioreactor unit may be used for feed addition control and monitoring. Each of the industrial scales may be dedicated as follows: a first scale ("scale one") to either Alkali addition linked to pH control or to process shot feed additions, a second scale ("scale two") to "Continuous Feed 1," and a third scale ("scale three") to "Continuous Feed 2."
[00284] In another example, seven automatic control peristaltic pumps per bioreactor may be used (e.g., two independent pump rack sets of seven and/or split as required per system) for further feed addition control and monitoring. The pumps may be dedicated to Alkali addition for pH, Continuous Feed 1, Continuous Feed 2, "Shot Feed 1," "Shot Feed 2," "Shot Feed 3," and "Shot antifoam" addition. The peristaltic pumps, for instance, may be configured for variable speed. The pump speed may be determined automatically by the control system to achieve a required addition feed rate entered by the operator. In manual mode, the pump speeds may be determined and set by the operator.
[00285] Moreover, in one embodiment, delivery rates may include configurable alarm limits to delimit the maximum and minimum delivery rate around the configured

setpoint(s). Additionally, feed rates may be automatically confirmed based on loss-in-weight measurements or via calibrated flow controllers.
[00286] By way of example, the antifoam addition, and Shot Feeds 1, 2 and 3 may be controlled by the controller as follows. An operator may turn on the pump at a variable speed selected by the operator. After priming the line to the point of entry to the bioreactor, the actual addition will be quantified by the pump totalizer. An external scale, for instance, may be used for the pump calibration. Once primed, the controller facility for dosing a single addition (repeated on multiple days during the fermentation) may be performed. Subsequently, the user inputs the quantity to dose.
[00287] As described herein, the SUBs may already have suitable ports to connect the following: medium fill and inoculation; alkali for pH control; variable rate Continuous Feed 1 (e.g., approximately 25% of batch volume); variable rate Continuous Feed 2 (e.g., approximately 13% of batch volume); shot 1 acidic (e.g., approximately 2% of batch volume); shot 2 alkali (e.g., approximately 1% of batch volume); shot 3 pH neutral (e.g., approximately 2% of batch volume); antifoam (e.g., approximately 0.1% of batch volume). A suitable dosing cart, scale, and/or pump tower unit may be used to enable the best use of floor space and also operator access to set up at start of batch.
[00288] In addition, medium and inoculation addition may be controlled manually by the operator, with use of the bioreactor load cells. Alkali (e.g., medium pillow bioprocess container in rigid tray) may be located on an existing shelf and monitored either using a scale or pump for monitoring and/or totalizing of additions.
[00289] Continuous Feed 1 (e.g., large upright bioprocess container in cylindrical rigid drum) may be located on a low level (or floor space) dedicated scale. For example, feedback process control to a feed rate set point may be implemented. The scale will be zeroed with an empty container. At a user settable lolo level alarm, an interlock to stop feedback control (e.g., will not attempt to add from an empty bioprocess container) may be used.
[00290] Continuous Feed 2 (e.g., medium pillow bioprocess container in rigid tray) may be located on a low level (or floor space) with dedicated scale. For instance, feedback process control to a feed rate set point may be implemented. Similar to the above, the scale may be zeroed with an empty container. At a user settable lolo level alarm, an interlock

may stop feedback control (e.g., will not attempt to add from an empty bioprocess container).
[00291] Shots 1, 2 and 3 (e.g., medium pillow bioprocess container in rigid tray) may share a dedicated existing shelf and may be monitored either using a scale (if not used for alkali) or pump for monitoring and/or totalizing of additions. The antifoam (e.g., small bioprocess container or glass aspirator) may be located on an existing shelf and connected to a dedicated pump for monitoring and/or totalizing of additions. The seven peristaltic pumps may be fitted with tubing, such as, in one embodiment, 3.2 x 8.0 mm silicone tubing or 1/4" x 7/16" c-flex or 6 mm x 12 mm silicone.
[00292] By way of example, a process may include the addition of three shots at defined quantities and times during a batch. The three shots are acid, alkali, and neutral and may be added in that sequence. The shot volume per addition may be relatively small (e.g. between 0.15 and 0.5% of target bioreactor starting volume). The same set of three shots are added on multiple days during a batch. When adding these shots, it may be necessary to first inhibit just the alkali output for pH control, which prevents alkali being added unnecessarily (and irreversibly) during the acid shot. This, however, may be counteracted by the alkali shot that may immediately follow.
[00293] Moreover, the C02 addition for pH can remain active throughout. To ensure the process is controlled within known boundaries, these shots may be added at a suitable rate so as not to breach the acceptable pH range, such as triggering the lolo and/or hihi alarms. Automation of the shot sequence may thus include: (1) user definable volume for each shot to be added, (2) inhibiting alkali addition for pH control immediately prior to first shot, (3) tubing prime step to ensure shot liquid position is at the point of entry to the bioreactor (e.g., stopped by operator based on visual check), (4) each shot being added in series ("option 1"); and all 3 shots being added simultaneously ("option 2"). If, during shot addition, the pH approaches the lolo or hihi alarm limits, then the addition sequence is paused to wait until pH lo or hi alarms are re-established. The controller may also re¬activate alkali addition for pH control at completion of the shot sequence and when pH within lo and hi alarms.
[00294] In one aspect, use of a scale for shot addition monitoring may be used. Three shots, for instance, may be stored in one or more separate bioprocess containers, which

may be able to be stacked in individual trays. This stack of trays may be placed on a single scale, in which case, shots being added in series (e.g., option 1) can be performed using the change in mass from the scale.
[00295] In another aspect, use of pump totalizers for shot addition monitoring may be implemented. The pumps may have dedicated tubing lines which can be calibrated for this tubing type. After priming and resetting the totalizer, the pumps may determine the correct quantities to add and may also data log this quantity. Either option 1 (added in series) or option 2 (added in parallel) is appropriate since each pump will be operating independently, as opposed to what would be done on a single scale. In at least that regard, with the option 2 approach, the low and high pH perturbation will be reduced and cancelled out by both the acidic and alkali shots entering the bioreactor together. The lolo and hihi pH monitoring sequence may still be required in this scenario, but rather than wait for pH to return within alarm range, response can be performed by stopping the acid shot (if pH approaching lolo alarm) or alkali shot (if pH approaching hihi alarm).
Bioreactor Pressure
[00296] In accordance with a further embodiment of the disclosure, the one or more controllers of the SUB control system may monitor and control the bioreactor pressure via a device mounted on the bioreactor headspace. At a user defined pressure alarm value, this will enable a control action to stop all gas additions as a safety interlock. Moreover, the controller may be configured so as to scale for negative and positive pressure.
[00297] In examples, for system consistency and improved pressure test capability, a digital display pressure sensor may be provided. Moreover, it is possible to add a bioreactor pressure control valve on a gas outlet, which will enable feedback pressure control of the bioreactor based on the digital display pressure sensor.
[00298] Since the SUB may not be a rated pressure vessel, custom designed SUB bioprocess containers can be installed with a disposable pressure transducer. In some embodiments, the pressure inside of the SUB bioprocess container should not exceed a certain pressure. Provision to alarm and data log the pressure may be required. The controller may be configured to shut gases off if a high pressure alarm sounds. In some embodiments, the controller may be configured to open a second gas outlet filter, such as by opening pinch valves, prior to shutting the gases off. A message should appear on the

screen saying bioprocess container is over pressurized, which may be logged. To initiate gases again a second prompt (e.g., "are you sure?") may be displayed for safety reasons.
Antifoam
[00299] In yet another embodiment of the disclosure, the one or more controllers of the SUB control system may implement at least one foam sensor and transmitter, which may be directly integrated in the SUB and determine the amount of antifoam to be added in mass, which may also be displayed to an operator on an interface. For example, the level or measurement of foam in the SUB may be measured and transmitted to the controller for further processing in order to maintain requisite levels of antifoam. Moreover, these readings may be displayed on an interface for an operator. Provision may be made for the user to set the required flowrate if using manual control. If using the controller, then a timed on/off method may be used. In an example, the period of on and off may be definable by the operator via a touch screen.
Level
[00300] In an additional embodiment of the disclosure, the one or more controllers of the SUB control system may integrate a level sensor and transmitter for detecting level values. These values, like many other measured values described herein, may also be displayed for the operator.
Auxiliary Input and Control Loops
[00301] In accordance with an embodiment of the disclosure, at least two auxiliary inputs for signal generation may be needed for each controller used for controlling the SUB control system. A channel, for example, can be used for connection of a bio mass sensor and transmitter output (e.g., Aber Instruments BM 200, redox sensors, etc.).
[00302] Additionally, for example, at least two auxiliary inputs for signal generation and feedback control may be implemented for each controller. A channel, here for example, could be used for connection of an optical DOT sensor (e.g., Mettler Toledo InPro6960i, etc.).
Software
[00303] In accordance with another embodiment of the disclosure, software and/or the set(s) of computer executable instructions for controlling the SUB control system can be

provided. For example, the application code for the one or more control procedures described herein may be developed from an established library of "routines" or modules (e.g., for scaling, motor control, calculation blocks, etc.). The routines may be tested, documented, developed, and verified beforehand. Moreover, input signals associated with an unstable medium may include a damping facility, either in-circuit or applied as a software function, in order to eliminate, for instance, spurious operation (e.g., process variable (PV) filters). Further, all setpoints/operational parameters (e.g.. alarm limits, alarm deadband parameters, etc.) may be accessible and adjustable via the control system, and software for allowing control and adjustment of those parameters may be implemented. In examples, the process setpoints/operational parameters may be entered into the control system in the engineering units to which they are defined and may be configurable during the batch production operation cycle.
[00304] In further examples, processing interlock capability for the system may be provided based on signal processing. Interlock may be provided between agitation and temperature control, temperature control and bioreactor level, bioreactor bioprocess container pressure and gas additions via mass flow control valves (MFCVs), feed addition balance and corresponding feed addition pump (e.g., low alarm for feed weight stopping pump), and shot feed addition pumps and pH lolo or hihi alarms.
Data and Alarms
[00305] Data, alarms, and/or various events may be captured on a network, such as network 190 of Figure 1. In the event of a failure of the IT Network, the data can continue to be saved to the application station. Moreover, the operator interface system may provide read-only access to historical data stored on the drive or in the event of failure from the local drive and the reporting system may be able to detect altered and/or corrupted electronic records.
[00306] For example, an automatic, electronic audit trail may be implemented to capture all changed data, date and time and author of the change. The audit trail must not be editable and must be inextricably linked to the electronic records whose data has been changed. The audit trail can be classed as an electronic record and may be treated with the same level of security as the data.

