Cell culture method

Abstract

The present disclosure provides an unsteady continuous perfusion cell culture method for producing proteins in a bioreactor. After entering the production stage, the cells are cultured in an unsteady state, so that the activity is reduced along with time. Productions of protein products using these cell culture methods are increased.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to provisional application No. 63 / 443,190, filed February 3, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure provides a non-steady-state continuous perfusion cell culture method for producing proteins in a bioreactor. After entering the production phase, cells are cultured in a non-steady-state, resulting in a decrease in viability over time. Using these cell culture methods, the production of protein products is increased. Background Art

[0004] A variety of cell culture methods are used to produce recombinant biopharmaceutical proteins from bioreactors. The protein product yields and processing times associated with these methods remain areas of development in the bioprocessing field. Changes in protein product manufacturing that increase productivity, improve product consistency, reduce starting material costs or processing times, or lower equipment costs can have significant economic benefits.

[0005] Bioprocessing can be carried out in batches (the most common fed-batch processing) or in a continuous perfusion system. Mammalian cells (such as Chinese hamster ovary (CHO) cells) are often used in bioprocessing. In a batch system, the protein product is harvested at the end of the culture run. In a perfusion system, fresh culture medium is regularly added to the cell culture, and the culture medium fluid containing the protein product is continuously harvested throughout the cell culture run. The perfusion system uses a retention device based on filtration to capture the protein product and / or cells in operation by, for example, tangential flow filtration, recirculating tangential flow filtration, or alternating tangential flow filtration.

[0006] In a steady-state perfusion system, the culture cell density is maintained essentially constant by removing excess cells with a harvesting solution, and cell growth continues. In contrast, in a non-steady-state perfusion system, cells are allowed to grow in a growth phase up to a user-defined maximum, after which cell growth slows and eventually stops to favor protein production.

[0007] The first step of bioprocessing relates to a series of amplification and expansion stages, which are intended to produce enough cell masses to inoculate production bioreactors. By thawing the bottle from the working cell bank (WCB) and by using, for example, a series of shake flasks, culture bags and / or amplification seed bioreactors (for example, N-3, N-2, where the numerals represent how many steps the bioreactor has from N (ultimate) or production bioreactors) to start the cell culture process. After growing in the seed culture bioreactor, the culture is transferred to the N-1 bioreactor, which can be, for example, a perfusion bioreactor. In the N-1 perfusion bioreactor, the culture is perfused with fresh culture medium to produce enough cell densities for inoculating the final culture step, i.e., production bioreactor (N). Operation (N) production bioreactor is maximized so that the effective production of the protein product is maximized.

[0008] There remains a need in the art to increase the productivity of perfusion systems used in bioprocessing. Summary of the Invention

[0009] The present disclosure provides a method for producing a protein product in a continuous perfusion mode in a bioreactor. The method includes a growth phase, followed by a production phase that is not operated under steady-state cell culture conditions. The growth phase includes, but is not limited to, the following steps: (a) cells and liquid culture medium expressing the protein product are inoculated into a bioreactor with a high cell density, and (b) the cells are grown to a first biomass set point at a set temperature and a gradually higher permeation rate; and the non-steady-state production phase includes, but is not limited to, the following steps: (c) when the first biomass set point is reached, switching to a lower temperature or a lower permeation rate to begin transitioning the culture to protein production, (d) the cells are grown at a set temperature and permeation rate until a second higher biomass set point is reached that promotes non-steady-state cell culture and high productivity, (e) the cells are cultured under the culture conditions described in (d) so that viability decreases over time, and (f) the protein product is collected from a harvest stream during the production phase.

[0010] The variable cell density (VCD) may decrease over time and / or the packed cell volume (PCV) may increase over time.

[0011] One or more cell releases can be performed during the production phase to ensure that the culture does not exceed the maximum viability. Once the cell growth exceeds the second biomass set point, release occurs. In this case, when the culture is in a non-steady state, the release rate in step (e) is reduced or dropped to zero. A manual non-zero constant release can be used in the non-steady state.

[0012] The high cell density of (a) can be about 200,000 cells / mL to about 5 million cells / mL, about 1 million cells / mL to about 5 million cells / mL, or about 1 million cells / mL, about 2 million cells / mL, or about 4 million cells / mL.

[0013] The first biomass set point can be about 50 million cells / mL to about 100 million cells / mL. The second biomass set point can be about 100 million cells / mL to about 150 million cells / mL.

[0014] The permeation rate of (b) can be from 0 to about 4.1 working volumes / day. In step (b), the permeation rate increases with the increase in biomass to support cell growth. The permeation rate of (d) can be from about 1.0 to about 4.2 working volumes / day.

[0015] The maximum VCD of (d) can be about 130 million to about 140 million cells / mL. The VCD can be reduced to about 10 million cells / mL to about 120 million cells / mL during the production phase.

[0016] The set temperature of (b) may be about 35.5°C to about 36.5°C, or for example about 36°C.

[0017] The set temperature transitions of (c) and (d) may be to lower the temperature to about 32.5°C-35.5°C.

[0018] The permeation rates of (c) and (d) that induce the transition can be reduced to about 1.0 to about 2.5 working volumes per day.

[0019] The cell discharge during the growth phase may be 0% to about 40%. The cell discharge during the growth phase may be 0% to about 3%. The cell discharge during the growth phase may be about 1.5%.

[0020] The cell viability in the growth phase can be about 90% to about 99%. The cell viability in the growth phase can be about 97%. In step (e), the viability is reduced over time to about 30% to about 80%, about 30% to about 75%, about 35% to about 70%, about 35% to about 60%, about 35% to about 50%, or about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45% or about 40% viability.

[0021] The PCV during the growth phase may increase to a value of about 2% to about 24%.The PCV during the production phase may be about 25% to about 50%, or about 40% to about 50%.

[0022] The duration of the growth phase can be from about 4 to about 12 days. The duration of the production phase can be from about 9 to about 41 days. The duration of the production phase can be about 10 days.

[0023] These methods may further comprise the downstream steps of subjecting the harvested protein product to capture chromatography, viral inactivation, and / or polishing steps.

[0024] The cells in these methods can be mammalian cells. The mammalian cells can be Chinese hamster ovary (CHO) cells.

[0025] The protein product may be, for example, an antibody product. The protein product may be a bispecific antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Daily trends over the course of the CM run for the first antigen binding protein are shown.

[0027] Figure 2 Daily trends over the course of the CM run for the second antigen binding protein are shown.

[0028] Figure 3 Daily trends over the course of the CM run are shown for a third antigen binding protein. DETAILED DESCRIPTION

[0029] This article provides a non-steady-state continuous perfusion culture process for the manufacture of biological preparations. Unlike traditional extended continuous perfusion technology, after reaching the maximum peak growth defined by the user, the cell culture is not operated under steady state. This process improves productivity (because cells remain in the production phase after the cell growth plateau) and reduces the culture duration, which improves productivity compared with typical perfusion culture processes. The production phase of the culture begins with the transition from the growth phase. Modify the culture conditions to favor product production rather than exponential cell growth. Due to the change in culture conditions, cells are transitioned from the growth phase to the production phase, and viable cell density and viability are forced to decline. Once the maximum value set is reached, the cell culture is transitioned to the production phase. This transition can be achieved by temperature conversion or other means. Alternatively, there may be a short-term biomass reinforcement at the beginning of the perfusion phase, wherein cell discharge is optionally performed to maintain the biomass set point, but after the reinforcement period, the biomass is allowed to decline far beyond the set point, resulting in no net cell growth. Discharge can be performed to ensure that the cell density does not exceed the maximum viable cell density (VCD). When using discharge, as the cell growth decreases, discharge also decreases. During the non-stationary portion of the production phase, cell diameter and biovolume (as measured by, for example, packed cell volume (PCV)) increase and biomass decreases due to the absence of net cell growth. It was found that under these conditions, cell diameter and biovolume (as measured by packed cell volume (PCV)) increased, indicating that the cells remained in the production phase.

[0030] According to the present disclosure, operating a production bioreactor under non-steady-state cell culture unexpectedly results in higher productivity (protein production) than operating under steady-state cell culture. This shortens the culture duration and therefore increases the utilization of the bioreactor and improves process economics. The production (N) bioreactor operation herein can be extended and flexible time periods, such as 15-35 days. Due to the reduction in discharge demand, the harvest yield in the culture is increased. For example, traditional steady-state culture continuously removes about 10% of the cells within a 20-day culture period. By operating the culture under non-steady state, the discharge can be reduced by half or more. Therefore, the non-steady-state operation according to the present disclosure produces an economically advantageous process. In many non-steady-state continuous manufacturing (CM), the protein product quality is similar, indicating that the process is in a controlled state and meets the requirements of government regulations.

[0031] As used herein, the term "viable cell density" or "VCD" refers to the number of viable cells present in a given volume of culture medium under a given set of experimental conditions (e.g., cells / mL). In the methods herein, during the growth phase preceding the production phase, the culture cell mass grows to approximately 60×10 6 cells / ml to about 143×10 6In some embodiments, the production phase may be initiated in continuous perfusion mode at a maximum VCD set point of 10 cells / ml, and then the production phase may be initiated in continuous perfusion mode, and the VCD may be allowed to decline throughout the remainder of the culture. During the production phase, the VCD may decline by at least 10%, at least 15%, at least 20%, or at least 25% from the maximum set point. During the production phase, the VCD may decline by at least 2% / day, at least 3% / day, at least 4% / day, or at least 5% / day. These methods may further include performing an initial purge during the production phase to prevent the VCD from exceeding the maximum set point. A second maximum VCD set point may be used during the decline period to prevent clogging of the filter device.

[0032] As used herein, the "growth phase" of a cell culture refers to a phase in which the viable cell density at any point in time is higher than at any previous point in time. Cells can be in the growth phase for at least 4 days.

[0033] As used herein, the "production phase" of a cell culture refers to the phase during which cells produce large quantities of protein and accumulate it for future processing. For the production phase, the perfusion culture can be run continuously for at least 7 days; at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days; or at least 35 days.

[0034] Dielectric constant (pF / cm) is a measure of the electrical susceptibility of bioelectricity. Compared with materials with low dielectric constants, materials with high dielectric constants (such as the (outer) cell membranes of (living) cells) are more polarized in response to applied electrical materials. The electric displacement field D generated by the applied electric field E is D=eE. More generally, dielectric constant is a thermodynamic function of state. It may depend on the frequency, amplitude and direction of the applied electric field. The SI unit of dielectric constant is farad / meter (F / M). Dielectric constant is measured with a dielectric constant probe (e.g., Hamilton Bonaduz AG, Switzerland). It can be measured online or manually offline. The dielectric constant increases from about 4pF / cm to a dielectric constant maximum of 60-115pF / cm during the growth phase. Due to non-steady-state cell culture operations, the dielectric constant decreases from the dielectric constant maximum during the production phase.

