Single-use cell culture vessel equipped with one or more in-situ online sensors

Abstract

Described herein is a small-volume bioreactor comprising a culture vessel and a reactor head plate, wherein the culture vessel has a working volume of 20 mL to 350 mL and is equipped with two or more in-situ sensors, and the reactor head plate is equipped with in-situ sensor ports to which at least one in-situ glucose sensor and one in-situ pH sensor are connected.

Description

[Technical Field]

[0001] The present invention relates to the field of mammalian cell culture. More specifically, the present invention relates to a disposable small-volume cell culture vessel, also called a single-use small-volume bioreactor (SUSVB), comprising one or more, in particular two or more, in-situ sensors, and its use in the small-volume culture of mammalian cells. [Background technology]

[0002] Acceleration of bioprocess development for biologics and vaccines can be enabled by automated high-throughput technologies, which can significantly reduce the resource burden of multifactorial statistical experiments required to control product quality attributes of complex biologics (Bareither, R., et al., Biotechnol. Bioeng. 110 (2013) 3126-3138).

[0003] Recently, Bareither, R. et al. provided proof-of-concept evaluation of an automated, disposable, small-scale reactor for high-throughput upstream process development by establishing a small-scale, stirred-tank, disposable 250 mL reactor similar to laboratory- and pilot-scale reactors for the process performance of industrial biopharmaceutical processes for therapeutic protein and monoclonal antibody production using CHO cell cultures, Pichia pastoris, and Escherichia coli (Biotechnol. Bioeng. 110 (2013) 3126-3138). This included similar growth, cell viability, product titer, and product quality. The technology was shown to be robust over multiple experiments and met the requirements for the ability to run high-cell-density processes (>400 g / L wet cell weight) using exponential feeding and a sophisticated event-triggered process.

[0004] Single-use cell culture vessels are well known in the art, such as the ambr® 15 and 250 systems commercially available from Sartorius Stedim Biotech.

[0005] US Patent Application Publication No. 2019 / 048305 (Patent Document 1) reports a perfusion bioreactor and method of using it to perform continuous cell culture, where the reactor includes a single sensor port (240) fitted with a single sensor (254).

[0006] US Patent Application Publication No. 2019 / 153381 (Patent Document 2) reports a perfusion bioreactor and related methods of use, in which the reactor includes a RAMAN sensor in the headspace, i.e., a culture medium non-contact sensor, as one sensing element.

[0007] US Patent No. 8,026,096 reported in vivo active erythropoietin produced in insect cells using bioreactors with working volumes of 2 liters or more. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent Application Publication No. 2019 / 048305 [Patent Document 2] U.S. Patent Application Publication No. 2019 / 153381 [Patent Document 3] U.S. Patent No. 8,026,096 [Non-patent literature]

[0009] [Non-Patent Document 1] Bareither, R., et al., Biotechnol. Bioeng. 110 (2013) 3126-3138 Summary of the Invention

[0010] Commercially available single-use small-volume bioreactors (SUSVBs) offer at best temperature, dissolved oxygen, and pH control, with the capacity for feed addition and sampling. However, there is no option for direct contact with a second in situ culture medium, i.e., a metabolite sensor. Therefore, if additional monitoring of glucose, for example, is required in addition to pH control, offline analysis by sampling must necessarily be performed. This increases the risk of culture contamination, among other things, in addition to the handling effort.

[0011] The inventors have found that by using small volume bioreactors according to the present invention, the timeline for analytical and biological process development can be shortened, and costs can be reduced, all without loss of portability to subsequent large-scale processes.

[0012] One aspect of the present invention is a small volume bioreactor that includes one or more in-situ sensors, where at least one in-situ sensor is for determining glucose.

[0013] One aspect of the present invention is a culture vessel (105), - an agitator comprising an agitator shaft (108) and one or more impellers (112; 109); an electrode or electrode-type sensor as a first in-situ sensor; - Gas sparger (127) and - a reactor head plate (104), - a motor coupling (103) for connecting the drive shaft of the motor to the agitator shaft (108)(122); - at least one gas inlet (116) and gas outlet (117), - supply port area (133), - an in-situ sensor port (130) for picking up an electrode or electrode-type sensor, a reactor head plate (104) comprising: wherein the small-volume bioreactor comprises a second in-situ sensor.

[0014] Below are embodiments of all aspects outlined above, and it is expressly stated that combinations of each embodiment with other embodiments, as well as combinations with each aspect, are encompassed as well, even if not stated.

[0015] In one preferred embodiment, the small volume bioreactor comprises two or more in-situ sensors, one for determining glucose and one for determining pH, hi one preferred embodiment, both sensors are submerged sensors, i.e., in direct contact with the culture medium.

[0016] In one embodiment, the pH sensor is a pH electrode.

[0017] In one embodiment, the glucose sensor is an electrochemical and / or enzyme-based sensor.

[0018] In one preferred embodiment, the small volume bioreactor comprises two or more in-situ sensors, one in-situ sensor for determining glucose and one in-situ sensor for determining pH, the pH sensor being a pH electrode and the glucose sensor being an electrochemical and / or enzyme-based sensor. In one preferred embodiment, both sensors are immersed sensors, i.e., in direct contact with the culture medium.

[0019] In one embodiment, the small volume bioreactor has a working volume of 20 mL to 350 mL. In one embodiment, the working volume is 25 mL to 300 mL. In one embodiment, the working volume is 50 mL to 280 mL. In one embodiment, the working volume is 55 mL to 270 mL.

[0020] In one embodiment, the small volume bioreactor is operated at a volume between 60 mL and 260 mL. In one embodiment, the small volume bioreactor is operated at a volume between 90 mL and 250 mL.