[00307] Additionally, electronic records associated with this application may, with the appropriate security access, be capable of being copied without adversely impacting the record. Dynamic process data directly derived from the bioreactor batch may be made available to a specified location on the above-described network for offline analysis. The transferred data may be linked into discrete files (or alternative applications) created by the user and generated for each batch to view the in-line process control parameters.
[00308] In further examples, alarms may be captured and annunciated (e.g., audible and visual) locally or generally. For example, "Product Critical Alarms" can be identified at the impact assessment phase that indicates a possible impact on product quality, "Process Alarms," whose limits are defined "Alarm Limits," when detected can indicate a transgression from normal operating parameters but not impacting product quality. "System Alarm," when detected can indicate a failure of a plant item or control system component to operate to expectation. In one embodiment, only certain users may acknowledge alarms based on user security rights.
[00309] Alarms may be individually inhibited via the operator interface and such instances may be logged as events. The SUB control system may also maintain an alarm log, identifying each and every alarm event and their associated time and date. Each alarm may display a meaningful identification (e.g., tag and description).
[00310] One of the numerous advantages of the SUB control system is that the overall mechanism can be provided to so as to customize various processes that are not only run on each SUB unit but also other types of bioreactors. With respect to feeds, for example, another advantage is that there may be continuous feed set point control of flow rate, alarms and ability to automatically stop addition when the feed bioprocess container is empty and there may be the ability to dose multiple shots in series, or in parallel (simultaneously), designed to be added in way that hi/lo pH feeds have a net neutral effect on the cell culture. With respect to automation, for instance, another advantage is that there may be automation that enables 2-click operation of multiple shot feeds to be added in a controlled way that is able to prevent exceeding hi/lo pH conditions in the bioreactor and automation that enables manipulation of pCO2 levels using in-line pCO2 measurement linked to CO2 gas flow and a CO2 stripping gas (such as air or nitrogen flow). With respect to sensors, for example, a further advantage is that in-line redox measurements could be

used to determine optimum cell culture conditions that minimize risk of antibody disassociation or damage (e.g., by better understanding or preventing highly reducing or oxidizing conditions during fermentation and harvest); in-line biomass (capacitance) has been used previously at pilot scale, which is a reading that could potentially be used to automatically start or adjust nutrient feed addition rates; and other in-line measurements of interest include glucose, lactate, glutamine, glutamate, ammonia and to perform in-line measurements of these, and other parameters.
[00311] The systems, devices, facilities, and/or 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 systems, devices, facilities, and/or methods are suitable for culturing suspension cells or anchorage-dependent (adherent) cells and/or tissues 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.
[00312] In some 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 systems, devices, facilities, and/or methods can be used for producing biosimilars.
[00313] As mentioned, in embodiments, systems, devices, facilities, and/or 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), synthesised by the eukaryotic cells in a large-scale manner. Unless stated otherwise herein, the systems, devices, facilities, and/or methods can include any desired volume or production capacity including but not limited to bench-scale, pilot-scale, and full production scale capacities.
[00314] Moreover and unless stated otherwise herein, the systems, devices, facilities, and/or methods can include any suitable reactor(s) including but not limited to stirred tank, airlift, fiber, micro fiber, 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 C02 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., 316L or any other suitable stainless steel) and Inconel, plastics, and/or glass.
[00315] In embodiments and unless stated otherwise herein, the systems, devices, facilities, and/or 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.
[00316] By way of non-limiting examples and without limitation, U.S. Publication Nos. 2013/0280797; 2012/0077429; 2011/0280797; 2009/0305626; and U.S. Patent Nos. 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.
[00317] 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, PC 12, BHK (baby hamster kidney cell), VERO, SP2/0, YB2/0, Y0, CI27, L cell, COS, e.g., COS1 and COS7, QCl-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 Biologies, Inc.). Eukaryotic cells can also be avian cells, cell lines or cell strains, such as for example, EBx® cells, EB14, EB24, EB26, EB66, or EBvl3.

[00318] 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).
[00319] 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.
[00320] 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 Certified™ 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, North Carolina, USA 27709.
[00321] 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.
[00322] 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)).
[00323] In one embodiment, the eukaryotic cell is an insect cell (e.g., Sf9, Mimic™ Sf9, Sf21, High Five™ (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).
[00324] In one embodiment, the cell is a bacterial or prokaryotic cell.
[00325] 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 OH 43210-1214.
[00326] 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, XLl-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.
[00327] 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).

[00328] 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.
[00329] 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-nl, DL-8234, interferon, Suntory (gamma-la), 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, molgramostirn, 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 VI11 (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(l-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, rBetVl, Der-pl/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), CML 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-DMl, 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 (gpEl+gpE2+MF-59), otitis media therapy, HCV vaccine (core antigen+ISCOMATRIX), hPTH(l-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.
[00330] In some embodiments, the polypeptide is adalimumab (HUMIRA), infliximab (REMICADE™), rituximab (RITUXAN™/MAB THERA™) etanercept (ENBREL™), bevacizumab (AVASTIN™), trastuzumab (HERCEPTIN™), pegrilgrastim (NEULASTA™), or any other suitable polypeptide including biosimilars and biobetters.
[00331] Other suitable polypeptides are those listed below and in Table 1 of US2016/0097074:

TABLE 1 Protein Product Reference Listed Drug
interferon gamma-lb Actimmune ®
alteplase; tissue plasminogen activator Activase ®/Cathflo ®
Recombinant antihemophilic factor Advate
human albumin Albutein ®
Laronidase Aldurazyme ®
Interferon alfa-N3, human leukocyte derived Alferon N ®
human antihemophilic factor Alphanate ®
virus-filtered human coagulation factor IX AlphaNine ® SD
Alefacept; recombinant, dimeric fusion protein LFA3-Ig Amevive ®
Bivalirudin Angiomax ®
darbepoetin alfa Aranesp ™
Bevacizumab Avastin™
interferon beta-la; recombinant Avonex ®
coagulation factor IX BeneFix ™
Interferon beta-lb Betaseron ®
Tositumomab BEXXAR®
antihemophilic factor Bioclate ™
human growth hormone BioTropin ™
botulinum toxin type A BOTOX®
Alemtuzumab Campath ®
acritumomab; technetium-99 labeled CEA-Scan®
alglucerase; modified form of beta-glucocerebrosidase Ceredase ®
imiglucerase; recombinant form of beta-glucocerebrosidase Cerezyme ®
crotalidae polyvalent immune Fab, ovine CroFab ™
digoxin immune fab [ovine] DigiFab ™
Rasburicase Elitek®
Etanercept ENBREL ®
epoietin alfa Epogen ®
Cetuximab Erbitux™
algasidase beta Fabrazyme ®
Urofollitropin Fertinex ™
follitropin beta Follistim™
Teriparatide FORTEO ®

TABLE 1 Protein Product Reference Listed Drug
human somatropin GenoTropin ®
Glucagon GlucaGen ®
follitropin alfa Gonal-F ®
antihemophilic factor Helixate ®
Antihemophilic Factor; Factor XIII HEMOFIL
adefovir dipivoxil Hepsera ™
Trastuzumab Herceptin ®
Insulin Humalog ®
antihemophilic factor/von Willebrand factor complex-human Humate-P ®
Somatotropin Humatrope ®
Adalimumab HUMIRA™
human insulin Humulin ®
recombinant human hyaluronidase Hylenex™
interferon alfacon-1 Infergen ®
eptifibatide Integrilin ™
alpha-interferon Intron A ®
Palifermin Kepivance
Anakinra Kineret ™
antihemophilic factor Kogenate ® FS
insulin glargine Lantus ®
granulocyte macrophage colony-stimulating factor Leukine ©/Leukine ® Liquid
lutropin alfa for injection Luveris
OspA lipoprotein LYMErix™
Ranibizumab LUCENTIS ®
gemtuzumab ozogamicin Mylotarg ™
Galsulfase Naglazyme ™
Nesiritide Natrecor ®
Pegfilgrastim Neulasta ™
Oprelvekin Neumega ®
Filgrastim Neupogen ®
Fanolesomab NeutroSpec ™ (formerly LeuTech®)
somatropin [rDNA] Norditropin ®/Norditropin Nordiflex ®

TABLE 1 Protein Product Reference Listed Drug
Mitoxantrone Novantrone ®
insulin; zinc suspension; Novolin L ®
insulin; isophane suspension Novolin N ®
insulin, regular; Novolin R ®
Insulin Novolin ®
coagulation factor Vila NovoSeven ®
Somatropin Nutropin ®
immunoglobulin intravenous Octagam ®
PEG-L-asparaginase Oncaspar ®
abatacept, fully human soluable fusion protein Orencia ™
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™
Abarelix (injectable suspension); gonadotropin-releasing hormone Plenaxis ™
antagonist
epoietin alfa Procrit ®
Aldesleukin Proleukin, IL-2 ®
Somatrem Protropin ®
dornase alfa Pulmozyme ®
Efalizumab; selective, reversible T-cell blocker RAPTIVA™
combination of ribavirin and alpha interferon Rebetron™
Interferon beta la Rebif®
antihemophilic factor Recombinate ® rAHF/
antihemophilic factor ReFacto ®
Lepirudin Refludan ®
Infliximab REMICADE ®
Abciximab ReoPro ™
Reteplase Retavase ™
Rituxima Rituxan™
interferon alfa-2a Roferon-A ®
Somatropin Saizen ®