[0035] Permeation rate (wv / day) is the measurement of the volume of culture medium that passes through and removes from the bioreactor system every day. Typically, this is measured relative to the volume of the bioreactor (as " bioreactor volume " (bv) or " working volume " (wv) / day). Permeation rate can be about 1.0 to about 4.2wv / day. During the production phase, the permeation rate is about 2.0.

[0036] Biomass specific permeation rate (pF / cm. day) is drawn by permeation rate (1 / day) divided by dielectric constant (pF / cm).It is the fresh feed rate with respect to the biomass in the culture.Biomass specific permeation rate and temperature are the process parameters that can be used for process design space being defined as steady state or unsteady state.This calculation rate is used for the process input in the operation of comparing single cell line, because it is combined into one with two process parameters.Therefore, by temperature and biomass specific permeation rate when observing biomass maximum, process design space can be defined.If repeat identical cell line and product when biomass maximum (relative ratio of perfusion rate and biomass maximum) under identical temperature and biomass specific permeation rate, then aspect protein production and biomass reduction rate (cell culture performance index), can expect similar result.

[0037] Packed Cell Volume (PCV) – The biological volume (sometimes referred to herein as cell mass or biomass) of cells in a culture, expressed as a percentage, relative to the total volume of the culture. PCV can increase due to an increase in cell number, cell diameter for the same number of cells, or a combination. When the cell discharge in a continuous culture is zero (indicating no cell growth) and the PCV continues to increase, this indicates that the cell diameter is increasing.

[0038] A "biomass capacitance probe" is a probe that can measure viable cell density and other capabilities. A biomass capacitance probe uses capacitance to measure the total viable cells in a culture. Living cells act as capacitors in an alternating electric field. The biomass capacitance probe can measure and report the charge from these cells.

[0039] Steady state and non-steady state

[0040] The CM culture design space consists of several process parameters that can produce conditions that favor cell growth over protein production (steady-state CM) or conditions that favor protein production over cell growth (non-steady-state CM). In steady-state culture, all culture set points and outputs remain unchanged during the culture period. This is only possible if cells grow at a rate (cell growth rate) that maintains VCD, viability, PCV, and dielectric constant, but this allows cell resources to be preferentially diverted from protein production to cell growth. Typically, in the field of continuous manufacturing, VCD, viability, and usually protein production rate are constant throughout the culture production period. Cell growth is equal to the cell removal rate, and cells both grow and produce protein.

[0041] However, in non-steady-state cultures, cell growth slows or stops completely to favor protein production. This results in a decrease in cell number and viability (due to some cell death) and an expansion in size, increasing cell diameter, to accommodate the increased protein production. Therefore, non-steady-state CM processes result in higher protein production rates and are therefore more economically attractive than traditional steady-state processes.

[0042] There are large and small non-steady-state operations. Performance indicators are measures of cell number, size and viability (i.e., VCD, viability, cell diameter, PCV and dielectric constant) and protein production rate. The process parameters that adjust the magnitude of their effect on the performance indicators are temperature and permeation rate relative to the biomass in the culture (biomass-to-permeation rate). Process parameters can be modified to meet the targets for the performance indicators. Fouling of the ATF filter due to high biomass, cell debris, culture viscosity or high permeation rate limits the maximum biomass and permeation rate. In addition, low temperatures and permeation rates with high biomass may prevent cell growth too quickly, which may end the culture before sufficient protein is produced. Therefore, the maximum biomass (target dielectric constant), temperature and permeation rate are optimized in the process design space to meet the protein production target while considering process robustness.

[0043] In steady-state operation, the dielectric constant / biomass is maintained at a single target level in the production phase. In unsteady-state operation as described herein, the dielectric constant / biomass is maintained at or below the maximum value in both parts of the production phase, maintained at a higher maximum value in the biomass intensification stage, and maintained at a second lower maximum value in the remaining operation. Under unsteady state, biomass needs to be maintained below the maximum value to reduce the possibility of ATF fouling. The two-step maximum method (biomass intensification, then reducing the dielectric constant maximum) can reduce the risk of ATF fouling / malfunction and has the advantage of higher productivity from a higher target dielectric constant.

[0044] Biomass intensification stage

[0045] In the present invention, the biomass intensification stage is used for the production of protein.This stage refers to before allowing biomass to reduce, and biomass is increased to set point in a short period of time.This intensification stage causes protein production higher in the remaining operation.The set point in the initial biomass stage is higher, and the cell that can be used for protein production in the remaining operation is just more, and this causes higher process productivity.This stage is optional in non-stable CM.The biomass intensification stage is similar to the growth stage, and is typically designed to occur right after the growth stage.Difference is that the biomass intensification stage has lower (production stage) temperature set point and different target dielectric constants, and this target dielectric constant is higher than the growth stage target dielectric constant.

[0046] Cell culture

[0047] The cell culture methods herein are performed in a production bioreactor in a continuous perfusion mode (typically using alternating tangential flow filtration technology), in non-steady state operation.Cell culture refers to a liquid culture medium containing a plurality of cells maintained or propagated under a set of controlled physical conditions.

[0048] Mammalian cells (such as CHO cells) can be cultured on a small scale, such as, for example, in a 100 ml container with about 30 ml of culture medium, a 250 ml container with about 35 to about 70 ml of culture medium, or a 500 ml container with about 100 to about 200 ml of culture medium. Alternatively, culture can be large-scale, such as, for example, a 1000 ml container with about 140 to about 300 ml of culture medium, a 3000 ml container with about 500 ml to about 2200 ml of culture medium, a 50 L container with about 4 L to about 30 L of culture medium, and a 200 L container with about 50 L to about 135 L of culture medium. Large-scale cell culture (such as for clinical manufacture of protein therapeutics) typically maintains several days or even weeks while the cells produce the desired protein.

[0049] The term "bioreactor" means any container that can be used for cell culture growth, such as a fluidized bed bioreactor, a hollow fiber bioreactor, a roller bottle, a shake flask, or a stirred tank bioreactor. A bioreactor can have any size as long as it can be used for cell culture; typically, the size of the bioreactor is suitable for the volume of the cell culture grown therein. Typically, the bioreactor will be at least 1 liter and can be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1500, 2000, 2,500, or 5,000 liters or larger, or any volume therebetween. A bioreactor can be 8,000, 10,000, 12,000, 18,000, 25,000 liters or larger, or any volume therebetween. The internal conditions of the bioreactor, including but not limited to pH and temperature, can be controlled during the incubation period. One of ordinary skill in the art will appreciate and will be able to select a suitable bioreactor for practicing the methods described herein.

[0050] The method disclosed herein can be carried out using a disposable bioreactor (single-use bioreactor or disposable bioreactor), which utilizes disposable bags to replace traditional culture containers. Transitioning to disposable technology minimizes the infrastructure requirements associated with traditional cell culture (such as steel / glass industrial-scale containers and related machines). Disposable bioreactors provide flexibility for manufacturing processes; and compared with traditional cell culture factories, site assembly, reconfiguration, sterilization and verification are faster, simpler and less costly. Disposable bioreactors typically utilize disposable plastic sterile bags supported by non-disposable support structures. The culture is stirred by an agitator in the bag or by shaking, and sensors measure and adjust various parameters of the culture, such as pH, temperature, oxygen, cell density, etc. Disposable bioreactors are commercially available from, for example, Xcellerex, General Electric (GE), Hyclone and Sartorius.

[0051] The bioreactor system maintains the conditions in the bioreactor to support cell culture. Suitable culture conditions for mammalian cells are known in the art. See, for example, Animal cell culture: A Practical Approach [animal cell culture: a practical method], edited by D. Rickwood, Oxford University Press [Oxford University Press], New York (1992). "Running" a bioreactor system means maintaining the conditions in the bioreactor system to support cell culture. A bioreactor "run" typically comprises the following steps: inoculating the prepared bioreactor with a seed culture, and allowing the culture to grow for a suitable or predetermined time until the culture terminates (usually by harvesting the contents of the bioreactor). For a production bioreactor (N bioreactor) operation, a seed bioreactor or an N-1 bioreactor is typically used to grow cells for inoculating a production bioreactor.

[0052] "Cultivation" refers to maintaining cells in a culture medium under conditions suitable for the survival and / or proliferation of cells other than multicellular organisms or tissues and suitable for producing protein products. Cell culture is typically operated in batch, fed-batch or perfusion mode. In batch mode, a fixed amount of culture medium and cells are added to the bioreactor at the beginning of the operation. During the cultivation process, the volume of culture medium in the reactor remains constant, while the nutrient content of the culture medium decreases. The cell concentration continues to increase during operation and may stabilize and decline as the nutrient content is exhausted and waste increases. Like batch culture, fed-batch culture starts with inoculated cells and a fixed amount of culture medium. Unlike batch culture, the volume of culture medium in the bioreactor increases as concentrated nutrients are added during the cultivation process.

[0053] "Growth" cell culture medium or feed medium refers to a cell culture medium that is typically used for cell culture during the exponential growth phase ("growth phase") and is sufficiently complete to support cell culture during this phase. Growth cell culture medium can also contain a selection agent that confers selectable marker resistance or viability that is incorporated into the host cell line. Such selection agents include, but are not limited to, geneticin (G4118), neomycin, hygromycin B, puromycin, bleomycin, methionine sulfenyl imide, methotrexate, cell culture medium without glutamine, cell culture medium lacking glycine, hypoxanthine and thymidine or a single thymidine. Growth cell culture medium is known in the art.

[0054] "Production" cell culture medium or feed medium refers to a cell culture medium typically used for cell culture during the transition period at the end of exponential growth and during the subsequent transition and / or production phase when protein production takes over. Such cell culture medium is sufficiently complete to maintain the desired cell density, viability, and / or product titer during this phase. Production cell culture media are known in the art.

[0055] As with batch culture, perfusion culture starts from the fixed inoculation of cells and culture medium.Different from batch culture and fed batch culture, fresh feed medium is added or perfused in bioreactor, and the spent culture medium of equivalent is taken out.In the case of methods described herein, perfusion is continuous perfusion.Retention device (such as tangential flow filtration (TFF) system, alternating tangential flow (ATF) system or recirculating tangential flow (RTF) system) can be used to remove spent culture medium and unwanted by-products from bioreactor.

[0056] RTF relies on the use of a recirculation device (most commonly a peristaltic pump) to move the cell culture in a unidirectional and parallel manner to the membrane surface to allow the removal of spent culture medium while retaining the cells in the bioreactor. The ATF system is similar to the RTF system, except that a pump is used to move the cell culture back and forth in the module (e.g., through a hollow fiber filter module) rather than flowing in only one direction. See, for example, U.S. Patent No. 6,544,424; Furey, 2002, Gen. Eng. News. [Gene Engineering News] 22 (7): 62-63. The benefit of ATF is the cleaning effect on the filter caused by the alternating flow. An exemplary method of the present disclosure includes running (N) bioreactors using an ATF perfusion system.