[0021] In one embodiment, the small-volume bioreactor has a total volume of 500 mL or less. In one embodiment, the small-volume bioreactor has a total volume of 450 mL or less. In one embodiment, the small-volume bioreactor has a total volume of 400 mL or less.

[0022] In one embodiment, the small volume bioreactor comprises a culture vessel (105) and a reactor head plate (104).

[0023] In one embodiment, the at least two in-situ sensors include at least a glucose or lactate sensor and a pH sensor.

[0024] In one embodiment, the feed port region comprises one to four inlets for liquid connected to individual feed lines (118, 119, 120, 121) and optionally a sparger gas inlet (116).

[0025] In one embodiment, two impellers (109, 112) are connected to the agitator shaft.

[0026] In one embodiment, the small volume bioreactor includes at least (i) one sparger gas inlet (116) and (ii) one headspace gas inlet (132).

[0027] In one embodiment, the glucose sensor determines the glucose concentration every 20 seconds and / or the glucose sensor provides a signal if there is a change in the glucose concentration. In one embodiment, the determined glucose concentration value is transmitted to a computer via wire or wirelessly. In one embodiment, the transmission of the glucose concentration is via Wi-Fi, RFID, or Bluetooth. In one embodiment, the glucose sensor has an operating range of up to 8 g / L glucose or up to 3 g / L glucose. In one embodiment, the glucose sensor is an electrochemical and / or enzyme-based sensor. In one embodiment, the glucose sensor is a screen-printed electrode coated with an immobilized enzyme. In one embodiment, the glucose sensor substrate is a USP (United States Pharmacopeia) Class VI polymer.

[0028] In one embodiment, the small volume bioreactor is a single-use bioreactor.

[0029] In one embodiment, the culture vessel (105) and the reactor head plate (104) are made from a non-metallic material, hi one embodiment, the culture vessel (105) and the reactor head plate (104) are made from plastic.

[0030] In one embodiment, the reactor head plate (104) further comprises a sampling port (102).

[0031] In one embodiment, the small volume bioreactor is sterilizable.

[0032] In one embodiment, the small-volume bioreactor is a radiation-sterilized small-volume bioreactor. In one embodiment, the small-volume bioreactor is a double-radiation-sterilized small-volume bioreactor. In one embodiment, the radiation is beta radiation and / or gamma radiation. DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description of the Invention In contrast to the early stages of biopharmaceutical manufacturing, it is nowadays well accepted that for the development of therapeutic proteins such as antibodies, the critical parameter is not only the product titer but also certain product quality attributes such as, but not limited to, the by-product profile or the glycosylation profile / pattern (see, e.g., Bareither, R. and Pollard, D., Biotech. Prog. 155 (2011) 217-224).

[0034] Therefore, these properties need to be evaluated and engineered as early as possible, best done during clone selection and process development. Thus, a none-time process, sequential experimental rounds using multiple parallel reactors, is typically required. Therefore, the method must be amenable to high throughput.

[0035] Due to ecological and economic constraints, large-scale cultivation cannot be used for process development, so suitable small-scale systems that have been proven to reliably reflect the future commercial (large-scale) process and process performance are employed.

[0036] One independent aspect of the present invention is a small volume bioreactor, the small volume bioreactor comprising: - With a working volume of 20 mL to 350 mL, - an agitator shaft (108) having at least one impeller (112) mounted thereon; - a feed pipe (107) comprising i) a sparger tube connected to a sparger (127) at its end, and ii) at least one feed line (118) having an opening at its end, - two or more baffles (114; 126) extending from the wall of the culture vessel (105) towards the centre of the culture vessel (105) (perpendicular to the wall of the culture vessel), A culture vessel (105); a reactor head plate (104); Equipped with The reactor head plate (104) comprises: - a coupling (122) for connecting the drive shaft of the motor to the agitator shaft (108); - a sparger gas inlet (116) connected to a sparge tube in the feed pipe (107); and, Optional connected to the headspace Gas inlet (132) and , - Culture volume vessel Gas outlet connected to headspace (117) and, - at least one inlet for the liquid in a feed line (118) that is part of the feed pipe (107); - one in-situ sensor port (130) fitted with a pH electrode (101); - a feed port area (133) including a sparger gas inlet and an inlet for at least one feed line (118); The culture vessel (105) and the reaction head plate (104) are both made substantially of non-metallic materials, and the small volume bioreactor is characterized by including an in-situ glucose sensor.

[0037] Embodiment 2. A small-volume bioreactor according to a separate aspect, wherein the small-volume bioreactor is a single-use small-volume bioreactor.

[0038] Embodiment 3. The small-volume bioreactor of an independent aspect and embodiment 2, wherein the small-volume bioreactor is a radiation-sterilized small-volume bioreactor.

[0039] Embodiment 4. The small-volume bioreactor of any one of an independent aspect or of Embodiment 2 or 3, wherein the small-volume bioreactor is a sterile small-volume bioreactor and has been sterilized twice using radiation.

[0040] Embodiment 5. The small volume bioreactor of any one of an independent aspect or of Embodiments 3 or 4, wherein the radiation is beta radiation and / or gamma radiation.

[0041] Embodiment 6. The small volume bioreactor of embodiment 4, wherein the first radiation is beta radiation and the second radiation is gamma radiation, or vice versa.

[0042] Embodiment 7. The small-volume bioreactor of an independent aspect or any one of Embodiments 2 to 6, wherein the glucose sensor is a screen-printed electrode coated with an immobilized enzyme.

[0043] Embodiment 8. The small-volume bioreactor of an independent aspect or any one of Embodiments 2 to 7, wherein the glucose sensor substrate is a USP Class VI polymer.

