Cell culture method

Abstract

The present disclosure provides unsteady-state continuous perfusion cell culture methods for protein production in bioreactors. Cells are cultured in unsteady-state conditions after entering the production phase, such that viability decreases over time. Protein product production using these cell culture methods is increased.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Provisional Application No. 63 / 443,190, filed February 3, 2023, which is incorporated herein by reference in its entirety.

[0002] The present disclosure provides unsteady-state continuous perfusion cell culture methods for protein production in bioreactors. Cells are cultured in unsteady-state conditions after entering the production phase, such that viability decreases over time. Protein product production using these cell culture methods is increased. [Background technology]

[0003] Various cell culture methods have been used to produce recombinant biopharmaceutical proteins in bioreactors. Protein product yield and process time associated with this method remain areas of development in the bioprocessing field. Manufacturing improvements that increase productivity, improve protein product consistency, reduce starting material costs or processing time, or reduce equipment costs could result in substantial economic benefits.

[0004] Bioprocessing can be carried out in batch systems (most commonly fed-batch processing) or continuous perfusion systems. Mammalian cells, such as Chinese hamster ovary (CHO) cells, are frequently used for bioprocessing. In batch systems, the protein product is harvested at the end of the culture run. In perfusion systems, the cell culture is periodically fed with fresh medium, and protein product-containing medium liquid is continuously harvested throughout the cell culture run. Perfusion systems use filtration-based retention devices, such as tangential flow filtration, recirculating tangential flow filtration, or alternating tangential flow filtration, to capture the protein product and / or cells from the culture.

[0005] In a steady-state perfusion system, the culture cell density is kept essentially constant by removing excess cells with a harvest fluid, and cells grow continuously. In contrast, in a transient perfusion system, cells are grown in a growth phase up to a user-defined maximum, after which cell growth slows and eventually stops in favor of protein production.

[0006] The first step in bioprocessing involves a series of scale-up and expansion phases designed to generate a sufficient cell mass for inoculation of a production bioreactor. The cell culture process begins by thawing a vial from a working cell bank (WCB) and growing the culture, for example, using a series of shake flasks, culture bags, and / or growth seed bioreactors (e.g., N-3, N-2, where the number indicates how many steps the bioreactor is from the N, final, or production bioreactor). After growth in the seed bioreactor, the culture is transferred to an N-1 bioreactor (which may be, for example, a perfusion bioreactor). In the N-1 perfusion bioreactor, the culture is perfused with fresh medium to generate a sufficient cell density for inoculation of the final culture step (production bioreactor (N)). The N-1 production bioreactor is operated to maximize efficient production of the protein product.

[0007] There remains a need in the art to improve the productivity of perfusion systems for bioprocessing. Summary of the Invention [Means for solving the problem]

[0008] The present disclosure provides a method for producing a protein product in a bioreactor in continuous perfusion mode. 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, (a) inoculating a bioreactor at a high cell density with cells expressing the protein product and a liquid medium, and (b) growing the cells at a set temperature and gradually increasing the permeate rate to a first biomass setpoint. The non-steady-state production phase includes, but is not limited to, (c) shifting the culture to a lower temperature or lower permeate rate when the first biomass setpoint is reached to begin shifting the culture to protein production, (d) growing the cells at a set temperature and permeate rate to a second, higher biomass setpoint that promotes non-steady-state cell culture and high productivity, (e) culturing the cells under the culture conditions described in (d) so that viability decreases over time, and (f) recovering the protein product from a harvest stream during the production phase.

[0009] The viable cell density (VCD) may decrease over time and / or the cell volume in solution (PCV) may increase over time.

[0010] One or more cell bleeds can be performed during the production phase to prevent the culture from exceeding the upper viability limit. Bleeding can occur if the cells increase beyond a second biomass setpoint. In this case, the bleed rate in step (e) is reduced or zero if the culture is in a non-steady state. In a non-steady state, a manual, non-zero constant bleed can be employed.

[0011] 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.

[0012] The first biomass setpoint can be between about 50 million cells / mL and about 100 million cells / mL, and the second biomass setpoint can be between about 100 million cells / mL and about 150 million cells / mL.

[0013] The permeate rate in (b) can be from 0 to about 4.1 working volumes per day. In step (b), the permeate rate increases as biomass increases to support cell growth. The permeate rate in (d) can be from about 1.0 to about 4.2 working volumes per day.