[0057] Typically, hollow fiber filters are used in RTF or ATF systems (although this is not required). When cell culture (including cell culture medium, cells (complete and lysis), soluble expressed recombinant proteins, host cell proteins, waste, etc.) are introduced into the filter, according to pore size or molecular weight cut-off (MWCO), the hollow fiber material can retain certain cell culture components (except the cells themselves) on the lumen side (inside), and allows certain components to pass through the filter (permeate) based on the pore size or molecular weight cut-off of the hollow fiber material. The retained material (retentate) is returned to the bioreactor. Fresh perfusion cell culture medium is added to the bioreactor, and the permeate is taken out from the filter at predetermined time intervals or continuously to maintain the required or constant bioreactor volume. The permeate can be discarded, stored in a storage tank, bag or tote or directly transferred to another unit operation, such as filtration, flocculation, centrifugation and / or other downstream purification methods, etc.

[0058] In various fields, hollow fiber has an internal diameter of about 0.5mm to about 1mm, and can have any suitable length (for example, about 30cm to about 110cm).Hollow fiber for microfiltration typically has the pore size in the range of 0.1 μm to 10 μm or 500 to 750kDa or larger molecular weight cut-off, and can be used for allowing protein to pass into permeate.Ultrafiltration hollow fiber typically has the pore size range or 300kDa or less molecular weight cut-off of 0.01 μm to 0.1 μm, and can be used for required protein being retained in the retentate and being returned to bioreactor.This can be used for example to concentrate recombinant protein product for gathering in the crops. Such filters are commercially available as Xampler UFP-750-E-4MA, Xampler UFP-30-E-4MA (GE Healthcare, Pittsburgh, PA) and Midikros TC modules T02-E030-10, T02-050-10, T02-E750-05, T02-M10U-06 (Spectrum Laboratories, Inc., Dominguez, CA), XCell (Repligen, Waltham, Massachusetts).

[0059] The cell culture fluid can be drawn from the bioreactor and into the filter module by a pumping system that moves the cell culture fluid through or along the filter (e.g., through the lumen side of the hollow fiber). Examples of cell pumping systems include peristaltic pumps, double diaphragm pumps, low shear pumps (Levitronix TM pumps, Zurich, Switzerland) and alternating tangential flow (ATF TM , Repligen, Inc., Waltham, MA). The permeate can be drawn from the filter using a peristaltic pump. In these examples, perfusion is achieved using an alternating tangential flow system.

[0060] The method of the present disclosure can be used as a part of a larger production process, whereby cells are cultivated in three or more different stages. For example, cells can be cultivated in one or more growth phases before the N-1 production stage, cultivated in the N-1 production stage, and then transferred to the (N) production stage under the conditions of maximizing protein production. Each stage can be carried out in its own bioreactor container or other containers suitable for cell culture. Alternatively, more than one stage can be carried out in a shared container. In one such example, the growth phase and the production stage are carried out in the same bioreactor container. In the commercial process of producing protein by mammalian cells, there are usually multiple, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9 or 10 growth phases, which occur in different culture vessels before the final production stage.

[0061] The duration of the N-1 period can range from, for example, 3 to 14 days and can be designed to maintain exponential growth of cells prior to inoculating the production (N) bioreactor.

[0062] Cell culture medium is a culture medium suitable for the growth of animal cells (such as mammalian cells) in in vitro cell culture. Cell culture medium preparations are well known in the art. Typically, cell culture medium is composed of buffers, salts, carbohydrates, amino acids, vitamins and trace essential elements. Cell culture medium may or may not contain serum, peptone and / or protein. Various tissue culture media (including serum-free and defined culture media) are commercially available, for example, any one or combination of the following cell culture media can be used: RPMI-1640 culture medium, RPMI-1641 culture medium, Dulbecco's modified Eagle's medium (DMEM), Eagle's minimal essential medium, F-12K culture medium, Ham's F12 culture medium (Ham's F12 Medium), Iscov's modified Dulbecco's medium, McCoy 5A culture medium, Leibovitz L-15 culture medium and serum-free culture medium such as EX-CELL TM 300 series (JRH Biosciences, Lenexa, Kansas), etc. Depending on the needs of the cells to be cultured and / or the desired cell culture parameters, the cell culture medium can be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, etc.

[0063] Cell culture medium can be serum-free, protein-free and / or peptone-free. "Serum-free" is applicable to cell culture medium that does not contain animal serum (such as fetal bovine serum). "Protein-free" is applicable to cell culture medium that does not contain protein (such as transferrins, protein growth factor IGF-1 or insulin) added by exogenous sources. Protein-free culture medium may contain or may not contain peptone. "Peptone-free" is applicable to cell culture medium that does not contain exogenous protein hydrolysate (such as animal and / or plant protein hydrolysate). Removing serum and / or hydrolysate from cell culture medium has the advantage of reducing batch variability and enhancing processing steps (such as filtration). However, when removing serum and / or peptone from cell culture medium, cell growth, viability and / or protein expression may weaken or be lower than optimal levels. Therefore, serum-free and / or peptone-free cell culture medium can be highly rich in amino acids, trace elements etc. Referring to, for example, U.S. Patent number 5,122,469 and 5,633,162.

[0064] Defined cell culture medium formulations are complex, containing amino acids, inorganic salts, carbohydrates, lipids, vitamins, buffers, and trace amounts of essential elements. Identifying the components necessary and beneficial for maintaining cell cultures with desired characteristics is an ongoing task. One approach to developing defined media is to supplement or enrich defined basal medium formulations to meet the needs of specific host cells or to satisfy desired performance parameters.

[0065] Induction production stage

[0066] Bioreactor process parameters that can be used to switch to non-steady-state conditions are permeation rate and temperature. The combination of permeation rate, temperature, and biomass can be used to promote protein production rather than cell growth and lead to non-steady-state CM operation. These parameters are determined empirically for each cell line to maximize productivity.

[0067] The cell culture temperature is typically 35°C to about 38°C. The cell culture typically contains at least one exponential growth phase and may contain a production phase. The growth phase can be carried out at a temperature higher than the production phase. For example, the growth phase can occur at a first temperature of about 35°C to about 37°C, and in order to induce a non-steady state, the production phase can occur at a second temperature lower than the first temperature, such as about 29°C to about 37°C, about 30°C to about 36°C, about 32°C to about 36°C, or about 30°C to about 34°C. In addition, chemical inducers for protein production, such as caffeine, sodium butyrate, and hexamethylenebisacetamide (HMBA), can be added before, during, or after the temperature transition. If the inducer is added after the temperature transition, the inducer can be added one to five days after the temperature transition, or alternatively one to two days after the temperature transition. The growth phase can also be carried out at a pH higher than the production phase.

[0068] The permeation rate can be reduced to about 1.0 to about 2.5 working volumes per day to induce a non-steady state.

[0069] In culture process, can use a variety of culture medium preparations, for example, to promote from one period (for example, vegetative period or growth stage) to change to another period (for example, production period or production stage) and / or optimize the condition (for example, the concentrated culture medium provided during perfusion culture) during cell culture. Growth medium preparation can be used to promote cell growth and minimize protein expression. Production medium preparation can be used to promote the production of target protein and the maintenance of cell, while minimizing cell growth. During the cell culture process, the consumed feed medium (typically containing more concentrated components, such as nutrients and amino acid whose culture medium) can be used to supplement and maintain active culture, particularly with the culture of fed-batch operation. " perfusion " culture medium refers to the cell culture that is specially developed for maintaining by perfusion method and is sufficiently complete to support the feed medium of cultivation during this process. Compared with basal medium and fed-batch feed medium preparation, perfusion medium preparation is typically more abundant, more concentrated and continuously fed with higher cumulative volume during culture, to adapt to higher cell density and for removing the method for spent culture medium. Perfusion medium can be used in both the growth stage and the production stage of cultivation. Such concentrated feed media and perfusion media typically contain those components that have been depleted and / or necessary to maintain the culture, and may be present in the concentrated cell culture medium, for example, at about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 20-fold, 30-fold, 50-fold, or more of their normal amount in the basal medium.

[0070] The culture pH is controlled at a preferred pH, typically about 6 to 7.4, preferably pH 6.85 to 7.2. In one embodiment, the pH is 6.90 to 6.95. The pH can be controlled by bubbling CO2 and 1M sodium carbonate. The dissolved oxygen is preferably 40 to 88 mmHg, more preferably 60-70 mmHg. Defoamer can be added at a frequency and volume suitable for the culture operation, and supplemental boluses of defoamer can be added as needed.

[0071] "Cell" includes any prokaryotic or eukaryotic cell. Cells include "host cells," also referred to as "cell lines," which are genetically engineered to express a protein of interest. Host cells are typically derived from a lineage from a primary culture that can be maintained in culture for an indefinite period of time. Genetically engineered host cells involve transfection, transformation, or transduction of cells with recombinant polynucleotide molecules, and / or otherwise altered (e.g., by homologous recombination and gene activation or fusion of recombinant cells with non-recombinant cells) to cause the host cells to express the desired protein. Methods and vectors for genetically engineering cells and / or cell lines to express a protein of interest are well known to those skilled in the art.

[0072] The host cell can be a eukaryotic cell, such as a mammalian cell. Any mammalian cell suitable for recombinant protein expression is suitable for use in the context of the present disclosure. Suitable mammalian cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, mouse myeloma (NS0, Sp2 / 0) cells, baby hamster kidney (BHK) cells, human embryonic kidney (293) cells, fibrosarcoma (HT-1080) cells, human embryonic retina (PER.C6) cells, hybrid kidney and B cells (HKB-11), CEVEC amniotic fluid cell production (CAP) cells, human liver (HuH-7) cells, and any other cells used for or applicable to clinical and / or commercial manufacturing. The most commonly used cell line is from CHO cells. CHO cells are widely used in the production of composite recombinant proteins. Dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXB11, and DG-44 are desirable CHO host cell lines because the efficient DHFR selectable and amplifiable gene expression system allows high-level recombinant protein expression in these cells (Kaufman RJ (1990), Meth Enzymol 185:537-566). The glutamine synthetase (GS) knockout CHOK1SV cell line, which utilizes glutamine synthetase (GS)-based methionine sulfenyl imide (MSX) selection, is also widely used. CHOK1 cells (ATCC CCL61) are also included. Key attributes and performance parameters of the cell lines can be measured to better inform decisions about performance at each step during manufacturing. These key attributes and parameters can be monitored in real time, near real time, and / or post-hoc. During cell culture, key parameters such as the levels of consumed culture medium components (e.g., glucose), metabolic byproducts that may accumulate in the culture (e.g., lactate and ammonia), and those related to cell maintenance and survival, such as dissolved oxygen content, can be measured. Key attributes such as specific productivity, viable cell density, pH, osmotic pressure, appearance, viability, aggregation, cell count, packed cell volume, product quality, yield percentage, and titer can be monitored during appropriate periods of the manufacturing process. Process and product impurities can also be monitored throughout the manufacturing process.