TABLE 1 Protein Product Reference Listed Drug
synthetic porcine secretin SecreFlo ™
Basiliximab Simulect ®
Eculizumab SOLIRIS (R)
Pegvisomant SOMAVERT ®
Palivizumab; recombinantly produced, humanized mAb Synagis ™
thyrotropin alfa Thyrogen ®
Tenecteplase TNKase ™
Natalizumab TYSABRI ®
human immune globulin intravenous 5% and 10% solutions Venoglobulin-S ®
interferon alfa-nl, lymphoblastoid Wellferon ®
drotrecogin alfa Xigris ™
Omalizumab; recombinant DNA-derived humanized monoclonal Xolair ®
antibody targeting immunoglobulin-E
Daclizumab Zenapax ®
ibritumomab tiuxetan Zevalin™
Somatotropin Zorbtive ™ (Serostim ®)
[00332] In embodiments, the polypeptide is a hormone, blood clotting/coagulation factor, cytokine/growth factor, antibody molelcule, fusion protein, protein vaccine, or peptide as shown in Table 2.
Table 2. Exemplary Products

Therapeutic Product Trade Name
Product type
Hormone Erythropoietin, Epoein-a Epogen, Procrit
Darbepoetin-a Aranesp
Growth hormone (GH), Genotropin, Humatrope, Norditropin,
somatotropin NovIVitropin, Nutropin, Omnitrope, Protropin, Siazen, Serostim, Valtropin
Human follicle-stimulating hormone Gonal-F, Follistim
(FSH) Ovidrel
Human chorionic Luveris
gonadotropin GlcaGen
Lutropin-a Geref

Glucagon ChiRhoStim (human peptide),
Growth hormone releasing SecreFlo (porcine peptide)
hormone (GHRH)
Secretin
Thyroid stimulating
hormone (TSH),
thyrotropin Thyrogen
Blood Factor VIIa Novo Seven
Clotting/Coagulation Factor VIII Bioclate, Helixate, Kogenate,
Factors Factor IX Recombinate, ReFacto
Antithrombin III (AT-III) Benefix
Protein C concentrate Thrombate III Ceprotin
Cytokine/Growth Type I alpha-interferon Infergen
factor Interferon-an3 (IFNan3) Alferon N
Interferon-βla (rIFN- β) Avonex, Rebif
Interferon-βlb(rIFN-β) Betaseron
Interferon-γlb (IFN γ) Actimmune
Aldesleukin (interleukin 2(IL2), epidermal theymocyte activating Proleukin
factor; ETAF Kepivance
Palifermin (keratinocyte growth factor; KGF) Regranex
Becaplemin (platelet-derived growth factor; PDGF)
Anakinra (recombinant IL1 antagonist) Anril, Kineret
Antibody molecules Bevacizumab (VEGFA Avastin
mAb) Erbitux
Cetuximab (EGFR mAb) Vectibix
Panitumumab (EGFR Campath
mAb) Rituxan
Alemtuzumab (CD52 Herceptin
mAb) Orencia
Rituximab (CD20 Humira
chimeric Ab) Trastuzumab (HER2/Neu Enbrel
mAb) Remicade
Abatacept (CTLA Ab/Fc Amevive
fusion) Raptiva
Adalimumab Tysabri
(TNFa mAb) Soliris
Etanercept (TNF receptor/Fc fusion) Orthoclone, OKT3

Infliximab (TNFa chimeric mAb) Alefacept (CD2 fusion protein)
Efalizumab (CD1 la mAb) Natalizumab (integrin a4 subunit mAb) Eculizumab (C5mAb) Muromonab-CD3
Other: Insulin Humulin, Novolin
Fusion Hepatitis B surface Engerix, Recombivax HB
proteins/Protein antigen (HBsAg)
vaccines/Peptides HPV vaccine Gardasil
OspA LYMErix
Anti-Rhesus(Rh) Rhophylac
immunoglobulin G Enfuvirtide Fuzeon
Spider silk, e.g., fibrion QMONOS
[00333] In embodiments, the protein is multispecific protein, e.g., a bispecific antibody as shown in Table 3.
TABLE 3: Bispecific Formats

Name (other
names,
sponsoring
organizations) BsAb format Targets Proposed
mechanisms of
action Developmen t stages Diseases(or
healthy volunteers)
Catumaxomab
(Removab®,
Fresenius
Biotech, Trion
Pharma,
Neopharm) BsIgG: Triomab CD3, EpCAM Retargeting of T cells to tumor, Fc mediated effector functions Approved in EU Malignant ascites in EpCAM positive tumors
Ertumaxomab (Neovii Biotech, Fresenius Biotech) BsIgG: Triomab CD3, HER2 Retargeting of T cells to tumor Phase I/II Advanced solid tumors
Blinatumomab (Blincyto®, AMG 103, MT 103, MEDI 538, Amgen) BiTE CD3, CD19 Retargeting of T cells to tumor Approved in USA
Phase II and III
Phase II Phase I Precursor B-cell
ALL
ALL
DLBCL
NHL

Name (other
names,
sponsoring
organizations) BsAb format Targets Proposed
mechanisms of
action Developmen t stages Diseases(or
healthy volunteers)
REGN1979 (Regeneron) BsAb CD3, CD20
Solitomab (AMG
110,MT110,
Amgen) BiTE CD3, EpCAM Retargeting of T cells to tumor Phase I Solid tumors
MEDI 565
(AMG 211,
MedImmune,
Amgen) BiTE CD3, CEA Retargeting of T cells to tumor Phase I Gastrointestinal adenocancinom a
R06958688 (Roche) BsAb CD3, CEA

BAY2010112 (AMG 212, Bayer; Amgen) BiTE CD3, PSMA Retargeting of T cells to tumor Phase I Prostate cancer
MGD006
(Macrogenics) DART CD3, CD123 Retargeting of T cells to tumor Phase I AML
MGD007
(Macrogenics) DART CD3, gpA33 Retargeting of T cells to tumor Phase I Colorectal cancer
MGD011
(Macrogenics) DART CD 19, CD3
SCORPION
(Emergent
Biosolutions,
Trubion) BsAb CD3, CD19 Retargeting of T cells to tumor
AFM11 (Affimed Therapeutics) TandAb CD3, CD19 Retargeting of T cells to tumor Phase I NHL and ALL
AFM12 (Affimed Therapeutics) TandAb CD19, CD16 Retargeting of NK cells to tumor cells
AFM13 (Affimed TandAb CD30, Retargeting of NK cells to Phase II Hodgkin's

Name (other
names,
sponsoring
organizations) BsAb format Targets Proposed
mechanisms of
action Developmen t stages Diseases(or
healthy volunteers)
Therapeutics) CD16A tumor cells Lymphoma
GD2 (Barbara Ann Karmanos Cancer Institute) T cells preloaded with BsAb CD3, GD2 Retargeting of T cells to tumor Phase I/II Neuroblastoma
and
osteosarcoma
pGD2 (Barbara Ann Karmanos Cancer Institute) T cells preloaded with BsAb CD3, Her2 Retargeting of T cells to tumor Phase II Metastatic breast cancer
EGFRBi-armed autologous activated T cells (Roger Williams Medical Center) T cells preloaded with BsAb CD3, EGFR Autologous activated T cells to EGFR-positive tumor Phase I Lung and other solid tumors
Anti-EGFR-
armed activated T-cells (Barbara Ann Karmanos Cancer Institute) T cells preloaded with BsAb CD3, EGFR Autologous activated T cells to EGFR-positive tumor Phase I Colon and pancreatic cancers
rM28 (University
Hospital
Tubingen) Tandem scFv CD28, MAPG Retargeting of T cells to tumor Phase II Metastatic melanoma
IMCgp100 (Immunocore) ImmTAC CD3,
peptide MHC Retargeting of T cells to tumor Phase I/II Metastatic melanoma
DT2219ARL
(NCI, University of Minnesota) 2 scFv linked to diphtheria toxin CD 19, CD22 Targeting of protein toxin to tumor Phase I B cell leukemia or lymphoma
XmAb5871 (Xencor) BsAb CD 19, CD32b
NI-1701 (Novlmmune) BsAb CD47, CD 19
MM-111 BsAb ErbB2,

Name (other
names,
sponsoring
organizations) BsAb format Targets Proposed
mechanisms of
action Developmen t stages Diseases(or
healthy volunteers)
(Merrimack) ErbB3

MM-141 (Merrimack) BsAb IGF-1R, 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 (MEHD7945A, Genentech, Roche) DAF EGFR, HER3 Blockade of 2
receptors,
ADCC Phase I and II Phase II Head and neck cancer Colorectal cancer
LY3164530 (Eli Lily) Not disclosed EGFR, MET Blockade of 2 receptors Phase I Advanced or
metastatic
cancer
MM-111
(Merrimack
Pharmaceuticals) HSA body HER2, HER3 Blockade of 2 receptors Phase II Phase I Gastric and esophageal cancers Breast cancer
MM-141,
(Merrimack
Pharmaceuticals) IgG-scFv IGF-1R, HER3 Blockade of 2 receptors Phase I Advanced solid tumors
RG7221
(RO5520985,
Roche) CrossMab Ang2, VEGF A Blockade of 2 proangiogenics Phase I Solid tumors

Name (other
names,
sponsoring
organizations) BsAb format Targets Proposed
mechanisms of
action Developmen t stages Diseases(or
healthy volunteers)
RG7716 (Roche) CrossMab Ang2, VEGFA Blockade of 2 proangiogenics Phase I Wet AMD
OMP-305B83 (OncoMed) BsAb DLL4/VEG F
TF2 (Immunomedics) Dock and lock CEA, HSG Pretargeting tumor for PET or radio imaging Phase II Colorectal, breast and lung cancers
ABT-981 (AbbVie) DVD-Ig IL-lα,IL-1β Blockade of 2 proinflammator y cytokines Phase II Osteoarthritis
ABT-122
(AbbVie) DVD-Ig TNF, IL-
17A Blockade of 2 proinflammator y cytokines Phase II Rheumatoid arthritis
COVA322 IgG-
fynomer TNF, IL17A Blockade of 2 proinflammator y cytokines Phase I/II Plaque psoriasis
SAR1 56597 (Sanofi) Tetravalen t bispecific tandem IgG IL-13, IL-4 Blockade of 2 proinflammator y cytokines Phase I Idiopathic
pulmonary
fibrosis
GSK2434735 (GSK) Dual-targeting domain IL-13, IL-4 Blockade of 2 proinflammator y cytokines Phase I (Healthy volunteers)
Ozoralizumab (ATM 03, Ablynx) Nanobody TNF, HSA Blockade of proinflammator y cytokine, binds to HSA to increase half-life Phase II Rheumatoid arthritis
ALX-0761 (Merck Serono, Ablynx) Nanobody IL-17A/F, HSA Blockade of 2 proinflammator y cytokines, binds to HSA to increase half-life Phase I (Healthy volunteers)