[0044] Embodiment 9. The small-volume bioreactor of any one of an independent aspect or embodiments 2 to 8, wherein the glucose sensor determines the glucose concentration and / or determines the change in glucose concentration every 20 seconds.

[0045] Embodiment 10. The small-volume bioreactor of any one of the independent aspects or embodiments 2 to 9, wherein the determined glucose concentration values ​​are transmitted wirelessly or by cable from the glucose sensor to the computer.

[0046] Embodiment 11. The small volume bioreactor of any one of the independent aspects or embodiments 2 to 10, wherein the reactor head plate (104) further comprises a sampling port (102).

[0047] Embodiment 12. The small volume bioreactor of any one of the independent aspects or embodiments 2 to 11, wherein an in-situ glucose sensor passes through the head plate (104) at or within the feed port area (133).

[0048] Embodiment 13. A method for culturing mammalian cells using the small volume bioreactor of any one of the independent aspects or embodiments 2 to 12.

[0049] Embodiment 14. A method for determining culture conditions using the small volume bioreactor of any one of the independent aspects or embodiments 2 to 12.

[0050] General definition The term "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include the different constant region genes as well as the myriad immunoglobulin variable region genes. Antibodies can exist in a variety of formats, including, for example, Fv, Fab, and F(ab)2, as well as monovalent, bivalent, trivalent, tetravalent, pentavalent, and hexavalent forms, and as monospecific, bispecific, trispecific, or tetraspecific antibodies, as single chain (scFv) or diabodies or triabodies.

[0051] A "polypeptide" is a polymer of amino acids linked by peptide bonds, whether produced naturally or synthetically. Polypeptides of fewer than about 20 amino acid residues are referred to as "peptides," while molecules consisting of two or more polypeptides or containing a single polypeptide of more than 100 amino acid residues are sometimes referred to as "proteins." Polypeptides may also contain non-amino acid components (e.g., carbohydrate groups, metal ions, or carboxylic acid esters). Non-amino acid components may be added by the cell in which the polypeptide is produced and may vary with the type of cell. Polypeptides are defined herein with respect to their amino acid backbone structure or the nucleic acid encoding them. Additives such as carbohydrate groups are generally not specified, but may be present nonetheless.

[0052] The term "in-situ" sensor refers to a sensor that is in direct physical contact with the culture medium.

[0053] Embodiments of a small volume bioreactor according to the present invention Commercially available single-use small-volume bioreactors (SUSVBs) at best only provide means for controlling temperature, dissolved oxygen, and pH, with only the pH value being controlled by an in-situ sensor. Furthermore, these SUSVBs have ports for the addition of nutrients or correction fluids, as well as the ability for sampling. Nevertheless, due to the limited diameter / size and area of ​​the head plate (104), a second in-situ metabolite sensor is not available. Therefore, if additional monitoring of glucose, for example, is required in addition to the in-situ pH electrode, this can only be done by offline analysis using sampling.

[0054] The inventors have found that it is possible to provide a sterilizable SUSVB with two in-situ sensors by attaching an additional glucose sensor in the same area used for the liquid and gas supply lines, i.e., the supply port area (133).

[0055] The provision of a glucose sensor in the SUSVB according to the present invention allows for continuous in-situ determination of glucose concentration, thereby reducing the sampling, i.e., sample taking, previously required for glucose determination. The presence of a glucose sensor in the SUSVB allows for the determination of glucose concentration in real time, i.e., without a time offset. This allows for more detailed monitoring of the growth behavior and metabolic state of the culture, i.e., allowing for corrective action to be taken earlier than sampling.

[0056] A particular advantage of the SUSVB according to the invention is that glucose determination can be carried out without interfering with the culture, which would be necessary for sampling.

[0057] Therefore, by directly incorporating an in-situ glucose sensor into SUSVB, one or more of the drawbacks such as low cell density, low product yield, environmental changes such as carbon dioxide, temperature and pH disturbances, metabolic stress, altered gene expression, changes in culture volume, and most importantly contamination can be avoided.

[0058] In one embodiment, the glucose sensor determines the glucose concentration every 20 seconds and / or the glucose sensor provides a signal if there is a change in the glucose concentration. In one embodiment, the determined glucose concentration value is transmitted to a computer by wire or wirelessly. In one embodiment, the transmission of the glucose concentration is by Wi-Fi, RFID, or Bluetooth.

[0059] Due to the presence of a glucose sensor in the SUSVB, it is now possible to control the glucose concentration online.

[0060] In one embodiment, the glucose sensor has an operating range of up to 8 g / L glucose or up to 3 g / L glucose.

[0061] In one embodiment, the glucose sensor is an electrochemical and / or enzyme-based sensor. In one embodiment, the glucose sensor is a screen-printed electrode coated with an immobilized enzyme. In one embodiment, the glucose sensor substrate is a USP Class VI polymer.

[0062] USP Class VI refers to the official United States Pharmacopeia (USP) Biocompatibility Class VI, which specifies the requirements for biocompatible plastic materials. Class VI is the class with the most stringent requirements, comparable to pharmaceutical marketing registration. It is also specified in German Industrial Standard DIN-ISO-10993.

[0063] Glucose detection by electrochemical and / or enzyme-based sensors is based on the oxidation of glucose by glucose oxidase, an enzyme that catalyzes the oxygen-independent oxidation of the C1 residue of glucose. The sensor detects the hydrogen peroxide thus produced.

[0064] In one embodiment, the glucose sensor is sterilizable by gamma irradiation.