[0073] Known techniques and commercially available equipment can be used to monitor and measure.Mass spectrometry, liquid chromatography with UV and / or mass spectrometry detection, capillary electrophoresis, etc. can be used to realize the detection of product quality attributes.Polyhydroxyethyl aspartate amide column such as size exclusion mode operation and coupled with ESI-MS can be used to characterize post-translational modification such as amino acid processing and glycosylation (Brady et al., (2008) J Am Soc Mass Spectro [American Mass Spectrometry Society], 19: 502-509).The real-time monitoring (see, for example, U.S. Patent Publication No. US20130303732) of the eluent from ion exchange chromatography can be carried out by monitoring the normalized LS / UV ratio of each fraction using a laser scattering detector and UV absorbance.

[0074] For example, variable cell density (VCD) and vigor (%) can be determined using Cedex HiRes (Roche, Basel, Switzerland). Glucose, lactate, and NH4+ concentration can be determined using Cedex BioHT (Roche, Basel, Switzerland). Titer can be determined by high performance liquid chromatography (HPLC) via affinity chromatography (Protein A, Waters, Milford, Massachusetts). The percentage of impurities can be determined using reduced capillary electrophoresis sodium dodecyl sulfate (rCE-SDS), non-reduced capillary electrophoresis sodium dodecyl sulfate (nrCE-SDS), ultra-high performance liquid chromatography (SE-UHPLC), acidic and alkaline charged variants (CEX-HPLC) using cation exchange chromatography.

[0075] The cell culture viscosity (including the components of the culture medium and the cells themselves) can be between about 1 and about 6 centipoise (e.g., about 2 to about 6 centipoise). The cell culture viscosity can be characterized using any suitable viscometer (e.g., a "cone and plate" viscometer). The cell culture density (including the components of the culture medium and the cells themselves) can be about 1 g / L to about 1.5 g / L. The cell culture density can be characterized, for example, using an automated cell counter, such as a Roche Cedex HiRes using the Trypan Blue Exclusion method.

[0076] Harvest

[0077] The methods provided herein may further include harvesting the protein product from the cell culture. In the harvesting step, the protein product is separated from the cell debris and production cells in the culture medium. If necessary, the bioreactor contents can be cooled for the harvesting step. For example, the temperature of the bioreactor contents can be reduced to less than 12°C (but greater than 0°C). Harvesting can be carried out by any suitable method, including acid precipitation, accelerated sedimentation (such as flocculation), gravity separation, centrifugation, sonic separation, filtration, including membrane filtration using an ultrafilter, microfilter, tangential flow filter (including using a tangential flow filter in alternative tangential flow and recirculating tangential flow), depth filter and alluvial filter. Depth filtration can be part of the harvesting process to additionally remove impurities. One or more depth filters of the same or different materials (natural and / or synthetic) can be used. Depth filters commonly used in biomanufacturing processes are typically made of cellulose or polypropylene fibers, diatomaceous earth or perlite, or charged resins. Depth filters can optionally include filter membrane layers of different pore sizes (such as 0.22 μm).

[0078] Downstream purification

[0079] The harvested protein can be further purified by one or more downstream purification processes to remove any impurities, such as remaining cell culture medium, cell extracts, host cell proteins, DNA, viruses, incorrectly expressed proteins, product-related impurities, etc. Downstream process operations can be performed in batch, semi-continuous and / or continuous modes. Two or more operations can be directly connected, for example, with a surge tank, storage tank, bag, or other suitable container suitable for receiving feed from at least one operation to another operation.

[0080] Capture chromatography is typically used as an initial purification step, followed by one or more intermediate and / or polishing chromatography steps.

[0081] Affinity chromatography is often used as an initial capture step for harvested recombinant proteins because it performs well in the purification of crude or clarified material. Affinity chromatography media can include, for example, substrate binding capture mechanisms, aptamer binding capture mechanisms, or cofactor binding capture mechanisms. For proteins containing an Fc component, substrate binding capture mechanisms such as Protein A, Protein G, Protein A / G, and Protein L can be used. A variety of Protein A affinity chromatography resins and materials are commercially available, including but not limited to MabSelect from GE Healthcare. TM , from Millipore Ultra Plus and Purolite APc+. An exemplary resin is MabSelect TMSuRe resin (GE Healthcare Life Sciences), which exhibits enhanced clearance of low molecular weight species (LMWS).

[0082] One or more intermediate chromatography and / or polishing chromatography steps remove any remaining contaminants and / or impurities. The intermediate chromatography and / or polishing chromatography steps utilize a chromatography medium, such as a resin, monolith, and / or membrane, containing an agent that can be used in a bind and elute mode (wherein the protein of interest binds to the chromatography medium and elutes after contaminants and impurities have passed through or been washed from the chromatography medium), a frontal or overload mode (wherein a solution containing the protein of interest is loaded onto the column until adsorption sites are occupied and the material with minimal affinity for the stationary phase (protein of interest) begins to elute), a flow-through mode (wherein the protein of interest flows through the chromatography material without binding, and contaminants and impurities bind to the chromatography medium), or any other suitable mode or combination of modes. Examples of the most common chromatography modes used in intermediate and / or polishing steps for biomanufacturing include, but are not limited to, ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multimodal anion exchange chromatography (MMC); and hydroxyapatite chromatography (HA). Each polishing chromatography unit operation may be run in the same or different configurations and / or in different modes.

[0083] Each chromatography unit can be operated as a single unconnected unit, multiple connected units, and / or combined units. For example, a single chromatography column can be operated in a staggered circulation system, in countercurrent loading (periodic countercurrent chromatography), or as a multi-column countercurrent solvent gradient purification process (MCSGP).

[0084] Chromatographic media are well known and common in the art and are commercially available from many sources. Cation exchange media include, but are not limited to, those containing carboxylic acid or sulfonic acid functional groups, such as, but not limited to, sulfonates, carboxylic acids, carboxymethylsulfonic acid, sulfoisobutyl, sulfoethyl, carboxyl, sulfopropyl, sulfonyl, sulfoxyethyl, or orthophosphate. CEX resins include, but are not limited to, Mustang S, Sartobind S, SO3 Monolith, SCeramic HyperD, Poros XS, Poros HS50, Poros HS20, SPSFF, SP-Sepharose XL (SPXL), CM Sepharose Fast Flow, SP Sepharose Fast Flow XL TM,SP-Sepharose HighPerformance TM 、Capto S、Capto SPImpRes TM 、 HS, XS、UNOsphere TM , FractoPrep TM , Fractogel Se HiCap, Fractogel SO3, or Fractogel COO. Anion exchange media include but are not limited to Source 15Q, Capto TM Q, Q-sepharose Fast Flow TM 、FractogelEDM TMEA TM , Fractogel EDM DEAE, TOYOPEARL Poros HQ TM , and POROS XQ TM Mixed-mode or multimodal media including Capto TM Adhere. Hydrophobic interaction chromatography materials include but are not limited to Fractogel TM EMD Propyl or Fractogel TM EMD phenyl column (Merck), Octyl Sepharose TM High-efficiency columns (Pharmacia LKB Biotechnology) with low or high substitution PhenylSepharose TM 6 Fast Flow Column (LKB Biotechnology Company), Phenyl Sepharose TM High-efficiency column (LKB Biotechnology Co., Ltd.), Macro-Prep TM Methyl or Macro-Prep TM Tert-butyl carrier (Bio-Rad), WP HI-Propyl (C3) TM Column (JTBaker) and Toyopearl TM Ether, phenyl or butyl column (TosoHaas).

[0085] Virus inactivation and virus filtration

[0086] Viral mitigation measures are critical to ensuring the safety of protein therapeutics. Viral contaminants can arise from a variety of sources, including the use of reagents of animal origin, adventitious viral contaminants in host cell lines, or system failures at GMP manufacturing sites. Viruses are classified as enveloped and non-enveloped. Enveloped viruses have a capsid surrounded by a lipoprotein membrane or "envelope," which is composed of host cell proteins and phospholipids, as well as viral glycoproteins that coat the virus as it buds from its host cell. This envelope allows the virus to recognize, bind to, enter, and infect the target host cell. Therefore, enveloped viruses are susceptible to inactivation methods. Non-enveloped viruses are more difficult to inactivate without harming the manufactured protein and are removed by filtration methods. Viral mitigation strategies can be performed once or multiple times throughout the downstream purification process.

[0087] There are many methods for virus inactivation and include heat inactivation / pasteurization, UV and gamma ray irradiation, the use of high-intensity broad-spectrum white light, the addition of chemical inactivators and surfactants. Low pH and solvent / detergent treatment are the most common virus inactivation methods in protein therapeutics manufacturing processes. Virus inactivation is typically performed after purification of the harvested fluid by affinity chromatography, particularly affinity chromatography using a substrate-binding ligand from Staphylococcus aureus, such as protein A chromatography, because elution from such chromatographic materials is typically performed at low pH. The acidified eluate is kept for a certain time, which has been determined to inactivate the virus concentration to the desired logarithmic order. This is followed by neutralization of the inactivated material. Exemplary low pH virus inactivation methods are described in U.S. application 63 / 168,608 and U.S. application 63 / 159,217. Exemplary detergent inactivation is described in International Patent Organization Publication No. WO 2020 / 190985.

[0088] Non-enveloped viruses are difficult to inactivate without damaging the recombinant product; however, such viruses can be removed by size-based filtration methods. A prefilter can be used in combination with a virus filter to help eliminate certain contaminants from the product pool or eluate stream before applying it to the virus filter, thereby maintaining continuous flow during the virus filtration operation. An exemplary process is described in International Patent Publication No. WO 2020 / 159838. Virus filtration can be performed using micro or nano filters, such as those available from (Asahi Kasei Corporation, Chicago, IL), (Sartorius AG, Göttingen, Germany), Pro (MilliporeSigma, Burlington, MA), Pegasus TMVirus filtration can occur at one or more steps downstream in the biomanufacturing process. Typically, virus filtration is performed before the UFDF operation, but it can also be performed after the UFDF operation.

[0089] UF / DF

[0090] The method further optionally includes concentrating the protein product using ultrafiltration and diafiltration (UFDF). The purified protein is subjected to an ultrafiltration and diafiltration operation, which includes concentrating or diluting the purified protein by ultrafiltration; buffer exchanging the purified concentrated / diluted protein to the desired formulation by diafiltration; and further diluting or concentrating the formulated purified protein by a second round of ultrafiltration until the target protein concentration is achieved. One or more stability-enhancing excipients can be added directly to the UFDF retentate feed tank containing the formulated, purified protein to obtain the formulated drug substance, or to the UFDF eluate pool. Filters used in UFDF operations are well known and common in the art and are commercially available from many sources. There are many types of materials available: regenerated cellulose Pellicon (Millipore Sigma, Danvers, MA), stabilized cellulose, Slice, ECO (Sartorius AG, Gottingen, Germany), polyethersulfone (PES) membrane, Omega (Pall Corporation, Port Washington, NY). Multiple filters can be used to achieve the production capacity required by the reservoir, skids, or physical setup of the UFDF system to achieve or be achieved the desired goals of the production process.