[0014] The maximum VCD for (d) is about 130 to 140 million cells / mL. The VCD may decrease to about 10 to 120 million cells / mL during the production phase.

[0015] The set temperature of (b) can be about 35.5 to about 36.5°C, for example, about 36°C.

[0016] The set temperature shifts (c) and (d) can be to lower the temperature to approximately 32.5 to 35.5°C.

[0017] The flow rate of (c) and (d) to induce the shift can be reduced to about 1.0 to about 2.5 working volumes / day.

[0018] Cell bleed during the growth phase can be 0% to about 40%. Cell bleed during the growth phase can be 0% to about 3%. Cell bleed during the growth phase can be about 1.5%.

[0019] The cell viability during the proliferation phase may be about 90% to about 99%. The cell viability during the proliferation phase may be about 97%. In step (e), the viability decreases 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%.

[0020] The PCV during the growth phase can increase from about 2% to about 24%. The PCV during the production phase can be about 25% to about 50%, or about 40% to about 50%.

[0021] The duration of the growth phase may be about 4 to about 12 days. The duration of the production phase may be about 9 to about 41 days. The duration of the production phase may be about 10 days.

[0022] The method may further comprise subjecting the harvested protein product to downstream capture chromatography, viral inactivation and / or polishing steps.

[0023] The cell in this method may be a mammalian cell. The mammalian cell may be a Chinese hamster ovary (CHO) cell.

[0024] The protein product may be, for example, an antibody product. The protein product may be a bispecific antibody. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 shows the daily trends in CM operation of the first antigen binding protein. [Figure 2] FIG. 1 shows the daily trends in CM operation of a second antigen binding protein. [Figure 3] FIG. 10 shows the daily trends in CM operation of a third antigen binding protein. DETAILED DESCRIPTION OF THE INVENTION

[0026] Provided herein is a transient continuous perfusion culture process for biologics manufacturing. Unlike conventional extended continuous perfusion techniques, cell cultures are not operated in a steady state after reaching a user-defined maximum peak growth. This process improves productivity by maintaining cells in the production phase after cell growth plateaus, thereby increasing productivity and shortening the culture period, resulting in higher productivity than typical perfusion culture processes. The production phase of a culture begins when cells transition from the growth phase. Culture conditions are altered to favor product production over exponential cell growth. This change in culture conditions forces a decrease in viable cell density and viability as cells transition from the growth phase to the production phase. Once a set maximum is reached, the cell culture shifts to the production phase. This shift can be achieved by a temperature shift or other means. A brief biomass ramp-up may occur at the beginning of the perfusion phase, optionally with a cell bleed to maintain the biomass setpoint. However, after the ramp-up period, the biomass declines significantly above the setpoint, resulting in no net cell growth. Bleeding may be performed to prevent the cell density from exceeding the maximum viable cell density (VCD). When bleeding is used, bleed decreases as cell growth decreases. During the non-steady-state portion of the production phase, there is no net cell growth, so cell diameter and biovolume, as measured, for example, by cell volume in solution (PCV), increase and biomass decreases. Under these conditions, cell diameter and biovolume, as measured by cell volume in solution (PCV), increase, indicating that the cells are in the production phase.

[0027] Operating a production bioreactor in a non-steady-state cell culture according to the present disclosure surprisingly results in higher productivity (protein production) than operating in a steady-state cell culture. This shortens the culture period, resulting in higher bioreactor utilization and improved process economics. The production (N-type) bioreactor operation described herein can be performed for extended, flexible periods, e.g., 15 to 35 days. The reduced need for bleed increases the yield during culture. For example, in a conventional steady-state culture, approximately 10% of the cells are continuously removed over a 20-day culture period. By operating the culture in a non-steady-state state, bleed can be reduced by more than half. Therefore, non-steady-state operation according to the present disclosure provides advantageous process economics. Protein product quality is similar between lots in non-steady-state continuous manufacturing (CM), demonstrating that the process is in a controlled state as required by government regulations.