Name (other
names,
sponsoring
organizations) BsAb format Targets Proposed
mechanisms of
action Developmen t stages Diseases(or
healthy volunteers)
ALX-0061
(AbbVie, Ablynx; Nanobody IL-6R, HSA Blockade of proinflammator y cytokine, binds to HSA to increase half-life Phase I/II Rheumatoid arthritis
ALX-0141
(Ablynx,
Eddingpharm) Nanobody RANKL, HSA Blockade of bone resorption, binds to HSA to increase half-life Phase I Postmenopausal bone loss
RG6013/ACE91 0 (Chugai, Roche) ART-Ig Factor IXa, factor X Plasma coagulation Phase II Hemophilia
[00334] The enablements described in detail above are considered novel over the prior art of record and are considered critical to the operation of at least one aspect of the disclosure and to the achievement of the above described objectives. The words used in this specification to describe the instant embodiments are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this specification: structure, material or acts beyond the scope of the commonly defined meanings. Thus if an element can be understood in the context of this specification as including more than one meaning, then its use must be understood as being generic to all possible meanings supported by the specification and by the word or words describing the element.
[00335] The definitions of the words or drawing elements described herein are meant to include not only the combination of elements which are literally set forth, but all equivalent structure, material or acts for performing substantially the same function in substantially the same way to obtain substantially the same result. In this sense it is therefore contemplated that an equivalent substitution of two or more elements may be made for any one of the elements described and its various embodiments or that a single element may be substituted for two or more elements in a claim.

[00336] Changes from the claimed subject matter as viewed by a person with ordinary skill in the art, now known or later devised, are expressly contemplated as being equivalents within the scope intended and its various embodiments. Therefore, obvious substitutions now or later known to one with ordinary skill in the art are defined to be within the scope of the defined elements. This disclosure is thus meant to be understood to include what is specifically illustrated and described herein, what is conceptually equivalent, what can be obviously substituted, and also what incorporates the essential ideas.
[00337] Furthermore, the functionalities described herein may be implemented via hardware, software, firmware or any combination thereof, unless expressly indicated otherwise. If implemented in software, the functionalities may be stored as one or more instructions on a computer readable medium, including any available media accessible by a computer that can be used to store desired program code in the form of instructions, data structures or the like. Thus, certain aspects may comprise a computer program product for performing the operations presented herein, such computer program product comprising a computer readable medium having instructions stored thereon, the instructions being executable by one or more processors to perform the operations described herein. It will be appreciated that software or instructions may also be transmitted over a transmission medium as is known in the art. Further, modules and/or other appropriate means for performing the operations described herein may be utilized in implementing the functionalities described herein.
[00338] The scope of this description is to be interpreted only in conjunction with the appended claims and it is made clear, here, that the named inventor believes that the claimed subject matter is what is intended to be patented.
Example 1
1,000 L Single-Use Bioreactor
[00339] In this example, a single-use bioreactor of 1,000 L according to the present disclosure is used. A SUB is gamma irradiated (i.e. supplied sterile and ready to use) and is placed into a shell (30). The shell (30) has a jacketed temperature control capable of heating and cooling the culture in combination with an appropriate controller system and thermo circulator. The SUB shell (30) has an integrated motor (motor) for agitating the

culture. This is compatible with the controller systems of Figure 36. The single-use bioreactor has an agitator, a sparger, a gas filter inlet ports for sparger, and an exhaust gas outlet filter port with bifurcating line. It also has seven feed addition ports. Ideally, two are subsurface discharging in the impeller region and one discharging above the impeller region. It also has two medium fill ports, one harvest port designed to enable harvest the complete contents of the single-use bioreactor, one sample port, one condenser or equivalent on the gas exit line, and at least six measurement probe ports. These sample and harvest ports have animal derived component free (ADCF) C-flex tubing to enable aseptic connection for addition and removal of liquids. In addition, it has gas filters.
[00340] It is also preferable to have a fill line or lines directed such that the liquid flows down the side of the SUB to avoid splashing and foaming during the fill operation.
Example 2
Reactor Geometry
[00341] This example relates to the effect of changing reactor geometry on scale up of mammalian cell culture processes using multivariate data analysis to compare different geometries and different fill volumes. This approach uncovered a surprising result when working at half volume, which may not have been spotted using conventional data analysis methods.
[00342] Mass transfer studies were performed with two manufacturing scale SUB systems and a miniature SUB system using the gassing-out approach. A scale independent kLaCh model developed according to the geometry described in U.S. Publication No. US 2011/0312087 (referred to herein as "Lonza Geometry") was used to predict kLaO2 in both SUBs. The results have been compared to results generated using the STR geometry described in U.S. Publication No. US 2011/0312087 from 10 to 20,000 L. The vessel geometry has a substantial impact on mass transfer.
[00343] Multivariate analysis of the data showed that there were substantial differences in cell culture performance between different STR-scaled vessels. The results of this testing are presented in Figures 11-35.
[00344] As described herein, Figure 11 shows the results of a comparison of the Van't Reit model built with data from single-use bioreactors that were designed at least partially

according to the geometry described in U.S. Publication No. US 2011/0312087 at six different scales.
[00345] As described herein, Figure 12 shows the results of a comparison of the Van't Reit model built with data from single-use bioreactors that were designed at least partially according to the Lonza Geometry at six different scales as compared to a single-use bioreactor that did not incorporate the Lonza Geometry (red diamonds).
[00346] As described herein, Figure 13 shows the results of a comparison of the Van't Reit model built with data from single-use bioreactors that were designed at least partially according to the Lonza Geometry at six different scales as compared to a single-use bioreactor that did not incorporate the Lonza Geometry (red diamonds), two single-use bioreactors at two different scales built at least partially according to the Lonza Geometry when half full (blue triangles), and two single-use bioreactors at two different scales built at least partially according to the Lonza Geometry when full.
[00347] Cell culture evaluations were also performed with a model cell line in the two single-use bioreactor systems discussed above and one stainless steel/glass. The results were compared to historical data obtained in 10 L STR and 10 L airlift vessels ("ALR"). A total of fifteen measurements were taken for sixteen days in all four of the vessel geometries. The data were analyzed using the principal component analysis which projects high dimensional data sets onto lower dimension space to aid in data interpretation. Principal component analysis (PCA) and the calculation of associated statistics was performed in MATLAB Version 7.11.0.584 (The MathWorks Inc) using the PLS Tool Box Version 6.2 (Eigenvector Research, Inc.). The results are summarized in Figures 14, 15, and 16. These data show that the first four principal components captured 63% of the variance of the dataset, as shown in Figure 15. The cell cultures performed similarly in the ALRs, the STRs, and SUB1 at full volume. However, SUB2, which does not possess Lonza's geometry, performed outside the 95% confidence interval, as shown in Figure 16. Furthermore, ALRs and STRs performed similarly in principal components one, two, and three, as shown in Figure 17.
[00348] The impact of operating at half volume was investigated for one vessel design at two different vessel volumes, as shown in Figures 18-19. Here, the data show that the cultures in SUB1 at two scales, which contains at least partially Lonza's geometry,

performed similarly at full volume with the STR cultures on all principal components. However, when at half volume, that those same SUB at two scales displayed substantial differences in performance on the first three principal components indicates dissimilarity in culture properties.
[00349] Multivariate data analysis showed that there was considerable difference in behavior of the cultures performed at half volume when compared to cultures performed in the conventional scale-down model. For example, in Figures 20-21, cultures in SUB2 were performed at three scales with two bioprocess container materials.
[00350] The experiments conducted in Example 2 highlight the importance of bioreactor design, including the single-use bioreactors that are the object of the present disclosure. For example, loadings for principal component one normally track growth and/or culture progression. Loadings for a model built with STR data alone followed this norm. However, when the tests were expanded to include all four vessels designs of ALR, STR, SUB1, and SUB2, growth and/or culture progression was relegated to principal component two.
[00351] Additionally, Example 2 shows that geometric similarity is indicative of performance. The analysis indicated that there was also a difference in behavior of the half-volume cultures in different size vessels. Specifically, SUB 1 and STR cultures cluster well at full volume but not at half volume. At full volume, SUB 1 has a high degree of geometric similarity to the STR. However, at half volume, just one of these geometric parameters has been altered. Furthermore, culture performance was radically altered. Interestingly, kxaCh performance was not altered. Half-volume SUBl's performance was not consistent across scales as shown by the data where half volume cultures don't form a cluster.
[00352] Furthermore, the selection of bioprocess container material has an impact on SUB 2 culture performance. This is additionally supported by Figures 22-35 where the principal components were assessed over time for the various fills, volumes, and bioprocess container materials.
[00353] This indicated a lack of scalability between half-volume cultures performed in different scale vessels, which was not apparent when the same vessels were run at full volume.