[0091] Biopharmaceuticals

[0092] The methods provided herein can be used to produce protein products, such as recombinant proteins. The recombinant proteins can be eukaryotic proteins, such as mammalian proteins.

[0093] The mammalian protein can be an antigen binding protein, such as an antibody, antibody fragment, antibody derivative, antibody analog, antibody construct, fusion protein, mutant protein, multispecific protein, bispecific protein, bispecific T cell engager or peptibody. The antibody can be a whole antibody, a single chain variable fragment, Fv, Fab, Fab', F(ab')2, a bispecific antibody, a diabody, a triabody, a tetrabody, Fd, dAb, a minibody or a maxibody.

[0094] "Multispecific proteins" and "multispecific antibodies" refer to proteins that are recombinantly engineered to simultaneously bind to at least two different antigens or at least two different epitopes on the same antigen. For example, multispecific proteins can be engineered to target immune effectors and cytotoxic or infectious agents against tumors. These multispecific proteins have been found to be useful in a variety of applications, such as in cancer immunotherapy by redirecting immune effector cells to tumor cells, modifying cell signaling by blocking signaling pathways, targeting tumor angiogenesis, blocking cytokines, and as pre-targeted delivery vehicles for drugs, such as chemotherapeutics, radiolabels (to improve detection sensitivity), and nanoparticles (directed to specific cells / tissues, such as cancer cells).

[0095] The most common and diverse multispecific proteins are those that bind two antigens and are referred to interchangeably herein as "bispecific proteins" and "bispecific antibodies." Multispecific proteins also include trispecific antibodies, tetravalent bispecific antibodies, multispecific proteins without antibody components (such as diabodies, triabodies, or tetrabodies), minibodies, and single-chain proteins capable of binding to multiple targets. Coloma, MJ, et al., Nature Biotech. 15 (1997) 159-163.

[0096] Bispecific proteins can be divided into two major categories: immunoglobulin G (IgG)-like molecules and non-IgG-like molecules. IgG-like molecules retain Fc-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). The Fc region helps improve solubility and stability and facilitates some purification operations. Non-IgG-like molecules are smaller, thereby enhancing tissue penetration. (Sedykh et al., Drug Design, Development and Therapy 18(12), 195-208, 2018; Fan et al., J Hematol & Oncology 8:130-143, 2015; Spiess et al., Mol Immunol 67, 95-106, 2015); Williams et al., Chapter 41, Process Design for Bispecific Antibodies in Biopharmaceutical Processing Development, Design and Implementation of Manufacturing Processes, Jagschies et al., eds., 2018, pp. 837-855. Bispecific proteins are sometimes used as a framework for additional components with binding specificities for different antigens or epitopes, thereby increasing the binding specificity of the molecule.

[0097] Bispecific proteins come in many forms, including, but not limited to, quadroma, knobs-in-hole, cross-monoclonal antibody (cross-Mab), dual variable domain IgG (DVD-IgG), IgG-single chain Fv (scFv), scFv-CH3 KIH, bifunctional Fab (DAF), half-molecule exchange, κλ-body, tandem scFv, scFv-Fc, diabody, single chain diabody (scdiabody), scdiabody-CH3, triabody, minibody, minibody, TriBi minibody, tandem diabody, scdiabody-HSA, tandem scFv-toxin, dual affinity retargeting molecule (DART), nanobody, nanobody-HSA, docking and locking (DNL), chain exchange engineered domain SEEDbody (SEEDbody), trifunctional bispecific antibody (Triomab), leucine zipper (LUZ-Y), Fab-arm exchange, DutaMab, DT-IgG, charged pair, Fcab, orthogonal Fab, IgG(H)-scFv, scFV-(H)IgG, IgG(L)-scFV, IgG(L1H1)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFV-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-Ig4 (quadruplex), Fab-scFv, scFv-CH-CL-scFV, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, intracellular antibody, ImmTAC, HSA body (HSABody), IgG-IgG, Cov-X body, scFv1-PEG-scFv2, single-chain bispecific antibody construct, single-chain bispecific T cell engager (scBiTE), bispecific T cell engager and half-life extended bispecific T cell engagers (HLE BITEs) (Fan supra; Spiess, supra; Sedykh, supra; Seimetz et al., Cancer Treat Rev 36(6)458-67, 2010; Shulka and Norman, Chapter 26, Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody-Drug Conjugates, in Process Scale Purification of Antibodies, 2nd ed., Uwe Gottswchalk, ed., p559-594, John Wiley & Sons, 2017; Moore et al., MAbs 3:6, 546-557, 2011).

[0098] The methods provided herein can be used to produce colony stimulating factors, erythropoiesis stimulating agents, HER receptors, cell adhesion molecules, growth factors, osteoinductive factors, insulin, coagulation proteins, colony stimulating factors, blood group antigens; growth hormones, growth hormone receptors, T cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transporters, homing receptors, addressins, regulatory proteins or immunoadhesins. The growth factor can be a nerve growth factor, a fibroblast growth factor, a transforming growth factor or an insulin-like growth factor. The colony stimulating factor can be a granulocyte colony stimulating factor (G-CSF). Such G-CSF molecules include but are not limited to (filgrastim) and (Pegfilgrastim). Also included are erythropoiesis-stimulating agents (ESAs), such as (Epoetin α), (darbepoetin alfa), (Epoetine delta), (methoxypolyethylene glycol-epoetin beta), MRK-2578, INS-22, (Epoetin ζ), (Epoetin beta), (Epoetin ζ), (epoetin alfa), epoetin alfa Hexal, (Epoetin α), (Epoetin theta), (Epoetin theta), (Epoetin theta), epoetin alpha, epoetin beta, epoetin zeta, epoetin theta and epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activator, GLP-1 receptor agonist, and any of the foregoing molecules or variants or analogs and biosimilars thereof.

[0099] The methods provided herein can be used to produce proteins that specifically bind to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGFs), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, coagulation proteins and coagulation-related proteins, colony stimulating factors (CSFs), other blood and serum proteins, blood group antigens; receptors, receptor-associated proteins, growth hormones, growth hormone receptors, T cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transporters, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0100] The methods provided herein can be used to produce CD proteins, including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174; HER receptor family proteins, including but not limited to HER2, HER3, HER4, and EGF receptor EGFRvIII; cell adhesion molecules, including but not limited to LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, and αv / β3 integrin; growth factors, including but not limited to vascular endothelial growth factor ("VEGF"), VEGFR2, growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, mullerian-inhibiting substance, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factor (such as NGF-β), platelet-derived growth factor (PDGF), fibroblast growth factor [including but not limited to aFGF and bFGF], epidermal growth factor (EGF), Cripto, transforming growth factor (TGF) (especially including TGF-α and TGF-β, including TGF-β1, TGF-β2 , TGF-β3, TGF-β4 or TGF-β5), insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and osteoinductive factors; insulin and insulin-related proteins, including insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding protein; coagulation proteins and coagulation-related proteins, especially factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators [such as urokinase and tissue plasminogen activator ("t-PA")], bombazine, thrombin, thrombopoietin and thrombopoietin receptors colony stimulating factors (CSFs), including but not limited to M-CSF, GM-CSF, and G-CSF; other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens; receptors and receptor-associated proteins, including but not limited to flk2 / flt3 receptors, obesity (OB) receptors, growth hormone receptors, and T-cell receptors; neurotrophic factors, including but not limited to bone-derived neurotrophic factor (BDNF) and neurotrophin-3, neurotrophin-4, neurotrophin-5, or neurotrophin-6 (NT-3, NT-4, NT-5, or NT-6); relaxin A chain, relaxin B chain, and prorelaxin; interferons, including, for example, interferon-α, interferon-β, and interferon-γ;Interleukins (ILs), including but not limited to IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor, IL-13, IL-13RA2 or IL-17 receptor, IL-1RAP, IL1-α and IL-1β; viral antigens, including but not limited to AIDS enveloped virus antigens; lipoproteins; calcitonin; glucagon; atrial natriuretic factor; pulmonary surfactant; tumor necrosis factor-α and tumor necrosis factor- β; enkephalinase; BCMA; Igκ; ROR-1; ERBB2; mesothelin; RANTES (regulated normal T cell expressed and secreted upon activation); mouse gonadotropin-related peptide; DNA enzyme; FR-α; inhibin; activin; integrin; protein A or D; rheumatoid factor; immunotoxin; bone morphogenetic protein (BMP); superoxide dismutase; surface membrane protein; decay-accelerating factor (DAF); AIDS envelope protein; transporter; homing receptor; MIC (MIC-a, MIC-B); ULBP 1-6; EPCAM; PSA; addressin; regulatory protein; immunoadhesin; antigen-binding protein; growth hormone; CTGF; CTLA4; eotaxin-1; MUC1; CEA; c-MET; claudin-18; GPC-3; EPHA2; FPA; LMP1; MG7; NY-ESO-1; PSCA; ganglioside GD2; ganglioside GM2; BAFF; OPGL (RANKL); myostatin Protein; Dickkopf-1 (DKK-1); Ang2; NGF; IGF-1 receptor; Hepatocyte growth factor (HGF); TRAIL-R2; c-Kit; B7RP-1; PSMA; P-cadherin; NKG2D-1; Programmed cell death protein 1 and ligand (PD1 and PDL1); Mannose receptor / hCGβ; Hepatitis C virus; Mesothelin dsFv; PE38 conjugate; Legionella pneumophila pneumophila)(lly); gpA33; B7H3; IFNγ; interferon gamma-induced protein 10 (IP10); IFNAR; TALL-1; thymic stromal lymphopoietin (TSLP); proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor; stem cell factor; Flt-3; calcitonin gene-related peptide (CGRP); OX40L; α4β7; platelet-specific protein (platelet glycoprotein Iib / IIIb (PAC-1)); transforming growth factor beta (TFGβ); zona pellucida sperm-binding protein 3 (ZP-3); TWEAK; platelet-derived growth factor receptor alpha (PDGFRα); sclerostin; and biologically active fragments or variants of any of the foregoing.

[0101] The methods provided herein can be used to produce abciximab, adalimumab, adelimumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosozumab, brentuximab, brodalumab, mocantuzumab, canakinumab, cetuximab, certolizumab pegol, canakinumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitazumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lerdizumab , ruximab, levofloxacin (lxdkizumab), mapatumumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, oreluzumab, ofatumumab, omalizumab, oprelleukin, palivizumab, panitumumab, pembrolizumab, pertuzumab, pexelizumab, ranibizumab, rituximab, rituximab, romiplostim, lomosozumab, sargramostim, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, visilizumab, volocizumab, zalumab, zalutumumab, and variants or analogs thereof, and biosimilars of any of the foregoing.