[0028] As used herein, the term "viable cell density" or "VCD" refers to the number of viable cells present in a given volume of medium (e.g., cells / mL) under given experimental conditions. In the methods herein, during the growth phase before the production phase, the cultured cell population is approximately 60 x 10 6 cells / ml ~ approx. 143×10 6 The cells are grown to a maximum setpoint of VCD in cells / ml, after which a production phase is initiated in continuous perfusion mode, with the VCD being reduced throughout the remainder of the culture. During the production phase, the VCD may be reduced by at least 10%, at least 15%, at least 20%, or at least 25% from the maximum setpoint. During the production phase, the VCD may be reduced by at least 2% per day, at least 3% per day, at least 4% per day, or at least 5% per day. The method may further include performing an initial bleed during the production phase to prevent the VCD from exceeding the maximum setpoint. A second maximum setpoint may be used during the reduction of the VCD to prevent clogging of the filtration device.

[0029] As used herein, the "growth phase" of a cell culture refers to a phase in which the viable cell density at any time point is higher than at any previous time point. Cells may be in the growth phase for at least 4 days.

[0030] As used herein, the "production phase" of a cell culture refers to the phase in which cells produce large amounts of protein that accumulate for future processing. During the production phase, perfusion culture can be carried out continuously for at least 7 days, at least 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days, or at least 35 days.

[0031] The dielectric constant (pF / cm) is a measure of bioelectric polarizability. Materials with a high dielectric constant, such as the (outer) cell membrane of a (living) cell, polarize more in response to an applied electric field than materials with a low dielectric constant, thereby storing more energy in the field. Typically, the electric displacement field D resulting from an applied electric field E is D = eE. More generally, the dielectric constant is a thermodynamic function of state. It may depend on the frequency, amplitude, and direction of the applied field. The SI unit for dielectric constant is farads per meter (F / M). Dielectric constant is measured using a dielectric constant probe (e.g., Hamilton Bonaduz AG, Switzerland). It can be measured in-line or manually offline. The dielectric constant increases from approximately 4 pF / cm during the growth phase to a maximum dielectric constant of 60–115 pF / cm. The dielectric constant decreases from its maximum value during the production phase due to unsteady-state cell culture operation.

[0032] The permeate rate (wv / day) is a measure of the volume of medium passed through and removed from a bioreactor system per day. Typically, it is measured relative to the volume of the bioreactor, such as "bioreactor volumes" (bv) or "working volumes" (wv) per day. The permeate rate can be from about 1.0 to about 4.2 WV / day. During the production phase, the permeate rate is about 2.0.

[0033] Biomass specific permeate velocity (pF / cm·day) is the permeate velocity (l / day) divided by the dielectric constant (pF / cm). This is the fresh feed rate relative to the biomass being cultured. Biomass specific permeate velocity, along with temperature, is a process parameter that can be used to define the process design space as steady-state or unsteady-state. This calculated rate is used to compare multiple process inputs implemented with a single cell line because it combines two process parameters into one. Thus, the process design space can be defined by observing the temperature and biomass specific permeate velocity at biomass maximum. Repeated runs of the same cell line and product at the same temperature and biomass specific permeate velocity at biomass maximum (the relative ratio of perfusion rate to biomass maximum) are expected to produce similar results in terms of protein production and biomass loss rate (a cell culture performance indicator).

[0034] Cell Volume in Solution (PCV) - The biovolume (also referred to herein as cell mass or biomass) of cultured cells relative to the total culture volume, expressed as a percentage. PCV can increase as a result of an increase in cell number, an increase in cell diameter for the same number of cells, or a combination thereof. If continuous culture has zero cell bleed (indicating no cell growth) and the PCV continues to increase, it indicates that the cell diameter is increasing.

[0035] "Biomass capacitance probe" refers specifically to a probe that can measure viable cell density. Biomass capacitance probes use capacitance to measure the total viable cells in a culture. Living cells act as capacitors in an alternating current electric field. Biomass capacitance probes can measure and report the charge from these cells.

[0036] Steady State and Unsteady State The CM culture design space consists of several process parameters that can result in conditions that favor cell growth over protein production (steady-state CM) or conditions that favor protein production over cell growth (unsteady-state CM). In steady-state culture, all culture set points and outputs remain the same throughout the culture period. This is only possible if the cells are growing at a rate (cell growth rate) that maintains VCD, viability, PCV, and dielectric constant, but in this case, cell resources are prioritized from protein production to cell growth. Typically, in continuous production technologies, VCD, viability, and often protein production rate remain constant throughout the production period of the culture. Cell growth is equal to the cell removal rate, and cells grow and simultaneously produce protein.