[00354] Single-use bioreactor geometry does matter when scaling processes up and should be a key consideration in a quality by design approach to minimizing differences in culture behavior during cell culture process scale up. Moreover, multivariate data analysis can provide useful supplemental insight in bioreactor process performance comparisons.
Example 3
A 1,000 L Bioreactor Set Up
[00355] The single-use bioreactors of the present disclosure are suitable for use in the production processes described in WO 2017/072201 A2, which is incorporated by reference in its entirety herein.
[00356] The bioprocess container shell was a jacketed stainless steel container, which supported the SUB container. The shell incorporated two doors that open outwards for operators to fit the SUB bioprocess container. These were fastened shut by clamps. The shell incorporated a water jacket at the bottom for regulation of temperature. This was connected to the controller of the present disclosure.
[00357] At the bottom of the shell there was a drain port for harvesting and two openings for control probes and sampling. For non-disposable probes the shell had shelving set at 15 degrees from horizontal to support the probes.
[00358] At the top of the bioprocess container holder there was a motor to which the SUB container impeller was connected via a magnetic coupling. The motor attached to the 200 liter shell could be moved, but in the motor attached to the 1000 liter shell was fixed. There was a gas filter holder, pressure sensor and manual pressure relief valve situated on the arm of the motor.
[00359] The SUB bioprocess container incorporated a pressure release valve which actuated if pressure exceeded 100 mbar. Both the pressure transmitter and the relief valve were connected to the SUB container via a 0.22 um filter.
[00360] The controller of the present disclosure contained: two Watson Marlow pumps one for acid and one for base control, rotameters for control of gas flow, a human machine interface (HMI), a thermocirculator and gas mass flow controllers (MFCs) built into the tower. The pH probes, dissolved oxygen tension (DOT) probes, temperature probes,

pressure sensor and vent heater were external to but connected to a controller of the present disclosure.
[00361] Set points were entered into the HMI screen for all control parameters. The controller used these values to regulate culture temperature, gas flow rates and pump speed. The HMI also displayed current values of all measured parameters.
[00362] Temperature measurement was performed using a ptlOO probe inserted into a pocket in the SUB container.
[00363] Inside the SUB container there was: (i) an agitator shaft with a choice of two impeller designs (see Figure 1A and Figure 1B); (ii) disposable optical pH and DOT probes; (iii) a combination sparger (option of micro (0.15 mm) macro (0.8 mm) holes; and (iv) surface and subsurface feed lines.
[00364] On the outside of the SUB bioprocess container there were C flex lines for inoculum, medium and feed additions and OPTA connections for gas filters and feed additions. At the bottom of the SUB bioprocess container there were four connections for non-disposable probes, a sample line, and an insert for a pt100 probe. The harvest line was at the bottom of the SUB bioprocess container.
[00365] Hydrophobic 0.22 urn gas filters came autoclaved separately and were connected to the SUB bioprocess container using OPTA connections. Each SUB bioprocess container had connections for two gas outlet filters, one pressure sensor filter, one filter for headspace aeration and filter each for micro and macro spargers.
[00366] The pressure filter was connected to the pressure sensor and the gas inlet and outlet filters were open before inflation was started.
[00367] The sparger and head space gas filters were connected to the controller of the present disclosure using silicon tubing, which in turn was connected to the main gas supplies via nylon tubing. The main gas supply pressures were set to 1.8 barg for all gases. The MFCs had a turn down ratio of 1:50 and range of up to 100 L/min. As a result an additional calibrated rotatmeter was required supply of the CO2 ballast because this flow rate was too low to control with the MFC.
[00368] For safety reasons it was important to ensure that gas outlet line and pressure sensor line were not kinked during inflation.

[00369] Inflation of the SUB bioprocess container was started slowly with a low gas flow rate. A scientist had to hold the SUB bioprocess container in place until the agitator shaft 8 and motor were magnetically coupled. To prevent damage to the SUB bioprocess container it had to be inflated such that no components inside the bioprocess container (agitator blades or dip tubes) touched the bioprocess container. Inflation had to be stopped once it was possible to couple the agitator and the motor.
[00370] The agitator magnetic coupling was then slowly lifted up to the motor. Once in place the SUB bioprocess container was rotated slowly into position to align the probe ports with the probe holders and to align the seal of the SUB bioprocess container with the middle of where the two doors met. When in final position the agitator shaft 8 was secured in place to the motor using a tri clamp. The filters were fitted into position on the filter holder. A vent heater was placed around the gas outlet filter. The SUB bioprocess container was then fully inflated. A continuous air flow (at the air cap described in the pilot fermentation process description (FPD)) was maintained through the sparger and headspace in order to keep the SUB bioprocess container inflated.
[00371] One standard pH and one standard DOT probe were calibrated prior to starting each batch using the standard calibration procedure used the Slough pilot plant. These probes were fitted into the probe sleeves with connections and autoclaved on a fluid cycle. The probes were fitted into the SUB bioprocess container using the connections and placed onto the probe holder shelf set at a 15° degree angle to the horizontal.
[00372] Once pH and DOT probes were fitted the medium or buffer as appropriate was filtered into the SUB bioprocess container using a pre irradiated 0.1 urn filter welded on onto the dip tube. The Bioprocess container holders tested did not have a load cell, so a floor balance was used to weigh in the medium / buffer. During medium fill / buffer fill a constant air flow (at the air cap described in the pilot FPD) was maintained to avoid liquid going into the gas inlet line.
[00373] Once the required volume was achieved the jacket was filled with DI water and temperature and agitation control was initiated. Following medium fill pH control was initiated based on the reusable probe using C02 to prevent the pH from drifting outside the acceptable range for medium hold. The disposable pH and DOT probes were then

activated. The pH and DOT probes were left to equilibrate overnight in the medium or buffer.
[00374] Sample bioprocess containers were welded onto the sample line situated next to the disposable pH and DOT probes in order to ensure the sample was representative of the environment experience by the probes. Samples were removed the day after the vessel was filled and analyzed for pH and pCO2. The results from these measurements were used to perform single point calibrations on the DOT and pH probes.
[00375] For inoculation an S200 cell bioprocess container was connected to the SUB bioprocess container using sterile c flex tubing attached to the dip tube line. The required volume of inoculum was pumped to the SUB bioprocess container using a calibrated Watson Marlow 600 series pump.
[00376] The feeds, alkali and antifoam were all welded onto the SUB bioprocess container using c flex tubing, each had dedicated lines. Alkali addition was via the Watson Marlow 100 series alkali pump built into the control tower. Alkali was added as required to control the pH. Antifoam was added manually using the second Watson Marlow 100 series pump built into the control tower.
[00377] Feeds were added using Watson Marlow 500 series pumps. Flow rates and addition volumes were determined using appropriately sized balances correcting for the density of the feeds. The flow rate of the continuous feeds SF70 and 400 g/L D glucose were adjusted on a daily basis according to the viable cell concentration (VCC) and glucose concentration of the culture. Shot feeds SF71, SF72 and SF73 were added according to the FPD.
[00378] Each day samples were taken as part of daily monitoring of the bioreactors to check cell concentrations, viabilities, metabolites and dissolved gases using sample bioprocess containers attached to the sample line.
[00379] One point adjustments for online pH probes were performed when necessary according to UKSL 182 using results from a calibrated offline pH probe (Mettle Toledo offline 405 DPAS SC K8S/120 with pHM220 meter).
EXAMPLE 4
Use of a Single Use Bioreactor in a Production System

[00380] In another example, this single-use bioreactor can also be used in the systems and methods disclosed in WO 2017/072201 A2, the entirety of which is incorporated by reference.
[00381] In WO 2017/072201 A2, bioreactors are used during both the inoculum expansion and production process steps. The single-use bioreactors of the present disclosure provide advantages to this system because they can be made ready for different runs more quickly and efficiently, thereby reducing bioreactor "down time" needed for cleaning and sterilizing.
[00382] This will allow the systems of WO 2017/072201 A2 to produce high quality, safe, and cost effective active pharmaceutical ingredients (APIs) and biopharmaceutical products in a more timely and cost-effective manner. For instance, there would be greater flexibility in vessel architecture and components used when designing processes to manufacture proteins and cells, significantly reduced operating costs (e.g., labor, utility, and maintenance), improved facility throughput as batch turnaround times are condensed, clean in place and steam in place operations.
[00383] As part of the process disclosed in WO 2017/072201 A2, there are purification steps. During the purification processes, numerous resins can be used during purification, including but not limited to, MabSelect SuRe / MabSelect SuRe LX / MabSelect SuRe pcc (GE Healthcare), Amsphere A and Amsphere A3 (JSR micro), Praesto AP and Praesto AC (Purolite), KanCapA (Pall), Toyopearl AF-rProtein A HC (Tosoh), Poros MabCapture A (Thermo-Fisher), and the like. Other purification material would be known to a person of ordinary skill in the art and this is by no means an exhaustive list.
[00384] It should be recognized that the one or more examples in the disclosure are non-limiting examples and that the present disclosure is intended to encompass variations and equivalents of these examples.
CLAIMS
1. A bioreactor comprising:
a bioprocess container made from a liquid impermeable and flexible shape-conforming material, the bioprocess container having a top, a bottom, and at least one side wall therebetween, the bioprocess chamber defining a hollow enclosure for receiving a culture media; and
a mixing device comprising a rotatable shaft coupled to at least one top impeller and at least one bottom impeller that extend into the hollow enclosure of the bioprocess container, the top impeller and the bottom impeller both being made from a polymer material.
2. A bioreactor as defined in claim 1, wherein the top impeller and the bottom impeller include a hydrophilic surface.
3. A bioreactor as defined in claim 1 or 2, wherein the polymer material comprises a polyolefin, such as polyethylene.
4. A bioreactor as defined in any of the preceding claims, wherein the polymer material comprises a low density polyethylene that has been modified to form a hydrophilic surface.
5. A bioreactor as defined in claim 4, wherein the low density polyethylene has been modified by being subjected to irradiation, photo or plasma induction, or oxidation.
6. A bioreactor as defined in any of the preceding claims, wherein the top impeller is a hydrofoil impeller.
7. A bioreactor as defined in any of the preceding claims, wherein the impeller to tank diameter ratio is from about 0.35 to about 0.55, such as from about 0.44 to about 0.46.
8. A bioreactor as defined in any of the preceding claims, wherein the top impeller flow number (Nq) is from about 0.4 to about 0.9.
9. A bioreactor as defined in any of the preceding claims, wherein the bottom impeller flow number (Nq) is from about 0.4 to about 0.9.
10. A bioreactor as defined in claim 1, further comprising at least one baffle being configured to extend adjacent to the side wall of the bioprocess container in a longitudinal direction, the baffle having a shape that extends radially inward from the side

wall an amount sufficient to affect fluid flow in the hollow enclosure during mixing of a culture media by the mixing device.
11. A bioreactor as defined in claim 10, wherein the baffle defines an inflatable fluid bladder, the baffle being capable of being inflated and deflated.
12. A bioreactor as defined in claim 10, wherein the baffle is integral with the bioprocess container.
13. A bioreactor as defined in claim 10, wherein the baffle is configured to be placed outside the hollow enclosure and wherein the side wall of the bioprocess container conforms around the shape of the baffle.
14. A bioreactor as defined in claim 10, wherein the bioreactor includes from about two to about six baffles, the baffles being spaced around a circumference of the hollow enclosure of the bioprocess container.
15. A bioreactor as defined in claim 10, wherein the bioprocess container has a diameter and wherein the baffle extends radially inward a distance of from about 3% to about 20%, such as from about 5% to about 15% of the diameter of the bioprocess container.
16. A bioreactor as defined in claim 10, wherein the baffle is made from a flexible polymer film.
17. A bioreactor as defined in claim 1, wherein the bioreactor further comprises at least one sparger.
18. A bioreactor as defined in claim 17, wherein the sparger comprises a ballast sparger, the ballast sparger comprising a gas tube that has a longitudinal portion and a lateral portion, the longitudinal portion extending vertically into the hollow enclosure of the bioprocess container, the lateral portion being located at an end of the longitudinal portion below the agitator, the lateral portion defining a plurality of holes for releasing a gas into a culture media contained within the bioprocess container.
19. A bioreactor as defined in claim 1, wherein the flexible film comprises a multi-layered film, the multi-layered film including an interior surface facing the hollow enclosure and an opposite exterior surface, the interior surface comprising a low density polyethylene that has been modified to form a hydrophilic surface.