[0065] In one embodiment, the entire glucose sensor has a total length of 40 to 500 mm. In one preferred embodiment, the glucose sensor has a total length of 75 to 350 mm. In one embodiment, the electrode or enzyme-coated area of ​​the glucose sensor has a width of 5 to 20 mm.

[0066] In one preferred embodiment, the SUSVB (see also Figures 1 to 7) comprises: - Working volumes of 20mL to 350mL, 25mL to 300mL, 50mL to 280mL, 55mL to 270mL, or 95mL to 255mL; - an agitator including an agitator drive shaft equipped with either two Rushton impellers (approximately 20 mm diameter, approximately 30 mm apart) for microbial culture or two pitched blade impellers (approximately 25 mm diameter, approximately 30 mm apart) for mammalian cell culture; - two or four equally spaced baffles (approximately 6.25 mm wide) arranged on the (vertical) side walls of the reactor and extending towards the center of the vessel, i.e. perpendicular to the inner wall of the culture vessel; a motor (150 rpm to 3,000 rpm, for example a brushless electric servo motor) directly coupled to the drive shaft of the agitator, which in a preferred embodiment is an electric DC motor (for example a RE-max 17 series manufactured by Maxon, Switzerland); - a reactor head plate having lines with sterile filters for the subsurface addition of gases and liquids and an additional outlet for vent gases; - a dissolved oxygen sensor fluorescent patch (e.g., manufactured by PreSens) embedded in the base of the reactor and controlled by the agitation and / or aeration cascade, with a fast response (<2 seconds) dO2 probe, in one preferred embodiment the measurement interval is between 10 and 15 seconds, in one preferred embodiment about 12 seconds; - a gel electrode for pH determination using a three-point calibration performed before autoclaving and a one-point calibration after adding the medium; - a temperature-controlled jacket filled with liquid or a temperature-controlled metal block (made of aluminum in one preferred embodiment) on top of which a SUSVB can be placed; - a control station (on top of which a SUSVB can be placed) containing a fluorescence reader for the dissolved oxygen sensor and temperature probe, individual sensors for vent gas analysis of oxygen (e.g. electrochemical detector) and carbon dioxide (e.g. infrared detector); - a recessed area at the base of the vessel wall for measuring the temperature; - Temperature control (clamp) plate (6°C) adapted to the head plate design for vent gas humidity control; - single bolus addition (e.g., at a volume of 10 μL to 10 mL), in one preferred embodiment up to four liquid feeds (e.g., for pH control reagents (acids and bases), nutrients) by individual pumps (e.g., syringe pumps) allowing continuous feed at about 150 μL / h (e.g., with different profiles, such as linear or exponential, and at flow rates of 20 nL / h to 20 mL / h); - a sparging tube (for the supply of gas (air)), which may have an open tube configuration with the gas outlet directly below the bottom impeller; a headspace gas inlet for gassing the culture medium headspace; - a multi-configurable inlet gas manifold (e.g., for air, pure air, oxygen, nitrogen, carbon dioxide mixes) with a pneumatic pulse valve for each gas stream (to control the composition / mix), a 1 second cycle with a minimum pulse time of 20 ms (to control the duration and spacing of the gas pulses), and a mass flow sensor downstream of the pulse valve allowing a flow range of 0.0013 mL / min to 550 mL / min; The present invention has one or more of the following:

[0067] The geometries of the different reactor sizes are shown in the table below (reproduced from Bareither, R., et al., Biotechnol. Bioeng. 110 (2013) 3126-3138; Table II): TIFF0007778076000001.tif92153TIFF0007778076000002.tif86131

[0068] A suitable SUSVB is at least equal to H L / D i - and D T / D i - ratio, impeller spacing, and baffles intended for use in large-scale fermenters. Input power is 0.01 kW / m 3 ~0.4kW / m 3 , k LThe a value should be in the range of 1 l / h to 15 l / h. Dissolved oxygen should be controllable above 20% air saturation, while the dissolved carbon dioxide (CO2) content should be in the range of 35 mmHg to 80 mmHg. The temperature should be in the range of 32°C to 38°C, and the pH value should be controlled in the range of pH 6.8 to 7.2. The feed should be in the range of 20 pg / cell / day to 90 pg / cell / day, and automation and different feeding methods (linear ramp, exponential, constant, bolus addition) for feed control should be provided. The working volume, which allows parallel processing and sampling, should be in the range of 20 mL to 300 mL, preferably 60 mL to 255 mL, for development and product quality analysis. The SUSVB should allow for the addition of nutrients triggered by sensors or feeding via a pH stat or dO stat.

[0069] Generally, for fed-batch cultivation of monoclonal antibody-expressing CHO cell lines, e.g., CHO K1 cell line, any medium can be used, such as commercially available CD-CHO medium or any other serum-free medium. 5 SUSVB is performed using standard conditions, such as an inoculation cell density of 1 x 10 viable cells / mL. SUSVB is performed by growing a shake flask seed culture (e.g., 1 x 10 viable cells / mL) every 3-4 days in a humidified incubator at 36.5 °C, 5% CO, within the growth phase. 6The inoculation volume should be greater than 10 ... For analysis, samples are removed at defined time points (e.g., daily) to determine cell viability, glucose, lactate, osmolality, pH, while dissolved gases (dO2, dCO2) are determined offline, e.g., using a blood gas analyzer.

[0070] Generally, a SUSVB according to the present invention comprises: - To determine the fermentation conditions, - To determine the parameters of a bioprocess, It can be used.

[0071] Generally, in one preferred embodiment, such single-use small-volume bioreactors (SUSVBs) are at least partially fabricated from non-metallic, non-glass, polymeric materials. As disclosed in U.S. Patent No. 9,938,493 (incorporated herein by reference in its entirety), known cell culture vessels are constructed from multiple layers, where the inner layer, i.e., the layer in contact with the cell culture, is fabricated from a polymeric material. Typically, at least this layer is made of polyethylene (PE) or ethyl-vinyl acetate (EVA).