[0037] However, in unsteady-state culture, cells grow slowly or even stop growing altogether in favor of protein production. This causes cells to decrease in number and viability (due to some cell death) and increase in size (cell diameter) to accommodate increased protein production. Unsteady-state CM processes therefore result in higher protein production rates and are more economically attractive than traditional steady-state processes.

[0038] Unsteady-state operation has a certain degree of magnitude. Performance indicators are cell number, size, viability (i.e., VCD, viability, cell diameter, PCV, and dielectric constant), and protein production rate. Process parameters that can be used to adjust the magnitude of their influence on performance indicators are temperature and the permeate velocity relative to the biomass in the culture (biomass-permeate velocity). Process parameters can be modified to achieve performance indicator targets. High biomass, cell debris, medium viscosity, and ATF filter fouling due to high permeate velocity constrain the maximum biomass and permeate velocity. In addition, increasing the temperature and permeate velocity at high biomass may cause cell growth to stop too quickly, resulting in the termination of the culture before sufficient protein is produced. Therefore, the maximum biomass (dielectric constant target), temperature, and permeate velocity are optimized within the process design space to achieve the protein production target while considering process robustness.

[0039] In steady-state operation, the permittivity / biomass is held at one target level during the production phase. In transient operation as described herein, the permittivity / biomass is maintained below a maximum value during both portions of the production phase, with a higher maximum value during the biomass expansion phase and a second lower maximum value for the remainder of operation. In transient operation, the biomass must be maintained below a maximum value to reduce the risk of ATF fouling. The two-stage maximization method (reducing the maximum permittivity after biomass expansion) addresses the risk of ATF fouling / failure and provides the benefit of higher productivity from a higher permittivity target.

[0040] Biomass increase phase This phase refers to increasing the biomass to a set point for a short period of time before allowing the biomass to decline. This expansion phase increases protein production for the remainder of the run. The higher the set point for the initial biomass phase, the more cells are available for protein production for the remainder of the run, improving process productivity. This phase is optional in non-steady-state CM. The biomass expansion phase is similar to the growth phase and is typically designed to occur immediately after the growth phase. The difference is that the set point for the biomass expansion phase is lower (production phase) and the target dielectric constant is higher than that of the growth phase.

[0041] cell culture The cell culture methods herein are carried out in a production bioreactor in a non-steady-state operation in continuous perfusion mode, typically using alternating tangential flow filtration technology. Cell culture refers to a liquid culture medium containing a plurality of cells that are maintained or grown under a controlled set of physical conditions.

[0042] Mammalian cells, such as CHO cells, can be cultured in small-scale cultures, e.g., 100 ml vessels containing about 30 ml of medium, 250 ml vessels containing about 35 to about 70 ml of medium, or 500 ml vessels containing about 100 to about 200 ml of medium. Alternatively, cultures can be cultured on a larger scale, e.g., 1000 ml vessels containing about 140 ml to about 300 ml of medium, 3000 ml vessels containing about 500 ml to about 2200 ml of medium, 50 L vessels containing about 4 L to about 30 L of medium, or 200 L vessels containing about 50 L to about 135 L of medium. Large-scale cell cultures, such as those used for clinical production of protein therapeutics, are typically maintained for several days or weeks while the cells produce the desired protein.

[0043] The term "bioreactor" refers to any vessel useful for growing cell cultures, such as a fluidized-bed bioreactor, hollow fiber bioreactor, roller bottle, shake flask, or stirred-tank bioreactor. Bioreactors can be of any size useful for culturing cells, typically sized appropriately for the volume of cell culture to be grown therein. Typically, bioreactors are at least 1 liter and can be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, or 5,000 liters or more, or any volume therebetween. Bioreactors can be 8,000, 10,000, 12,000, 18,000, 25,000 liters or more, or any volume therebetween. Internal conditions of the bioreactor, including, but not limited to, pH and temperature, can be controlled during the culture period. Those skilled in the art will know and be able to select suitable bioreactors for use in practicing the methods described herein.