20. A bioreactor as defined in claim 19, wherein the low density polyethylene has been modified by being subjected to irradiation, photo or plasma induction, or oxidation.
21. A bioreactor as defined in claim 1, further comprising at least one feed line that extends into the hollow enclosure for feeding fluids into the bioprocess container, the feed line including a subsurface fluid outlet positioned adjacent the agitator, the fluid outlet being associated with a fluid control device that only permits fluid to flow out of the fluid outlet and prevents fluid flow in an opposite direction.
22. A bioreactor as defined in claim 1, further comprising at least one feed line positioned at the top of the bioprocess container, the feed line including a supersurface fluid discharge positioned above a volume of culture media residing in the bioprocess container, the supersurface fluid discharge being located such that a fluid flowing through the fluid discharge makes direct contact with a culture media contained within the bioprocess container.
23. A bioreactor as defined in claim 22, wherein the top impeller forms a circumference when rotated and wherein the supersurface fluid discharge of the feed line is positioned above the circumference of the top impeller such that fluids flowing through the fluid discharge contact a culture media within the circumference.
24. A bioreactor as defined in claim 1, wherein the bioprocess container is in fluid communication with a drain line located at the bottom of the bioprocess container, and wherein a fluid collecting device is positioned inbetween the hollow enclosure of the bioprocess container and the drain line, the fluid collecting device having a shape configured to induce a vortex flow of fluids from the bioprocess container into the drain line.
25. A bioreactor as defined in claim 24, wherein the hollow enclosure of the bioprocess container has a volume and wherein the drain line has a cross-sectional area and wherein the cross-sectional area of the drain line is proportional to the volume of the hollow enclosure, the drain line having a cross-sectional area of from about 0.3 mm2 to about 0.7 mm2 per liter of volume of the hollow enclosure.
26. A bioreactor comprising:
a bioprocess container made from a liquid impermeable and flexible shape-conforming material, the bioprocess container having a top, a bottom, and at least one side

wall therebetween, the bioprocess chamber defining a hollow enclosure for receiving a culture media;
at least one inlet port for feeding materials into the hollow enclosure of the bioprocess container;
a mixing device comprising a rotatable shaft coupled to at least one agitator, the shaft and agitator extending into the hollow enclosure of the bioprocess container; and
at least one baffle being configured to extend adjacent to the side wall of the bioprocess container in a longitudinal direction, the baffle having a shape that extends radially inward from the side wall an amount sufficient to affect fluid flow in the hollow enclosure during mixing of a culture media by the mixing device.
27. A bioreactor as defined in claim 26, wherein the baffle defines an inflatable fluid bladder, the baffle being capable of being inflated and deflated.
28. A bioreactor as defined in claim 26 or 27, wherein the baffle is integral with the bioprocess container.
29. A bioreactor as defined in any of claims 26-28, wherein the baffle is configured to be placed outside the hollow enclosure and wherein the side wall of the bioprocess container conforms around the shape of the baffle.
30. A bioreactor as defined in any of claims 26-29, wherein the baffle is configured to be placed inside the hollow enclosure of the bioprocess container.
31. A bioreactor as defined in any of claims 26-30, wherein the bioreactor includes from about two to about six baffles, the baffles being spaced around a circumference of the hollow enclosure of the bioprocess container.
32. A bioreactor as defined in any of any of claims 26-31, wherein the bioprocess container has a diameter and wherein the baffle extends radially inward a distance of from about 3% to about 20%, such as from about 5% to about 15% of the diameter of the bioprocess container.
33. A bioreactor as defined in any of any of claims 26-32, wherein the baffle is made from a flexible polymer film.
34. A bioreactor as defined in any of any of claims 26-33, wherein the bioreactor further comprises at least one sparger.
35. A bioreactor as defined in claim 34, wherein the sparger comprises a ballast sparger, the ballast sparger comprising a gas tube that has a longitudinal portion and a

lateral portion, the longitudinal portion extending vertically into the hollow enclosure of the bioprocess container, the lateral portion being located at an end of the longitudinal portion below the agitator, the lateral portion defining a plurality of holes for releasing a gas into a culture media contained within the bioprocess container.
36. A bioreactor as defined in claim 35, wherein the bioreactor contains a second sparger.
37. A bioreactor as defined in any of claims 26-36, wherein the agitator comprises an impeller.
38. A bioreactor as defined in claim 37, wherein the bioreactor includes a first impeller and a second impeller, the impellers being spaced along the rotatable shaft.
39. A bioreactor as defined in any of claims 26-38, wherein the shape-conforming material of the bioprocess container comprises a flexible film.
40. A bioreactor as defined in claim 39, wherein the flexible film comprises a multi-layered film, the multi-layered film including an interior surface facing the hollow enclosure and an opposite exterior surface, the interior surface comprising a low density polyethylene that has been modified to form a hydrophilic surface.
41. A bioreactor as defined in claim 40, wherein the low density polyethylene has been modified by being subjected to irradiation, photo or plasma induction, or oxidation.
42. A bioreactor as defined in any of claims 26-41, further comprising at least one feed line that extends into the hollow enclosure for feeding fluids into the bioprocess container, the feed line including a subsurface fluid outlet positioned adjacent the agitator, the fluid outlet being associated with a fluid control device that only permits fluid to flow out of the fluid outlet and prevents fluid flow in an opposite direction.
43. A bioreactor as defined in claim 42, wherein the fluid control device comprises a one-way valve.
44. A bioreactor as defined in any of claims 26-43, further comprising at least one feed line positioned at the top of the bioprocess container, the feed line including a supersurface fluid discharge positioned above a volume of culture media residing in the bioprocess container, the supersurface fluid discharge being located such that a fluid flowing through the fluid discharge makes direct contact with a culture media contained within the bioprocess container.

45. A bioreactor as defined in claim 44, wherein the agitator forms a circumference when rotated and wherein the supersurface fluid discharge of the feed line is positioned above the circumference of the agitator such that fluids flowing through the fluid discharge contact the culture media within the circumference.
46. A bioreactor as defined in any of claims 26-45, wherein the bottom of the bioprocess container has a dome-shape.
47. A bioreactor as defined in any of claims 26-46, further comprising a load cell in operative association with the bioprocess container for indicating a mass of a culture media contained within the hollow enclosure.
48. A bioreactor as defined in any of claims 26-47, wherein the bioprocess container includes a plurality of ports for connecting to a plurality of supply lines for feeding fluids to the bioprocess container and wherein each port and corresponding supply line include matching indicators for assisting a user in connecting the supply lines to the respective ports.
49. A bioreactor as defined in claim 48, wherein the matching indicators comprise color such that each port and corresponding supply line are color coded.
50. A bioreactor as defined in claim 48 or 49, wherein the ports comprise universal connectors, the ports having a first end and a second end, the first end for forming a reconnectable attachment to a respective supply line.
51. A bioreactor as defined in any of claims 26-50, wherein the bioprocess container is in fluid communication with a drain line located at the bottom of the bioprocess container, and wherein a fluid collecting device is positioned inbetween the hollow enclosure of the bioprocess container and the drain line, the fluid collecting device having a shape configured to induce a vortex flow of fluids from the bioprocess container into the drain line.
52. A bioreactor as defined in claim 51, wherein the hollow enclosure of the bioprocess container has a volume and wherein the drain line has a cross-sectional area and wherein the cross-sectional area of the drain line is proportional to the volume of the hollow enclosure, the drain line having a cross-sectional area of from about 0.3 mm2 to about 0.7 mm2 per liter of volume of the hollow enclosure.
53. A bioreactor as defined in claim 48, 49 or 50, wherein each of the supply lines includes a fluid filter positioned upstream from the corresponding ports.