[0072] As used herein, the terms "single-use cell culture vessel" and "single-use small-volume bioreactor (SUSVB)" may be used interchangeably and refer to a cell culture vessel having a working volume of 300 mL or less, made from a single-layer or multi-layer polymeric material, for culturing mammalian or bacterial cells for the production of biological substances. The cell culture vessel or small-volume bioreactor used in accordance with the present invention may be of any shape. As used herein, the term "single-use" means that the cell culture vessel or small-volume bioreactor is used only once for culturing cells. This does not refer to the fact that the cell culture vessel or small-volume bioreactor is sterilized more than once before use. Therefore, before filling the cell culture vessel or small-volume bioreactor with culture medium or cells, it is treated with microorganism-killing radiation, such as beta or gamma radiation. This must be done to kill all microorganisms present in the device that may interfere with or affect the growth of the cultured cells.

[0073] In one embodiment, the occurrence of a prolonged lag phase in the culture of mammalian cells in a single-use cell culture vessel (SUSVB) according to the present invention using serum-free medium is prevented by treating the vessel, i.e., the SUSVB, with an inert gas prior to the application of sterilizing radiation. In one embodiment, the single-use cell culture vessel (SUSVB) is made at least in part from a polymeric material. In one embodiment, the sterilizing radiation is beta or gamma irradiation.

[0074] Experimental results The following summarizes experimental results obtained using exemplary SUSVBs in accordance with the present invention. These are presented by way of example only and should not be construed as limiting. The true scope of the present invention is set forth in the following claims.

[0075] A SUSVB according to the present invention including an in-situ glucose sensor was compared with a standard SUSVB without an additional glucose sensor, i.e., with a single in-situ sensor. The results of each experiment are summarized in the table below. TIFF0007778076000003.tif73146

[0076] RE01 / RE05 / RE09 were comparative experiments, each involving offline glucose analysis using a small-volume bioreactor, i.e., a 10-day culture using only one in-situ sensor sterilized once with beta radiation.

[0077] RE02 / RE04 / RE06 / RE07 / RE08 / RE10 / RE12 were each cultured for 16 days using SUSVB according to the present invention, i.e., two in-situ sensors sterilized twice with beta and gamma rays.

[0078] In the SUSVB according to the present invention, two different in-situ glucose sensors were used: a high glucose sensor and a low glucose sensor. The difference between both sensor types is the different type of membrane layer on the sensor. This difference results in different diffusion times of glucose to the enzyme in the sensor.

[0079] RE02 / 06 / 07 / 10 each used a low glucose sensor with a measurement range of 0 to 3 g / L, and RE04 / 08 / 12 each used a high glucose sensor with a measurement range of 0 to 8 g / L.

[0080] Feed 1 (containing glucose) was stopped in vessel RE02 / 04 / 06 / 07 / 08 / 10 / 12 on day 9 to allow glucose levels to fall into the measurement range of the glucose sensor and initiate glucose control via the sensor.

[0081] An exemplary glucose curve obtained for RE02 is shown in Figure 8. The green curve is the glucose concentration measured with the in-situ sensor. Each steep step on the green curve (Tue 30 Apr; Mon 06 May; Thu 09 May) is a recalibration step for the sensor. It can be seen that the glucose sensor exhibits low drift. No recalibration was required for 10 days. The red line is the cumulative volume / amount of glucose solution delivered. The pink line represents the operating time / flow rate of the glucose solution feeding pump. The circled time points represent bolus feeds without glucose. On day 9, the feeding of Feed 1, which also contains glucose, was stopped. The black dots represent control glucose values ​​obtained by sampling and Cedex BioHT offline analysis.

[0082] One aspect reported herein is a SUSVB for culturing animal cells. In one embodiment, the SUSVB comprises: a) a single-use small-volume culture vessel according to the invention suitable for receiving a culture medium and animal cells to be cultured therein; b) agitation system; c) glucose sensors; d) a gas inlet at the bottom of the culture vessel; and e) at least one inlet for adding correction and / or feeding solutions; Includes.

[0083] One embodiment reported herein comprises the steps of: a) culturing cells containing nucleic acids encoding the polypeptide in a SUSVB according to the invention; b) recovering the polypeptide from the culture medium or the cells, and c) optionally purifying the polypeptide, thereby producing the polypeptide; The present invention relates to a method for producing a polypeptide, particularly an antibody, comprising:

[0084] One aspect reported herein is a method for culturing animal or bacterial cells, characterized in that the animal or bacterial cells are cultured in the SUSVB reported herein, thereby optionally producing a product.

[0085] One embodiment reported herein is the use of SUSVB for the production of polypeptides or antibodies or viruses.

[0086] In one embodiment, the ratio of the impeller diameter d to the diameter D of the SUSVB when the agitation system is placed in the culture vessel is in the range of 0.2 to 0.8, in another embodiment in the range of 0.3 to 0.6, in a further embodiment in the range of 0.31 to 0.39, or in one embodiment about 0.34. In a further embodiment, the pitch of the agitator blades of the axially conveying impeller is between 10 and 80 degrees relative to the shaft axis, in another embodiment between 24 and 60 degrees, or in a further embodiment between 40 and 50 degrees. In one embodiment, all impellers have a ratio of the conveying element diameter d to the culture vessel diameter D of 0.32 to 0.35.

[0087] In one embodiment, the purification is a multi-step chromatographic process, hi another embodiment, the purification comprises affinity chromatography, cation exchange chromatography, and anion exchange chromatography.