[0102] The methods provided herein can be used to produce blinatumomab, catumaxomab, ertuinomab, solituzumab, targomiRs, rugilumab (ABT981), vanucelezizumab (RG7221), nonolutumab (ABT122), ozoralixumab (ATN103), floteuzumab (MGD006), pertuximab (AMG112, MT112), linfumimab (FBTA05), (ATN-103), AMG211 (MT111, Medi-1565), AMG330, AMG420 (B1836909), AMG-110 (MT110), MDX-447, TF2, rM28, HER2Bi-aATC, GD2Bi-aATC, MGD006, MGD007, MGD009, MGD010, MGD011 (JNJ64052781), IMCgp100, indium-labeled IM P-205, DH7945A), RG7802, RG7813 (RO6895882), RG7386, BITS7201A (RG7990), RG7716, BFKF8488A (RG7992), MCLA -128, MM-111, MM141, MOR209 / ES414, MSB0010841, ALX-0061, ALX0761, ALX0141; BII034020, AFM13, AFM11, SAR156597, FBTA05, PF06671008, GSK2434735, MEDI3902, MEDI0700, MEDI7352, and variants or analogs thereof, and biosimilars of any of the foregoing.

[0103] Other terms

[0104] Although various embodiments in the specification are presented using "comprising" language, in many cases, "consisting of" or "consisting essentially of" language may also be used to describe related embodiments. The present disclosure contemplates embodiments described as "comprising" a feature to include embodiments "consisting of" or "consisting essentially of" the feature. The term "a" or "an" refers to one or more; the terms "a", "one or more" and "atleast one" are used interchangeably herein. Unless the context clearly requires otherwise, the term "or" should be understood to cover items in alternative or combination. The term "and / or" should be understood to cover each item in the list (individually), any combination of the items in the list, and all the items in the list together.

[0105] When about is used in conjunction with a numerical value, the present disclosure contemplates a range around that numerical value. For example, about can mean ±1%, ±2%, ±5%, ±10%, etc. When referring to units such as temperature or pH, about can also mean ±0.1 units, ±0.2 units, ±0.3 units, ±0.4 units, ±0.5 units, ±1 unit.

[0106] When describing the range of values, the present disclosure contemplates the individual values ​​found within the range. For example, "cell aggregate size between about 20 μm and about 200 μm" can be, but is not limited to, 40 μm, 60 μm, 100 μm, etc., and any value between such values. Within any range described herein, the endpoints of the range are included in the range. However, the present disclosure also contemplates the same range excluding lower and / or higher endpoints. When a value is stated as "about" a value, the exact value is also contemplated, either individually or as the endpoints of a range.

[0107] As used herein, "may be," "may," "can be," or "can" refer to something that the inventor contemplates as functional and obtainable as part of the provided subject matter.

[0108] From the entirety of this application (including the drawings and detailed description), additional features and variations of the present invention will be apparent to those skilled in the art. The entire document is intended to be described as a unified disclosure, and it should be understood that all combinations of features described herein (even if described in separate sections) are contemplated, even if the combination of these features is not found in the same sentence, paragraph or section of this document.

[0109] Examples

[0110] While the following examples describe specific embodiments, numerous changes and modifications will occur to those skilled in the art. It is therefore intended that the present invention be subject to only such limitations as appear in the claims.

[0111] Example 1

[0112] Experiment 1

[0113] The first experiment illustrates a non-steady-state cell culture under the first condition. In the first experiment, process conditions were present that induced a non-steady-state cell culture.

[0114] CHO cells expressing antigen-binding proteins were cultured at 45 x 10 5 A 100 L disposable bioreactor (Xcellerex, Marlborough, MA) was inoculated with 60 L of serum-free chemically defined basal medium at a density of 10 cells / ml. After inoculation, the target volume in the bioreactor was adjusted to 100 L of basal medium at a set point pH of 6.90 and an initial temperature of 36.0° C. and maintained for 24 hours.

[0115] The bioreactor was equipped with two ATF 6 (0.2 μm) alternating tangential flow devices (Repligen, Waltham, Massachusetts). On day 1, the culture was continuously perfused with a serum-free, chemically defined perfusion medium and continued to operate at a pH of 6.90 and a temperature of 36.0°C. The permeate flow rate was adjusted daily to reach a maximum working volume (WV) of 2.0 per day on day 6, see Table 1. The cells were retained in the retentate and returned to the bioreactor to establish and / or maintain cell density. Any recombinant product passed through the permeate flow and was discharged to waste during the growth phase (days 0-6). The biomass specific permeate rate was 0.0211 cm / pF.day at the end of the growth phase.

[0116] Measure cell count, viability, cell diameter, pH, pCO2, pO2, dielectric constant, glucose, lactate, ammonia, osmotic pressure, packed cell volume, bioreactor titer, permeate titer, and harvest titer daily. Remove bulk bioreactor supernatant and harvest samples for PQ. Add antifoam directly to the bioreactor to control foam.

[0117] Table 1 Permeate flow rate schedule

[0118] Number of days WV / day 1 0.5 2 0.8 3 1.2 4 1.5 5 1.8 6-28 2.0-2.2

[0119] Table 2: Process parameters of Experiment 1

[0120]

[0121] The culture was maintained until a target dielectric constant of 95 pF / cm was achieved (day 6), at which time a temperature shift from 36.0°C to 34.0°C was performed to control cell growth and increase protein production (Table 3). A capacitance probe was used to monitor cell biomass and trigger a temperature shift at the target dielectric constant value. The capacitance probe was also used to increase and maintain biomass by controlling cell purge to remove cells from the bioreactor during the growth and biomass intensification phases. The minimum cell packing was ≥25%.

[0122] Biomass Intensification: Starting on day 6 and after the temperature shift marking the start of the production phase, biomass increased to a maximum of 105 pF / cm. This high cell density maintained high protein production rates during the run at the start of the production phase. At this maximum biomass, the biomass-specific permeability was 0.0190 pF / cm.

[0123] Non-steady-state CM: This is a run in which the combination of process parameters (ie, temperature, biomass, and permeation rate (or alternatively, biomass to permeation rate)) results in a non-steady-state CM, where the target dielectric constant and cell flux are no longer maintained.

[0124] Experiment 1, Condition 1: This condition was performed during the production phase, so the recombinant product was continuously harvested from the permeate stream into sterile harvest bags. From day 8 to day 20, the temperature set point was maintained at 34.0°C, and the permeate rate was maintained at 2.0–2.2 wv / day. Cell culture was performed in non-steady-state operation, resulting in decreased VCD and viability, while increasing cell diameter and PCV. Initially, the dielectric constant was maintained at a target value of 95 pF / cm while the cells were drained. After approximately one week, as expected due to the reduction in biomass during non-steady-state operation, the dielectric constant decreased to below this maximum value of 95 pF / cm. The benefits of reducing VCD, viability, PCV, dielectric constant, drain, and cell growth rate were stable and high protein production rates.

[0125] Experiment 1, Condition 2: On day 21, the culture temperature was increased to 36.0°C, establishing a target dielectric constant of 65 pF / cm and maintaining it by cell purging. The cell culture was also operated in a non-steady state, but the rate of biomass reduction in the culture was reduced by increasing the temperature. The permeation rate was maintained at 2.2 wv / day. The biomass-to-permeation rate was 0.0338 pF / cm.day. The recombinant product in the permeate stream was continuously harvested into sterile harvest bags. Culture was continued under these conditions until day 28, at which time the culture was terminated. The protein production rate initially remained as high as in Condition 1, but the production rate decreased over the past few days. This indicates that relaxing the process parameters in Condition 1 results in lower productivity.

[0126] Experiment 1 results

[0127] The first experiment demonstrated biomass intensification and non-steady-state operation, which resulted in high protein production rates. Thus, the effects of biomass intensification and Condition 1 appeared to determine production rates for most of the run. During the last few days of cultivation, production rates declined due to Condition 2, which had a higher temperature and reduced the rate of biomass loss.

[0128] Experiment 2

[0129] The second experiment started with steady-state conditions, followed by non-steady-state conditions with decreased vitality.

[0130] In the second experiment, the process design space was explored to divide the order of magnitude of stable versus unstable CM operation and each type of CM operation. Several conditions of continuously reducing temperature and increasing dielectric constant were tested during the production phase. The purpose was to determine which combination of temperature and biomass specific permeation rate (relative to the permeation rate of biomass in culture) could maintain cell growth and result in maintaining high viability (steady state culture) rather than conditions where viability began to decrease (unsteady state culture). No biomass intensification step was tested in this experiment.

[0131] CHO cells were cultured at 45 x 10 5 A 100-L bioreactor was inoculated with 60 L of serum-free, chemically defined basal medium at a density of 10 cells / ml. After inoculation, the target volume in the bioreactor was adjusted to 100 L of basal medium at a set-point pH of 6.90 and an initial temperature of 36.0°C and maintained for 24 hours. The permeate flow rate was adjusted daily to achieve a maximum working volume (WV) of 2.0 per day on day 6 (see Table 3).

[0132] Table 3: Process parameters of Experiment 2

[0133]

[0134] The culture was maintained until the target dielectric constant of 75 pF / cm was achieved (day 6), Table 3. The minimum cell packing volume was ≥25%. The biomass specific permeation rate was 0.0293 pF / cm.day.

[0135] Experiment 2, Condition 1: The culture was maintained at 36.0°C, with a target dielectric constant of 75 pF / cm and a permeation rate of 2.2 wV / day until day 8. Upon reaching the target dielectric constant, the biomass-specific permeation rate remained at 0.0293 pF / cm.day.

[0136] Experiment 2, Condition 2: On day 8, a temperature shift from 36.0°C to 35.5°C was performed to control cell growth and promote protein production. The target dielectric constant (75 pF / cm) and permeation rate (2.2 wV / day) were maintained until day 12. Upon reaching the target dielectric constant, the biomass-specific permeation rate remained at 0.0293 pF / cm.day.

[0137] Experiment 2, Condition 3: On day 13, the temperature was lowered to 35.0°C, and the target dielectric constant was increased to 85 pF / cm and maintained until day 17. The permeation rate was maintained at 2.2 wV / day. After reaching the target dielectric constant, the biomass-specific permeation rate increased to 0.0259 pF / cm.day.

[0138] Experiment 2, Condition 4: On day 18, the temperature was lowered to 34.5°C and the target dielectric constant was increased to 95 pF / cm. The permeation rate was maintained at 2.2 wV / day. After reaching the target dielectric constant, the biomass-specific permeation rate increased to 0.0232 pF / cm-day.

[0139] Experiment 2, Condition 5: On day 26, the temperature was maintained at 34.5°C and the target dielectric constant was increased to 115 pF / cm. The permeation rate was maintained at 2.2 wV / day. After reaching the target dielectric constant, the biomass-specific permeation rate increased to 0.0191 pF / cm.day.