54. A bioreactor as defined in any of the preceding claims, wherein the bioprocess container includes a middle portion, the middle portion having an aspect ratio of from about 0.8 to about 1.5, such as from about 1 to about 1.2.
55. A bioreactor as defined in claim 36, wherein the ballast sparger defines a first plurality of holes for releasing a gas into a culture media and the second sparger defines a second plurality of holes for releasing a gas into the culture media, the second plurality of holes having a smaller diameter than the first plurality of holes.
56. A bioreactor as defined in any of claims 26-55, wherein the rotatable shaft comprises at least one impeller made from a hydrophilic polymer material, the at least one impeller being collapsible.
57. A bioreactor system comprising:
a bioprocess container made from a liquid impermeable and flexible shape-conforming material, the bioprocess container having a top, a bottom, and at least one side wall therebetween, the bioprocess chamber defining a hollow enclosure for receiving a culture media;
a plurality of inlet ports for feeding materials into the hollow enclosure of the bioprocess container;
a drain line positioned at the bottom of the bioprocess container for draining fluids from the bioprocess container;
a mixing device comprising a rotatable shaft coupled to at least one agitator, the shaft and agitator extending into the hollow enclosure of the bioprocess container;
at least one sensor in operative association with the bioprocess container for monitoring at least one parameter within the hollow enclosure, the at least one sensor comprising a pH sensor, a dissolved carbon dioxide sensor, a dissolved oxygen sensor, or a load cell; and
a controller in communication with the at least one sensor, the controller being configured to receive information from the at least one sensor and, based on the information, to control a fluid supply for varying a flow rate of a fluid from the fluid supply into the hollow enclosure of the bioprocess container for maintaining the at least one parameter of a culture media contained within the hollow enclosure within preset limits.
58. A bioreactor system as defined in claim 57, further comprising a carbon
dioxide gas supply in fluid communication with the bioprocess container and a liquid alkali

supply also in fluid communication with the bioprocess container, the at least one sensor comprising a pH sensor and wherein the controller is configured to regulate pH levels of a culture media within the preset limits by adding amounts of carbon dioxide gas from the carbon dioxide gas supply for selectively lowering the pH or by adding amounts of an alkali from the liquid alkali supply for selectively increasing the pH.
59. A bioreactor system as defined in claim 58, wherein the system includes a first pH sensor and a second pH sensor, each pH sensor being in communication with the controller.
60. A bioreactor system as defined in claim 57, further comprising an oxygen gas supply and wherein the at least one sensor comprises a dissolved oxygen sensor and wherein the controller regulates dissolved oxygen levels within a culture media within present limits by periodically adding amounts of oxygen gas from the oxygen gas supply to a culture media within the hollow enclosure of the bioprocess container based on information received from the dissolved oxygen sensor.
61. A bioreactor system as defined in claim 57, further comprising a carbon
dioxide gas supply and wherein the at least one sensor comprises a dissolved carbon
dioxide sensor and wherein the controller regulates dissolved carbon dioxide levels within
a culture media within present limits by periodically adding amounts of carbon dioxide gas
from the carbon dioxide gas supply to a culture media within the hollow enclosure of the
bioprocess container based on information received from the dissolved carbon dioxide
sensor.
62. A bioreactor system as defined in any of claims 57-61, further
comprising a thermal jacket surrounding the bioprocess container, the thermal jacket being
in fluid communication with at least one of a heated fluid or a chilled fluid, the bioreactor
system further comprising a temperature sensor for sensing a temperature of a culture
media contained within the bioprocess container, the temperature sensor being in
communication with the controller, and wherein the controller is configured to receive
information from the temperature sensor and, based on the information, control flow of a
fluid into the thermal jacket for increasing or decreasing the temperature of a culture media
contained in the bioprocess container for maintaining a culture media within preset
temperature limits.

63. A bioreactor system as defined in any of claims 58-62, further comprising a tachometer for monitoring a rotational speed of the rotatable shaft coupled to the at least one agitator, the tachometer being in communication with the controller, the controller being in communication with a motor that rotates the shaft, the controller being configured to control the motor in a manner that rotates the shaft at a predetermined speed based on information received from the tachometer.
64. A bioreactor system as defined in any of claims 58-63, wherein the controller comprises one or more microprocessors.
65. A bioreactor system as defined in any of claims 58-64, wherein the system includes a pH sensor and a dissolved oxygen sensor that are both in communication with the controller and wherein the controller receives information from the pH sensor and the dissolved oxygen sensor and controls a flow of different fluids into the bioprocess container for maintaining pH levels and dissolved oxygen levels of a culture media contained within the bioprocess container within preset limits.
66. A bioreactor comprising:
a bioprocess container made from a liquid impermeable and flexible shape-conforming material, the bioprocess container having a top, a bottom, and at least one side wall therebetween, the bioprocess chamber defining a hollow enclosure for receiving a culture media;
a mixing device comprising a rotatable shaft coupled to at least one agitator, the shaft and agitator extending into the hollow enclosure of the bioprocess container; and
at least one feed line that extends into the hollow enclosure for feeding fluids into the bioprocess container, the feed line including a subsurface fluid outlet positioned adjacent the agitator, the fluid outlet being associated with a fluid control device that only permits fluid to flow out of the fluid outlet and prevents fluid flow in an opposite direction.
67. A bioreactor as defined in claim 66, further comprising at least one baffle being configured to extend adjacent to the side wall of the bioprocess container in a longitudinal direction, the baffle having a shape that extends radially inward from the side wall an amount sufficient to affect fluid flow in the hollow enclosure during mixing of a culture media by the mixing device.
68. A bioreactor as defined in claim 67, wherein the baffle defines an inflatable fluid bladder, the baffle being capable of being inflated and deflated.

69. A bioreactor as defined in claim 67, wherein the baffle is configured to be placed outside the hollow enclosure and wherein the side wall of the bioprocess container conforms around the shape of the baffle.
70. A bioreactor as defined in claim 67, 68 or 69, wherein the bioreactor includes from about two to about six baffles, the baffles being spaced around a circumference of the hollow enclosure of the bioprocess container.
71. A bioreactor as defined in claim 66, wherein the bioreactor further comprises at least one sparger.
72. A bioreactor as defined in claim 71, wherein the sparger comprises a ballast sparger, the ballast sparger comprising a gas tube that has a longitudinal portion and a lateral portion, the longitudinal portion extending vertically into the hollow enclosure of the bioprocess container, the lateral portion being located at an end of the longitudinal portion below the agitator, the lateral portion defining a plurality of holes for releasing a gas into a culture media contained within the bioprocess container.
73. A bioreactor as defined in claim 66, wherein the shape-conforming material of the bioprocess container comprises a flexible film.
74. A bioreactor as defined in claim 73, wherein the flexible film comprises a multi-layered film, the multi-layered film including an interior surface facing the hollow enclosure and an opposite exterior surface, the interior surface comprising a low density polyethylene that has been modified to form a hydrophilic surface.
75. A bioreactor as defined in claim 74, wherein the low density polyethylene has been modified by being subjected to irradiation, photo or plasma induction, or oxidation.
76. A bioreactor as defined in claim 66, wherein the fluid control device comprises a one-way valve.
77. A bioreactor as defined in claim 66, further comprising a second feed line positioned at the top of the bioprocess container, the second feed line including a supersurface fluid discharge positioned above a volume of culture media residing in the bioprocess container, the supersurface fluid discharge being located such that a fluid flowing through the fluid discharge makes direct contact with a culture media contained within the bioprocess container.

78. A bioreactor as defined in claim 77, wherein the agitator forms a circumference when rotated and wherein the supersurface fluid discharge of the feed line is positioned above the circumference of the agitator such that fluids flowing through the fluid discharge contact the culture media within the circumference.
79. A bioreactor as defined in claim 66, wherein the bioprocess container includes a plurality of ports for connecting to a plurality of supply lines for feeding fluids to the bioprocess container and wherein each port and corresponding supply line include matching indicators for assisting a user in connecting the supply lines to the respective ports.
80. A bioreactor as defined in claim 79, wherein the ports comprise universal connectors, the ports having a first end and a second end, the first end for forming a reconnectable attachment to a respective supply line.
81. A bioreactor as defined in claim 66, wherein the bioprocess container is in fluid communication with a drain line located at the bottom of the bioprocess container, and wherein a fluid collecting device is positioned inbetween the hollow enclosure of the bioprocess container and the drain line, the fluid collecting device having a shape configured to induce a vortex flow of fluids from the bioprocess container into the drain line.
82. A bioreactor as defined in claim 66, wherein the hollow enclosure of the bioprocess container has a volume and wherein the drain line has a cross-sectional area and wherein the cross-sectional area of the drain line is proportional to the volume of the hollow enclosure, the drain line having a cross-sectional area of from about 0.3 mm2 to about 0.7 mm2 per liter of volume of the hollow enclosure.
83. A bioreactor as defined in claim 66, wherein the agitator comprises at least one impeller made from a hydrophilic polymer material, the at least one impeller being collapsible.
84. A bioreactor comprising:
a bioprocess container made from a liquid impermeable and flexible shape-conforming material, the bioprocess container having a top, a bottom, and at least one side wall therebetween, the bioprocess chamber defining a hollow enclosure for receiving a culture media;

a mixing device comprising a rotatable shaft coupled to at least one agitator, the shaft and agitator extending into the hollow enclosure of the bioprocess container; and
at least one feed line positioned at the top of the bioprocess container, the feed line including a supersurface fluid discharge positioned above a volume of culture media residing in the bioprocess container, the supersurface fluid discharge being located such that a fluid flowing through the fluid discharge makes direct contact with a culture media contained within the bioprocess container without contacting the side wall.
85. A bioreactor as defined in claim 84, further comprising at least one baffle being configured to extend adjacent to the side wall of the bioprocess container in a longitudinal direction, the baffle having a shape that extends radially inward from the side wall an amount sufficient to affect fluid flow in the hollow enclosure during mixing of a culture media by the mixing device.
86. A bioreactor as defined in claim 85, wherein the baffle defines an inflatable fluid bladder, the baffle being capable of being inflated and deflated.
87. A bioreactor as defined in claim 85, wherein the baffle is configured to be placed outside the hollow enclosure and wherein the side wall of the bioprocess container conforms around the shape of the baffle.
88. A bioreactor as defined in claim 85, 86, or 87, wherein the bioreactor includes from about two to about six baffles, the baffles being spaced around a circumference of the hollow enclosure of the bioprocess container.
89. A bioreactor as defined in claim 84, wherein the bioreactor further comprises at least one sparger.
90. A bioreactor as defined in claim 89, wherein the sparger comprises a ballast sparger, the ballast sparger comprising a gas tube that has a longitudinal portion and a lateral portion, the longitudinal portion extending vertically into the hollow enclosure of the bioprocess container, the lateral portion being located at an end of the longitudinal portion below the agitator, the lateral portion defining a plurality of holes for releasing a gas into a culture media contained within the bioprocess container.
91. A bioreactor as defined in claim 84, wherein the shape-conforming material of the bioprocess container comprises a flexible film.
92. A bioreactor as defined in claim 91, wherein the flexible film comprises a multi-layered film, the multi-layered film including an interior surface facing the hollow