[0088] In one embodiment, the culture is a semi-continuous culture.

[0089] In one embodiment, the polypeptide is an antibody or antibody derivative.

[0090] In one embodiment, the culture medium is an aqueous medium suitable for culturing prokaryotic and eukaryotic cells. In another embodiment, the culture medium is a Newtonian liquid. In one embodiment, the stirring system is operated with a power input of 0.01 W / kg to 1 W / kg. In a further embodiment, the stirring system is operated with a power input of 0.04 W / kg to 0.5 W / kg. In yet another embodiment, the flow induced by the stirring system in the culture medium is turbulent. In another embodiment, the culture medium has a viscosity of 3 mPas*s or less. In another embodiment, the viscosity is 2 mPas*s or less.

[0091] In one embodiment, the SUSVB is a submerged gas stirred tank reactor.

[0092] In another embodiment, the animal cell is a mammalian cell. In yet a further embodiment, the cell is a CHO cell, a BHK cell, an NS0 cell, a COS cell, a PER.C6 cell, an Sp2 / 0 cell, an HEK 293 cell, or a hybridoma cell.

[0093] To achieve high product titers and good product quality, the operating mode of the SUSVB according to the present invention plays an important role in addition to, for example, cell line development, medium composition and SUSVB size determination.

[0094] A distinction can be made between operating modes batch or batch processes, fed-batch or feeding processes, continuous processes with or without cell retention (e.g. perfusion or chemostat), as well as semi-continuous processes (e.g. internal or external dialysis).

[0095] The SUSVB has an upper portion, a middle portion, and a lower portion, and the longitudinal axis of the SUSVB extends from the center of the middle portion or upper portion to the center of the middle portion or lower portion.

[0096] The SUSVB has a substantially circular cross section when viewed perpendicular to its longitudinal axis. The diameter may be the same at the top and bottom, or the bottom may have a smaller diameter than the top.

[0097] The upper portion of the SUSVB may further comprise a gas release outlet means and / or one or more inlet means.

[0098] The lower portion of the SUSVB may further comprise one or more liquid medium inlet means and / or gas inlet means.

[0099] At least the lower or central portion of the SUSVB may further comprise a heat exchange jacket attached to the outer wall of the culture vessel.

[0100] The conveying elements of the agitator system are configured to rotate by a shaft, which is connected to an appropriate mechanism for inducing its rotation. The shaft extends along the longitudinal axis of the SUSVB, and therefore has a vertically oriented axis of rotation. The shaft does not extend to the bottom of the SUSVB, but rather to a point well above the bottom of the SUSVB and also to a point well above the optional gas sparger at the bottom of the SUSVB. The shaft is operably connected to a drive shaft by an appropriate connecting mechanism. In addition to the means for connecting the shaft to the drive shaft, the shaft may also include additional means for individually connecting the impeller to the shaft. The impeller of the agitator system is typically connected to the shaft at a position below the surface of the culture medium in the SUSVB when the agitator system is immersed in the culture medium. The surface is determined when the culture medium is stationary, i.e., not circulating.

[0101] In one embodiment, the SUSVB of the present invention is a baffled SUSVB. In another embodiment, the SUSVB of the present invention includes two or four baffles. "Baffle" refers to a plate disposed within the culture vessel in the same direction as the shaft axis and extending radially into the culture vessel toward the agitator. The baffle is generally rectangular in shape. In one embodiment, the baffle is disposed at a distance bd to the inner wall of the SUSVB. In another embodiment, the baffles are spaced at equal distances from each other around the inner circumference of the SUSVB.

[0102] The components of the SUSVB are sized so that they can perform their intended functions, i.e., so that the SUSVB can take up culture medium and the agitation system can mix the medium and disperse added compounds. Thus, the agitation system has a diameter that allows unobstructed rotation within the SUSVB.

[0103] High cell density culture (e.g., perfusion culture) can be performed using the SUSVB reported herein.

[0104] In one embodiment, the culture is carried out at a rotation speed of the stirring system that can achieve a constant power input to the culture medium that is independent of the Reynolds number, i.e., a rotation speed of the stirring system that provides a turbulent culture medium flow within the culture vessel during culture. Using the SUSVB according to the present invention, it is possible to culture shear-sensitive mammalian cells at low rotation speeds of the stirring system.

[0105] The shape of the SUSVB culture vessel is not limited. In one embodiment, the culture vessel is a cylindrical vessel. In another embodiment, the culture vessel is a stirred tank reactor-like vessel. The culture vessel may have any dimensions. In one embodiment, the culture vessel has a working volume of 55 mL to 265 mL, and in one preferred embodiment, a working volume of 20 mL to 350 mL.

[0106] Generally, submerged gas culture vessels are used in cell culture. In such cases, one-stage, two-stage, or three-stage axial conveying agitator systems are mainly used. When one-stage or two-stage axial conveying agitator systems are used, they generate a flow profile that is essentially parallel to the rotation axis of the employed agitator.

[0107] In one embodiment, the agitator comprises 1 to 5 axially-conveying elements, or in another embodiment 1 to 3 axially-conveying elements, or in an embodiment 1 or 2 axially-conveying elements, or only a single axially-conveying element. In one embodiment, one axially-conveying element is located in the upper four-fifths of the agitator shaft determined from the agitator head and is located at a distance of h 4 / 5 In one embodiment, one axially-conveying element is arranged at a maximum distance of 0.8h and / or one axially-conveying element is arranged at a maximum distance of 0.2h. In a further embodiment, the axially-conveying elements together form a single element. In one embodiment, all axially-conveying elements have the same diameter. In another embodiment, the axially-conveying elements are independently selected from propeller agitators, pitched blade agitators, or inclined blade agitators.