[0140] In experiment 2, conditions 1-3, the cell culture parameters maintained steady state, and the production rate was low. In experiment 2, conditions 4-5, as temperature and biomass increased (biomass decreased relative to permeation rate), the culture began to show the characteristics of non-steady-state CM (i.e., vigor decreased). The culture in conditions 4-5 still had certain cell growth (as demonstrated by cell release), so the non-steady-state characteristics were not as extreme as those tested in experiment 1. Experiment 2 illustrates the transition between steady state and non-steady-state CM design space. The cell culture performance indicators that showed the first signs of non-stable characteristics in conditions 4-5 were vigor decrease and cell release rate decrease. Compared with steady-state conditions 1-3, the production rate under conditions 4-5 also increased. Due to the steady-state properties (high cell growth rate) of many test conditions, the production rate in experiment 2 was about half of that in experiment 1.

[0141] Experiment 3

[0142] The third experiment illustrates non-steady-state conditions without a biomass intensification phase.

[0143] Experiment 3 replicated the growth phase of Experiment 2 and Condition 1 without the biomass intensification phase. The goal was to determine the effect of the biomass intensification phase on cell culture performance parameters.

[0144] CHO cells were grown as described in Experiment 1, except that no biomass fortification was performed. A 6-day growth phase at 36.0°C and a 12-day production phase at 34.0°C (Condition 1) were performed at the same dielectric constant (95 pF / cm) and permeation rate (2.0). The biomass-specific permeation rate for both the growth phase and Condition 1 was 0.0210 pF / cm.

[0145] Although Experiment 3 was a non-steady-state CM operation, the rate of viability decline was not as drastic, and the cell growth rate (as indicated by the discharge rate) exceeded that of Experiment 1. The productivity in this experiment was lower compared to Experiment 1. This phenomenon can be attributed to the lack of a biomass intensification phase.

[0146] Table 4: Process parameters of Experiment 3

[0147]

[0148] Experiment 4

[0149] The fourth experiment replicated the first and demonstrated the reproducibility of non-steady-state cell culture.

[0150] Experiment 4 was conducted as a non-steady-state CM culture with different target dielectric constants and permeation rates. The biomass-to-permeation rate was lower than in Experiment 1, which had a higher target dielectric constant and a lower permeation rate. This experiment demonstrates another example of high productivity in non-steady-state CM cultures, similar to Experiment 1.

[0151] CHO cells were grown as described in Experiment 1 with a higher dielectric constant (115 pF / cm) at a lower permeation rate (1.8 wv / day) during the growth phase, see Table 5. The biomass specific permeation rate was 0.0157 pF / cm.

[0152] Table 5: Process parameters of Experiment 4

[0153]

[0154] On day 7, the temperature was reduced to 34.0°C, and the dielectric constant and permeation rate remained unchanged for biomass intensification. The biomass specific permeation rate was 0.0157 pF / cm.

[0155] On the 9th day, the dielectric constant decreased to 105 pF / cm and the biomass-specific permeability was 0.0171 pF / cm.

[0156] Example 1 Results

[0157] Experiment 1 demonstrated high protein production throughout the low temperature production phase. Despite the set point changes, the production rate was similar throughout the process, see Figure 1AF. Thus, the biomass intensification phase and condition 1 appear to be responsible for the high rate of protein production (shown in the Production Mass Totalizer). In contrast, the effect of condition 2 was only observed in the last few days, as the effects of the conditions took several days to become apparent in the cell culture. Therefore, the biomass intensification phase ideally needs to occur immediately after the growth phase, as this step largely determines the protein production rate for most of the culture period.

[0158] The product quality of two examples of non-steady-state CM with a biomass intensification phase (Experiments 1 and 4) was tested in two batches per run and compared to a single batch of a discontinuous perfusion process of the same cell line / molecule. Samples were collected from each run to determine the product quality attributes of the recombinant protein produced. These PQAs were compared to discontinuous perfusion cultures producing antigen-binding proteins. The PQAs were similar between batches of non-steady-state CM runs. There appeared to be no adverse effect on PQAs between different CM runs or between CM and the discontinuous perfusion process.

[0159] Table 6: Product quality attributes of two samples from Experiment 1 compared to a discontinuous perfusion process operated at steady state (Control).

[0160]

[0161] Each CM bioreactor experimental run had two production batches. The product quality of the two batches within each experimental run was compared. The product quality attributes were similar between the two CM batches in Experiment 1 and between the two CM batches in Experiment 2. For most attributes, the product quality between the two different CM runs was also quite similar. A discontinuous perfusion process was performed to produce the same protein from the same cell line. This process also operates under non-steady state conditions, but with different culture conditions (e.g., temperature, perfusion rate). The product was retained in the reactor and then perfused out within the last three days. Despite the differences in process conditions and formats, the product quality attributes from the perfusion run were similar to those from the two CM runs.

[0162] Experiment 3 had similar growth phases and first conditions as Experiment 1, without biomass intensification. The conditions of Experiment 3 resulted in lower yields compared to Experiment 1, see Figure 1 AF. This can be attributed to the lack of a biomass intensification stage in Experiment 3. Therefore, the presence of a biomass intensification stage is important to ensure high productivity.

[0163] Experiment 4 had both a growth phase and a biomass intensification phase, followed by a production phase at the same temperature as Experiments 1 and 3. The dielectric constant and permeate set point were adjusted to achieve a lower biomass specific permeation rate. The productivity of Experiments 1 and 4 was similar due to the similar growth, biomass intensification, and production phases, see Figure 1 AF. This suggests that all three stages are important for establishing high productivity in non-steady-state CM cultures.

[0164] The purpose of Experiment 2 was to determine which temperature and biomass to permeation rate set points could sustain cell growth and result in high viability (steady-state culture), and at which temperatures viability began to decrease (non-steady-state culture). Experiment 2 demonstrated that there is a process design space where conditions favor cell growth and a different design space favoring protein production. Under the first three conditions, steady-state conditions were observed, and under the last two conditions, non-steady-state conditions existed, as indicated by a decrease in viability, see Figure 1 AF.

[0165] Overall results of Example 1

[0166] Several examples of the aforementioned non-steady-state CM conditions exist. Different levels of biomass reduction and corresponding differences in production rates were observed as a result of testing different combinations of process parameters. The importance of the biomass intensification phase was demonstrated in Experiment 3 by removing it from the culture and observing its effect on production rate. The transition between steady-state CM and non-steady-state CM was demonstrated by gradually increasing biomass and decreasing temperature.

[0167] Experiments 1 and 4 demonstrated non-steady-state CM with a biomass intensification phase.

[0168] Viable cell density – VCD – increases during the growth phase (days 0–6) and the biomass intensification phase (days 6–8), peaks on day 8, and then steadily decreases during the remainder of the production phase (due to non-steady-state operation).

[0169] Vitality – Vitality also begins to decrease in a similar manner to VCD.

[0170] Dielectric constant – The dielectric constant decreases in the non-steady state, but usually exhibits non-steady-state characteristics later, in contrast to VCD and viability (which decrease immediately).

[0171] Total Emission – The slope of the total emission is a measure of cell growth. If the slope is horizontal, there is no cell growth. A sharp drop in viability corresponds to a lower emission rate and a lower cell growth rate.

[0172] Packed Cell Volume – In non-steady-state cultures, PCV increases even as VCD decreases. This demonstrates that cell diameter (and therefore cell volume) increases in non-steady-state cultures.

[0173] Production Mass Totalizer – The slope of the Production Mass Totalizer represents the rate of protein production. This slope is steeper in non-steady-state CM cultures than in steady-state CM cultures.

[0174] Example 1 Conclusion

[0175] Experiment 1-4 helps to define the non-stable production stage. Studied the process parameters affecting non-stable behavior and the influence thereof on cultivating. In addition, illustrated the effect of the biomass reinforcement stage in improving productivity. In experiment 1, there is the biomass reinforcement stage, and the first condition causes non-stable operation, and the second condition attempts to switch this process back to stable operation. In experiment 2, by manipulating biomass level and temperature, cultivation is progressively changed to non-stable from stable state. In experiment 3, repeat to cause the process parameters of non-stable (that is, condition 1 of experiment 1 without the biomass reinforcement stage). This causes lower productivity, and this is owing to not having the biomass reinforcement stage. In experiment 4, repeat non-stable process (that is, condition 1 of experiment 1 with biomass reinforcement stage) with process parameters similar to experiment 1. This produces the productivity similar to experiment 1, and has confirmed non-stable operation.

[0176] Operating cell cultures under non-steady-state conditions (declining VCD, viability) results in higher productivity than under steady-state conditions. This reduces culture duration and can improve plant utilization, thereby improving process costs. Due to the reduced discharge volume, harvest yield from the culture increases. Harvest quality is higher due to the higher productivity and high permeation rate. Therefore, non-steady-state operation results in an economically advantageous process. The product quality of the two non-steady-state CM batches was similar, demonstrating that the process was in control.

[0177] Example 2

[0178] Example 2 illustrates non-steady-state cell culture on different cell lines and antigen-binding proteins. This demonstrates the reproducibility of these methods. The conditions in this example have non-steady-state cell culture.

[0179] CHO cells expressing the second bispecific T cell engager were used at 45 x 10 5 A 100 L disposable bioreactor (Xcellerex, Marlborough, MA) was inoculated with 60 L of serum-free, chemically defined basal medium at a density of 10 cells / ml. After inoculation, the target volume in the bioreactor was adjusted to 100 L of basal medium at a set-point pH of 6.95 and an initial temperature of 36.0° C. and maintained for 24 hours as described in Example 1.

[0180] The bioreactor was equipped with two ATF 6 (0.2 μ) alternating tangential flow devices (Refine, Pine Brook, NJ). On day 1, the culture was continuously perfused with a serum-free, chemically defined perfusion medium (pH 6.90, temperature 36.0° C.). The permeate flow rate was adjusted daily to reach a maximum working volume (WV) of 1.8 per day on day 5, see Table 7. The process parameters for the experiment are shown in Table 8. The cells were retained in the retentate and returned to the bioreactor to establish and / or maintain biomass. Any recombinant product passed through in the permeate flow and was discharged to waste during the growth phase (days 0-6). The biomass specific dielectric constant was 0.0157 pF / cm.day.

[0181] Cell count, viability, cell diameter, pH, pCO2, pO2, dielectric constant, glucose, lactate, ammonia, osmolality, packed cell volume, bioreactor titer, permeate titer, and harvest titer were measured daily. Large amounts of bioreactor supernatant and harvest samples were taken for PQ.

[0182] Table 7. Permeate flow rate schedule.

[0183] Number of days WV / day 1 0.4 2 0.7 3 1.2 4 1.5 5-28 1.8

[0184] Table 8: Process parameter set points

[0185]

[0186] The purpose of this experiment was to demonstrate non-steady-state operation of different molecules and to test the effects of different process conditions. The growth phase, biomass intensification phase, and Condition 1 in this example were identical to those in Experiment 4 in Example 1. Condition 2 employed a temperature increase to 35°C and a 5% manual cell drain to test the effects on cell culture performance. In the third condition, manual drain was discontinued, and the effects on the culture were observed.