enclosure and an opposite exterior surface, the interior surface comprising a low density polyethylene that has been modified to form a hydrophilic surface.
93. A bioreactor as defined in claim 92, wherein the low density polyethylene has been modified by being subjected to irradiation, photo or plasma induction, or oxidation.
94. A bioreactor as defined in claim 84, further comprising a second feed line positioned at the top of the bioprocess container, the second feed line including a subsurface fluid outlet, the fluid outlet being associated with a fluid control device that only permits fluid to flow out of the fluid outlet.
95. A bioreactor as defined in claim 94, wherein the fluid control device comprises a one-way valve.
96. A bioreactor as defined in claim 84, wherein the agitator forms a circumference when rotated and wherein the supersurface fluid discharge of the feed line is positioned above the circumference of the agitator such that fluids flowing through the fluid discharge contact the culture media within the circumference.
97. A bioreactor as defined in claim 84, wherein the bioprocess container includes a plurality of ports for connecting to a plurality of supply lines for feeding fluids to the bioprocess container and wherein each port and corresponding supply line include matching indicators for assisting a user in connecting the supply lines to the respective ports.
98. A bioreactor as defined in claim 97, wherein the ports comprise universal connectors, the ports having a first end and a second end, the first end for forming a reconnectable attachment to a respective supply line.
99. A bioreactor as defined in claim 84, wherein the bioprocess container is in fluid communication with a drain line located at the bottom of the bioprocess container, and wherein a fluid collecting device is positioned inbetween the hollow enclosure of the bioprocess container and the drain line, the fluid collecting device having a shape configured to induce a vortex flow of fluids from the bioprocess container into the drain line.
100. A bioreactor as defined in claim 99, wherein the hollow enclosure of the bioprocess container has a volume and wherein the drain line has a cross-sectional area and wherein the cross-sectional area of the drain line is proportional to the volume of the

hollow enclosure, the drain line having a cross-sectional area of from about 0.3 mm2 to about 0.7 mm2 per liter of volume of the hollow enclosure.
101. A bioreactor as defined in claim 84, wherein the agitator comprises at least
one impeller made from a hydrophilic polymer material, the at least one impeller being
collapsible.
102. A method for producing a single use bioreactor comprising:
constructing a bioprocess container from a liquid impermeable and flexible shape-
conforming material, the bioprocess container having a top, a bottom, and at least one side
wall therebetween, the bioprocess chamber defining a hollow enclosure for receiving a
culture media, the hollow enclosure having a volume of from about 10 liters to about
20,000 liters, the bioprocess container including a plurality of inlet ports for feeding
materials into the hollow enclosure of the bioprocess container, each inlet port having a
diameter;
inserting a mixing device into the hollow enclosure of the bioprocess container, the mixing device comprising a rotatable shaft coupled to at least one agitator;
inserting at least one sparger into the hollow enclosure of the bioprocess container, the sparger comprising a gas tube that has a longitudinal portion and a lateral portion, the longitudinal portion extending vertically into the hollow enclosure, the lateral portion being located at an end of the longitudinal portion below the agitator, the lateral portion defining a plurality of holes for releasing a gas into a culture media contained within the bioprocess container, the plurality of holes having a diameter;
connecting a drain line to the bottom of the bioprocess container, the drain line having a cross-sectional area; and
wherein the diameter of the inlet ports, the diameter of the plurality of holes on the sparger, and the cross-sectional area of the drain line are proportional to the volume of the hollow enclosure, and wherein the drain line has a cross-sectional area of from about 0.3 mm2 to about 0.7 mm2 per liter of volume of the hollow enclosure.
103. A bioreactor comprising:
a bioprocess container made from a liquid impermeable and flexible shape-conforming material, the bioprocess container having a top, a bottom, and at least one side wall therebetween, the bioprocess chamber defining a hollow enclosure for receiving a culture media;

at least one inlet port for feeding materials into the hollow enclosure of the bioprocess container;
a mixing device comprising a rotatable shaft coupled to a plurality of agitators, the shaft and agitators extending into the hollow enclosure of the bioprocess container;
a cell retention chamber in fluid communication with the hollow enclosure of the bioprocess container;
a filtrate outlet in fluid communication with the cell retention chamber, the filtrate outlet including a biofilter that is permeable to liquids but impermeable to biological materials contained in a culture media, the filtrate outlet for removing liquids from the cell retention chamber; and
a flow regulator that is configured to alternate flow of a culture media between the hollow enclosure of the bioprocess container and the cell retention chamber.
104. A bioreactor as defined in claim 103, wherein the flow regulator is in communication with a pressurized gas source and a vacuum source, the flow regulator being configured to alternatively apply a vacuum or a gas pressure to a fluid contained in the cell retention chamber for recycling fluids back and forth between the hollow enclosure of the bioprocess container and the cell retention chamber.
105. A bioreactor as defined in claim 104, wherein the flow regulator includes a reciprocating diaphragm that alternates between applying pressure and a suction force to the fluid contained in the cell retention chamber.
106. A bioreactor as defined in any of claims 103-105, further comprising at least one baffle configured to extend adjacent to the side wall of the bioprocess container in a longitudinal direction, the baffle having a shape that extends radially inward from the side wall an amount sufficient to affect fluid flow in the hollow enclosure during mixing of a culture media by the mixing device.
107. A bioreactor as defined in claim 106, wherein the baffle defines an inflatable fluid bladder, the baffle being capable of being inflated and deflated.
108. A bioreactor as defined in claim 106 or 107, wherein the baffle is configured to be placed outside the hollow enclosure and wherein the side wall of the bioprocess container conforms around the shape of the baffle.
109. A bioreactor as defined in claim 103, wherein the bioreactor further comprises at least one sparger.

110. A bioreactor as defined in claim 109, wherein the sparger comprises a ballast sparger, the ballast sparger comprising a gas tube that has a longitudinal portion and a lateral portion, the longitudinal portion extending vertically into the hollow enclosure of the bioprocess container, the lateral portion being located at an end of the longitudinal portion below the agitator, the lateral portion defining a plurality of holes for releasing a gas into a culture media contained within the bioprocess container.
111. A bioreactor as defined in claim 110, wherein the bioreactor contains a second sparger.
112. A bioreactor as defined in claim 103, wherein the shape-conforming material of the bioprocess container comprises a flexible film.
113. A bioreactor as defined in claim 112, wherein the flexible film comprises a multi-layered film, the multi-layered film including an interior surface facing the hollow enclosure and an opposite exterior surface, the interior surface comprising a low density polyethylene that has been modified to form a hydrophilic surface.
114. A bioreactor as defined in claim 113, wherein the low density polyethylene has been modified by being subjected to irradiation, photo or plasma induction, or oxidation.
115. A bioreactor as defined in claim 103, further comprising at least one feed line that extends into the hollow enclosure for feeding fluids into the bioprocess container, the feed line including a subsurface fluid outlet positioned adjacent to one agitator, the fluid outlet being associated with a fluid control device that only permits fluid to flow out of the fluid outlet and prevents fluid flow in an opposite direction.
116. A bioreactor comprising:
a bioprocess container made from a liquid impermeable and flexible shape-conforming material, the bioprocess container having a top, a bottom, and at least one side wall therebetween, the bioprocess chamber defining a hollow enclosure for receiving a culture media;
at least one inlet port for feeding materials into the hollow enclosure of the bioprocess container; and
a mixing device comprising a rotatable shaft coupled to at least one agitator, the shaft and agitator extending into the hollow enclosure of the bioprocess container, the agitator being collapsible onto the rotating shaft.

117. A bioreactor as defined in claim 116, wherein the agitator comprises an impeller comprising at least one blade element, the blade element being foldable towards the rotatable shaft.
118. A bioreactor as defined in claim 116 or 117, further comprising collapsible baffle elements projecting from the rotating shaft.
119. A bioreactor as defined in claim 117, wherein the rotatable shaft is coupled to a first impeller and a second impeller, both impellers including at least one blade element that are foldable towards the shaft.
120. A bioreactor as defined in any of claims 116-119, wherein the shaft and agitator are made from a hydrophilic polymer.
121. A bioreactor as defined in claim 120, wherein the hydrophilic polymer comprises a polyethylene polymer that has been modified by being subjected to irradiation, photo or plasma induction, or oxidation.
122. A bioreactor as defined in any of claims 116-121, wherein the rotatable shaft comprises a metallic reinforcing rod surrounded by a shaft sleeve, the shaft sleeve being comprised of a polymeric material.
123. A bioreactor as defined in claim 122, further comprising a retaining ring positioned on the shaft of the mixing device, the retaining ring including an agitator engaging position and an agitator disengaging position for holding the agitator in an upright position or in a collapsed position respectively.
124. A bioreactor as defined in claim 123, wherein the retaining ring moves from the agitator disengaging position to the agitator engaging position when the metallic reinforcing rod is inserted into the shaft sleeve.
125. A bioreactor as defined in claim 122 or 123, wherein the metallic reinforcing rod includes multiple sections attached together.
126. A bioreactor as defined in claim 122, 123, 124 or 125, wherein the metallic reinforcing rod has a top and wherein a magnetic member is located at the top of the metallic reinforcing rod, the magnetic member being configured to magnetically engage a motor.
127. A bioreactor as defined in any of claims 116-126, further comprising at least one baffle being configured to extend adjacent to the side wall of the bioprocess container in a longitudinal direction, the baffle having a shape that extends radially inward from the

side wall an amount sufficient to affect fluid flow in the hollow enclosure during mixing of a culture media by the mixing device.
128. A bioreactor as defined in claim 127, wherein the baffle defines an inflatable fluid bladder, the baffle being capable of being inflated and deflated.
129. A bioreactor as defined in claim 127 or 128, wherein the baffle is configured to be placed outside the hollow enclosure and wherein the side wall of the bioprocess container conforms around the shape of the baffle.
130. A bioreactor as defined in claim 116, wherein the bioreactor further comprises at least one sparger.
131. A bioreactor as defined in claim 130, wherein the sparger comprises a ballast sparger, the ballast sparger comprising a gas tube that has a longitudinal portion and a lateral portion, the longitudinal portion extending vertically into the hollow enclosure of the bioprocess container, the lateral portion being located at an end of the longitudinal portion below the agitator, the lateral portion defining a plurality of holes for releasing a gas into a culture media contained within the bioprocess container, the lateral portion engaging the shaft of the mixing device for stabilizing the shaft.