[0108] In a further embodiment, all conveying elements rotate at the same number of revolutions per unit time around the agitator shaft axis when the agitator is operated in SUSVB. In one embodiment, the conveying elements are permanently joined together and the agitator consists of one part, i.e., all elements are driven by the same rotating shaft and rotate at the same number of revolutions per unit time around the agitator shaft axis.

[0109] The ratio d / D of the agitator diameter (d) to the culture vessel diameter (D) is 0.2 to 0.8 in one embodiment, 0.3 to 0.6 in another embodiment, and 0.33 to 0.5 in yet another embodiment. In another embodiment, the ratio h / d of the blade height (h) to the agitator diameter (d) is 0.5 to 5, 1 to 4 in another embodiment, and 1 to 3 in yet another embodiment. In yet another embodiment, the ratio b / d of the impeller blade width (b) to the agitator diameter (d) is 0.05 to 0.3, and 0.1 to 0.25 in another embodiment.

[0110] The phrase "from to" means a range inclusive of the recited limits.

[0111] In one embodiment, the stirrer diameter (d) is selected from 5 mm, 6 mm, 7 mm, 8 mm, 10 mm, 12 mm, 14 mm, 16 mm, 18 mm and 20 mm.

[0112] In one embodiment, the impeller blade width (b) is selected from 0.42 mm, 0.60 mm, 0.89 mm, 1.08 mm, 1.33 mm.

[0113] In one embodiment, one radially-conveying element is an anchor impeller.

[0114] The term "approximately" means that a given value is the center point of a range that extends ±10% around that value. If the value is a percentage value, "approximately" also means ±10%, but cannot exceed the value 100%.

[0115] In one embodiment, the axially-conveying element is a pitched blade impeller.

[0116] The ratio h of the height of the axially-conveying elements and / or the width of the blades of the radially-conveying elements SB / b is between 0.5 and 4, in another embodiment between 0.8 and 3, and in a further embodiment between 1 and 2. In another embodiment, the pitch of the agitator blades of the pitched blade impeller is between 10 and 80 degrees relative to the agitator shaft axis, in a further embodiment between 24 and 60 degrees, and in one embodiment between 40 and 50 degrees.

[0117] In one embodiment, the radially-conveying elements have 1 to 8 blades, in another embodiment 1 to 4 blades, and in a further embodiment 4 blades. In one embodiment, the axially-conveying elements have 1 to 10 blades, in another embodiment 2 to 6 blades, and in a further embodiment 4 blades. In another embodiment, the radially-conveying elements and the axially-conveying elements have the same number of blades.

[0118] In one embodiment, the agitator has a height of 20 mm to 500 mm.

[0119] In one embodiment, the agitator and the SUSVB form a functional unit, i.e., the agitator is located within the culture vessel of the SUSVB and can rotate within the culture vessel without spatial limitations.

[0120] In one embodiment, the SUSVB according to the present invention is a stirred tank reactor-like SUSVB.

[0121] In a further embodiment, the culture vessel is an aerated or submerged gas-agitated reactor-like vessel.

[0122] In one embodiment, the culture vessel includes two (114, 126) or four (114, 126, 125, 128) baffles. In another embodiment, the baffles are spaced equal distances from each other around the circumference of the inner surface of the culture vessel.

[0123] The ratio d / D of the agitator diameter (d) to the culture vessel diameter (D) is 0.2 to 0.8 in one embodiment, 0.3 to 0.6 in another embodiment, and 0.33 to 0.5 in yet another embodiment. In another embodiment, the ratio H / D of the fill height (H) of the culture vessel to the culture vessel diameter (D) is 1.0 to 2.5, 1.1 to 2.0 in a further embodiment, and 1.4 to 1.8 in yet a further embodiment. In one embodiment, the culture vessel has a working volume of 20 mL to 350 mL.

[0124] In one embodiment, the ratio of the height difference (Δh) of the two axially conveying elements to the culture vessel diameter (D) is at least 0.75.

[0125] The following examples, sequences and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0126] Abbreviation: The abbreviations used in this application have the following meanings (see also Figure 13): b: width of the blade of the radially conveying element d: Agitator total outer diameter d w : Shaft diameter h: height of the agitator blade of the radially conveying element h m : Height of fastening sleeve h SB : Height of axial conveying element h u : Height of reducer Δh: difference in height between two conveying elements in the axial direction l: length of the agitator blade of the axially conveying element α: blade pitch of the blades of the axially conveying element z: Number of agitator blades per agitator d i : inner distance between the agitator blades of the radially conveying elements h 4 / 5 :4 / 5 height from the top of h K: Stirrer head, i.e., the highest point of the stirrer when not attached to the rotating shaft D: Culture container inner diameter H: Filling height of the culture vessel. [Brief explanation of the drawings]