[0187] Cultures were maintained until a target dielectric constant of 115 pF / cm was achieved (Day 6), Table 8. This dielectric constant was selected to achieve high VCD (>1E8 cells / mL) during the biomass intensification phase. The minimum cell packing volume was ≥25%. The biomass specific permeability was 0.0157 pF / cm.day. Manual cell draining was not used to maintain the desired dielectric constant. The biomass specific dielectric constant was 0.0157 pF / cm.day.

[0188] Condition 1: The culture temperature was lowered to 34.0°C and the dielectric constant was reduced to 105 pF / cm. This reduction in dielectric constant helped reduce the biomass in the culture and reduced the risk of ATF failure. The permeation rate was maintained at 18 wv / day until day 14. No manual cell draining was used to achieve or maintain the desired dielectric constant. The biomass-specific dielectric constant was 0.0171 pF / cm.day.

[0189] Condition 2: The culture temperature was raised to 35.0°C. The dielectric constant was maintained at 105 pF / cm by manual cell draining at 5%. The permeation rate was maintained at 18 wV / day until day 20. The biomass-specific dielectric constant was 0.0171 pF / cm.day.

[0190] Condition 3: The culture temperature, dielectric constant, and permeation rate remained the same as in Condition 2, but manual cell release was not used. The biomass-specific dielectric constant was 0.0171 pF / cm.day.

[0191] During conditions 1 to 3, the recombinant product in the permeate stream was continuously harvested into sterile harvest bags. The culture was terminated on day 28.

[0192] Example 2 Results

[0193] Condition 1 resulted in non-steady-state CM as expected, characterized by decreased VCD, viability, and high protein productivity.

[0194] The purge in Condition 2 was used to remove a portion of the cells and cell debris from the culture, and therefore, despite the higher temperature, the VCD decreased in Condition 2. The PCV (and cell diameter) of this cell line increased significantly compared to the cell line in Example 1. Based on experience, a PCV of 30%-35% is known to increase the risk of ATF failure in the setup being used. Cell purge in Condition 2 was necessary to help maintain or lower the PCV, reduce the risk of ATF failure, and extend the duration of cell culture.

[0195] Due to the temperature increase starting on day 15 and the lack of manual cell release in condition 3 starting on day 21, the VCD gradually and continuously increased, and there was no decrease in viability. This condition demonstrates that the temperature increase will convert the culture from an aggressive, non-steady-state culture that is conducive to protein production to a steady-state culture with cell growth. As expected, due to the temperature increase that is conducive to cell growth, the protein production rate in condition 3 was lower than in condition 1. The PQs of the two batches run in this CM were similar (see Table 9). This demonstrates that the process is in control and that the process conditions tested are suitable for cell culture.

[0196] The growth, biomass intensification, and Condition 1 in this Example 2 were identical to those in Experiment 4 of Example 1. Similar to Example 1, in Example 2, VCD and viability decreased and PCV increased. The rates of change in viability, VCD, and PCV differed from those observed in Example 1 because different cell lines / molecules had different magnitudes of response to process set points. The production rates were very similar between the two bispecific T cell engagers. In Example 2, viability decreased and PCV increased compared to Example 1 of Experiment 4 due to the differences in cell lines and bispecific T cell engagers used.

[0197] Table 9: Product quality attributes of two batches from the continuous perfusion bioreactor

[0198]

[0199] The quality of the two batches from the CM run was similar, which demonstrated that the process was in control.

[0200] Example 2 Conclusion

[0201] In this experiment, non-steady-state CM, along with a biomass intensification phase, was demonstrated in different cell lines (producing different molecules). A transition to steady-state CM operation was also demonstrated. This suggests that this cell line exhibits unique cell culture performance characteristics across both process design spaces, favoring protein production over cell growth in the non-steady-state space and favoring cell growth over protein production in the steady-state CM space. By varying the process temperature, the process can be tuned from one type of operation to the other.

[0202] Example 3

[0203] Example 3 illustrates non-stationary cell culture on a third different cell line and antigen binding protein, demonstrating the reproducibility of these methods. The cell density and dielectric constant in this example were lower than in the previous two examples.

[0204] CHO cells expressing the second bispecific T cell engager were used at 38 x 10 5 A 50 L disposable bioreactor (Hyclone) was inoculated with 25 L of serum-free chemically defined basal medium at a density of 10 cells / ml. After inoculation, the target volume in the bioreactor was adjusted to 45 L of basal medium at a set point pH of 6.90 and an initial temperature of 36.0°C and maintained for 144 hours.

[0205] The bioreactor was equipped with an ATF 6 (0.2 μ) alternating tangential flow device (Refine, Pine Brook, New Jersey). On day 1, the culture was continuously perfused with a serum-free, chemically defined perfusion medium. The permeate flow rate was adjusted daily to reach a maximum working volume (WV) of 1.8 on day 6, see Table 10. The process parameters for the experiment are shown in Table 10. The cells were retained in the retentate and returned to the bioreactor to establish and / or maintain biomass. Any recombinant product passed through the permeate stream and was discharged to waste during the growth phase (days 0-6).

[0206] Cell count, viability, cell diameter, pH, pCO2, pO2, dielectric constant, glucose, lactate, ammonia, osmolality, packed cell volume, bioreactor titer, permeate titer, and harvest titer were measured daily. Large amounts of bioreactor supernatant and harvest samples were taken for PQ.

[0207] Table 10 Permeate flow rate schedule

[0208] Number of days WV / day 2 0.4 3 0.5 4 0.75 5 1.0 6 1.8

[0209] Table 11: Process parameter set points

[0210]

[0211] The cultures were maintained until a target dielectric constant of 70 pF / cm was achieved (day 6).

[0212] Example 3 Condition 1: The culture temperature was lowered to 34.0°C to control cell growth and promote protein production. The dielectric constant was reduced to 70 pF / cm, and the permeation rate was maintained at 1.8 wv / day until the 15th day.

[0213] Example 3 Condition 2: The culture temperature was raised to 34.0°C, and the dielectric constant and permeation rate were reduced to 60 pF / cm. During both Conditions 1 and 2, the recombinant product in the permeate stream was continuously harvested into a sterile harvest bag. Culture was continued under these conditions until termination.

[0214] Condition 3. The culture temperature was raised to 36.0°C, and the dielectric constant and permeation rate remained the same as those in Condition 1. During both Conditions 1 and 2, the recombinant product in the permeate stream was continuously harvested into sterile harvest bags. Culture was continued under these conditions until termination.

[0215] Example 3 Results

[0216] The experiments demonstrated that biomass intensification and non-steady-state conditions initiated early in cell culture at low temperatures led to high protein production rates. Lowering the dielectric constant in condition 2 further increased protein production, while viability decreased. Increasing both temperature and dielectric constant in condition 3 allowed the culture to maintain viability.

[0217] Example 3 Conclusion

[0218] In summary, dielectric constant and temperature are levers that can be used to increase protein production.

[0219] Summary of process parameters in the examples

[0220] Tables 12 and 13 below summarize exemplary process parameter values ​​contemplated herein that may be used to improve protein production.

[0221] Table 12: Average and range of process parameter values

[0222]

[0223] Table 13: Average values ​​and ranges of daily variations in process parameters

[0224]

[0225] All documents mentioned in this application are hereby incorporated by reference in their entirety.

Claims

1. A method for producing a protein product in a bioreactor in continuous perfusion mode, the method comprising a growth phase followed by a production phase not operating under steady-state cell culture conditions, The growth stage includes the following steps: (a) inoculating a bioreactor with cells expressing the protein product and liquid culture medium at a high cell density, and (b) growing the cells at a set temperature and progressively higher infiltration rates to a first biomass set point; and The non-steady-state production stage includes the following steps: (c) when the first biomass set point is reached, transitioning to a lower temperature or a lower infiltration rate to begin shifting the culture toward protein production, (d) growing the cells at a set temperature and permeation rate until a second, higher biomass set point is reached that promotes non-steady-state cell culture and high productivity, (e) culturing the cells under the culture conditions described in (d) such that viability decreases over time, and (f) collecting the protein product from a harvest stream during the production stage.

2. The method of claim 1, wherein the variable cell density (VCD) decreases over time or the packed cell volume (PCV) increases over time.

3. The method of claim 1 , wherein one or more cell releases are performed such that the culture does not exceed the biomass set points.

4. The method of claim 3, wherein release occurs once the cells increase beyond the second biomass set point and the release rate is reduced or dropped to zero.

5. The method of any preceding claim, wherein the high cell density of (a) is from about 200,000 cells / mL to about 5,000,000 cells / mL.

6. The method of any preceding claim, wherein the first biomass set point is about 50 million cells / mL to about 100 million cells / mL.

7. The method of any preceding claim, wherein the second biomass set point is about 100 million cells / mL to about 150 million cells / mL.

8. The method of any preceding claim, wherein the permeation rate of (b) is 0 to about 4.1 working volumes per day.

9. The method of any preceding claim, wherein the permeation rate of (d) is from about 1.0 to about 4.2 working volumes per day.

10. The method of claim 2, wherein the VCD maximum of (d) is about 130 million to about 140 million cells / mL.

11. The method of any preceding claim, wherein the set temperature of (b) is from about 35.5°C to about 36.5°C.

12. The method of any preceding claim, wherein the temperature transition of (c) is to a temperature of about 32.5°C to about 35.5°C.

13. The method of any preceding claim, wherein the cell discharge during the growth phase is from about 0% to about 3%.

14. The method of any preceding claim, wherein the cell viability during the growth phase is about 90% to about 99%.

15. The method of claim 14, wherein the cell viability in step (e) is reduced to about 30% to about 80% viability.

16. The method of any preceding claim, wherein the PCV of the growth phase is increased to about 2% to about 24%.

17. The method of claim 16, wherein the PCV of the production stage is about 25% to about 50%.

18. A method as claimed in any preceding claim, wherein the duration of the growing phase is about 4 to 12 days.

19. The method of any preceding claim, wherein the production phase lasts for about 10 days before cell growth ceases.

20. The method of any preceding claim, wherein the duration of the production phase after cell growth has ceased is from about 9 to about 41 days.

21. The method of any preceding claim, further comprising the downstream step of subjecting the harvested protein product to capture chromatography, viral inactivation and / or polishing steps.

22. The method of any preceding claim, wherein the cells are mammalian cells.

23. The method of any preceding claim, wherein the mammalian cells are Chinese Hamster Ovary (CHO) cells.

24. The method of any preceding claim, wherein the protein product is an antigen binding protein.

25. The method of claim 24, wherein the antigen binding protein is an antibody, an antibody fragment, an antibody derivative, an antibody analog, an antibody construct, a fusion protein, a mutein, a multispecific protein, a bispecific protein, a bispecific T cell engager, or a peptibody.

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