[0127] [Figure 1] FIG. 1 is a side view of an exemplary single-use small-volume bioreactor (SUSVB) according to the present invention, including a glucose sensor (111). [Figure 2] FIG. 1 is a side view of an exemplary SUSVB according to the present invention with dimensional and volume annotations. [Figure 3] FIG. 1 is a top view of an exemplary SUSVB according to the present invention showing a glucose sensor (111) attached to the supply port area (133). [Figure 4] FIG. 1 is a top view of an exemplary SUSVB according to the present invention including a glucose sensor (111). [Figure 5] FIG. 1 is an enlarged view of a portion of a SUSVB according to the present invention including a glucose sensor (111). [Figure 6]FIG. 1 is a side view of the lower portion of an exemplary SUSVB according to the present invention, including a glucose sensor (111) embedded in culture medium (129). [Figure 7] FIG. 1 is a schematic diagram of an exemplary glucose sensor (111). [Figure 8] FIG. 11 is a time plot of the online determined glucose concentration (green line), the offline determined glucose concentration (filled circles), the volume of glucose solution added (red line), and the glucose pump operation (purple line) of culture RE02. [Figure 9] FIG. 11 is a time plot of the online determined glucose concentration (green line), the offline determined glucose concentration (filled circles), the volume of glucose solution added (red line), and the glucose pump operation (purple line) of culture RE06. [Figure 10] FIG. 11 is a time plot of the online determined glucose concentration (green line), the offline determined glucose concentration (filled circles), the volume of glucose solution added (red line), and the glucose pump operation (purple line) for culture RE10. [Figure 11] 10 is a plot of antibody concentration over time for fermentations RE01-RE02 and RE04-RE12. [Figure 12] Schematic diagrams of various embodiments of the combined agitator according to the invention, where b: width of the agitator blade, d: diameter of the agitator, dw: diameter of the rotating shaft, h: height of the agitator blade of the radially conveying agitator, hm: height of the fastening sleeve, hSB: height of the axially conveying agitator, hu: height of the reducer, l: length of the agitator blade of the axially conveying agitator, α: blade pitch of the axially conveying agitator, z: number of agitator blades per agitator, di: inner distance between the agitator blades of the radially conveying agitator, h4 / 5: height from the top 4 / 5 of h, K: agitator head. [Example]

[0128] Example 1 Culture conditions for RE01 to RE12 The culture was grown to a starting cell density of approximately 1.5 x 10 7The culture was performed at 170 mL total volume and 170 cells / mL. The culture medium was a serum-free, chemically defined medium. The culture temperature was 35°C, the gas supply rate was 5-5.5 mL / min, the agitation rate was 450-500 rpm, and the pH was set at pH 7. pH control was performed by adding 1 M sodium carbonate solution or CO2 at the maximum gas supply rate. Antifoam was added at the start of the culture and during the culture as needed. Feed 1, containing glucose, was added continuously at a predetermined rate until the culture was stopped. Feed 2, without glucose, was added as a bolus feed on days 1, 3, and 6.

[0129] In the cultures using the in-situ glucose sensor according to the present invention, sensor-dependent glucose feeding was initiated (Feed 3) after the end of Feed 1 once the determined in-situ glucose concentration had fallen below the threshold, during which the threshold was lowered.

[0130] 8-10 show plots of exemplary cultures, and FIG. 12 shows the product concentration profile.

Claims

1. - have a working volume of between 20 mL and 350 mL; - comprising an agitator shaft (108) on which is mounted at least one impeller (112); - comprising i) a feed pipe (107) comprising a sparger tube connected to a sparger (127) at its end, and ii) at least one feed line (118) having an opening at its end, - comprising two or more baffles (114; 126) extending perpendicularly from the wall of the culture vessel (105) towards the centre of said culture vessel (105), The culture vessel (105), a coupling (122) for connecting the drive shaft of a motor to said agitator shaft (108); a sparger gas inlet (116) connected to the sparge tube in the feed pipe (107) and, optionally, a gas inlet (132) connected to the headspace; a gas outlet (117) connected to the headspace of the culture vessel; at least one inlet for liquid in a feed line (118) that is part of said feed pipe (107); - one in-situ sensor port (130) fitted with a pH electrode (101); a feed port area (133) comprising said sparger gas inlet and said inlet of said at least one feed line (118); a reactor head plate (104) comprising:

1. A small volume bioreactor comprising:

1. A small-volume bioreactor, wherein the culture vessel (105) and the reactor head plate (104) both consist essentially of non-metallic materials, the sparge tube is part of the feed pipe (107) and the at least one feed line (118) is part of the feed pipe (107), and the small-volume bioreactor includes an in-situ glucose sensor.

2. 10. The small-volume bioreactor of claim 1, wherein the small-volume bioreactor is a single-use small-volume bioreactor.

3. 3. The small-volume bioreactor of claim 1 or 2, wherein the small-volume bioreactor is a radiation-sterilized small-volume bioreactor.

4. 4. The small volume bioreactor of claim 1, wherein the small volume bioreactor is a sterile small volume bioreactor and has been sterilized twice using radiation.

5. 5. The small volume bioreactor of claim 3 or 4, wherein the radiation is beta radiation and / or gamma radiation.

6. 5. The small volume bioreactor of claim 4, wherein the first radiation is beta radiation and the second radiation is gamma radiation, or vice versa.

7. 7. The small volume bioreactor of claim 1, wherein the glucose sensor is a screen-printed electrode coated with an immobilized enzyme.

8. 8. The small volume bioreactor of claim 1, wherein the glucose sensor substrate is a USP Class VI polymer.

9. 9. The small volume bioreactor of claim 1, wherein the glucose sensor determines the glucose concentration and / or determines changes in glucose concentration every 20 seconds.

10. 10. The small volume bioreactor of claim 1, wherein the determined glucose concentration value is transmitted from the glucose sensor to a computer wirelessly or by cable.

11. 11. The small volume bioreactor of any one of claims 1 to 10, wherein the reactor head plate (104) further comprises a sampling port (102).

12. 12. The small volume bioreactor of any one of claims 1 to 11, wherein the in-situ glucose sensor passes through the head plate (104) at or within the feed port area (133).

13. 13. A method for culturing mammalian cells using a small volume bioreactor according to any one of claims 1 to 12.

14. 13. A method for determining culture conditions using a small volume bioreactor according to any one of claims 1 to 12.

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