[0044] The disclosed methods can be performed using single-use bioreactors, also known as disposable bioreactors, which utilize disposable bags instead of traditional culture vessels. Transitioning to single-use technology minimizes infrastructure requirements, such as the steel / glass industrial-scale vessels and associated machinery required for traditional cell culture. Single-use bioreactors offer flexibility in the manufacturing process, and on-site assembly, reconstitution, sterilization, and validation are faster, easier, and less costly than traditional cell culture plants. Single-use bioreactors typically use a disposable plastic sterile bag supported by a non-disposable support structure. The culture is agitated by agitators or agitators within the bag, and sensors measure and regulate various parameters of the culture medium, such as pH, temperature, oxygen, and cell density. Single-use bioreactors are commercially available from, for example, Xcellerex, GE, Hyclone, and Sartorius.

[0045] A bioreactor system maintains conditions within the bioreactor to support cell culture. Culture conditions suitable for mammalian cells are known in the art. See, for example, Animal Cell Culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). "Operating" a bioreactor system means maintaining conditions within the bioreactor to support cell culture. "Running" a bioreactor typically involves inoculating a prepared bioreactor with a seed culture and growing the culture for an appropriate or predetermined time until the culture is terminated, usually by harvesting the bioreactor contents. In the operation of a production bioreactor (N-type bioreactor), a seed bioreactor or N-1-type bioreactor is typically used to grow the cells used to inoculate the production bioreactor.

[0046] "Culturing" refers to maintaining cells in a culture medium under conditions suitable for cell survival and / or growth, separate from a multicellular organism or tissue, and producing a protein product. Cell cultures are typically operated in batch, fed-batch, or perfusion mode. In batch mode, a fixed amount of culture medium and cells are placed in a bioreactor at the beginning of a run. During cultivation, the medium volume in the reactor remains constant while the nutrient content of the medium is simultaneously reduced. The cell concentration increases during the run, reaches a steady state as the nutrient content is depleted and waste increases, and may then decrease. Fed-batch cultures, like batch cultures, begin with an inoculation of cells and a fixed amount of culture medium. Unlike batch cultures, the volume of medium in the bioreactor increases as concentrated nutrients are added during cultivation.

[0047] "Growth" cell culture medium or feed medium refers to a cell culture medium typically used in cell culture during the period of exponential growth, the "growth phase," and that is sufficiently complete to support cell culture during this phase. Growth cell culture medium may also contain a selection agent that confers resistance or survival to a selection marker incorporated into the host cell line. Such selection agents include, but are not limited to, geneticin (G4118), neomycin, hygromycin B, puromycin, zeocin, methionine sulfoximine, methotrexate, glutamine-free cell culture medium, cell culture medium lacking glycine, hypoxanthine, and thymidine, or cell culture medium lacking only thymidine. Growth cell culture media are known in the art.

[0048] "Production" cell culture medium or feed medium refers to a cell culture medium typically used in cell cultures during the transition phase when exponential growth ends and during the subsequent transition and / or production phases when protein production becomes dominant. Such cell culture media are sufficiently complete to maintain a desired cell density, viability, and / or product titer during this phase. Production cell culture media are known in the art.

[0049] Perfusion culture, like batch culture, begins with a fixed inoculum of cells and culture medium. Unlike batch and fed-batch culture, fresh feed medium is added, or perfused, to the bioreactor and an equal amount of spent medium is removed. In the case of the methods described herein, the perfusion is continuous. A retention device, such as a tangential flow filtration (TFF) system, an alternating tangential flow (ATF) system, or a recirculating tangential flow (RTF) system, can be used to remove spent medium and unwanted by-products from the bioreactor.

[0050] RTF uses a recirculation means, most commonly a peristaltic pump, to move the cell culture in one direction and parallel to the membrane surface, removing spent medium while retaining the cells in the bioreactor. ATF systems are similar to RTF systems, except that instead of flowing in only one direction, there is a pump that moves the cell culture back and forth within a module (e.g., a hollow fiber filter module). See, e.g., U.S. Pat. No. 6,544,424; Furey (2002) Gen. Eng. News. 22(7), 62-63. An advantage of ATF is the filter cleaning effect induced by alternating flow. An exemplary method of the present disclosure includes operating a (N-type) production bioreactor using an ATF perfusion system.

[0051] Typically, hollow fiber filters are used in RTF or ATF systems (although this is not required). When cell culture media, including cells (whole and lysed), soluble recombinant proteins, host cell proteins, waste products, etc., is introduced into the filter depending on the pore size or molecular weight cutoff (MWCO), the hollow fiber material retains certain cell culture components (in addition to the cells themselves) on the luminal side (inside) and allows certain components (permeate) to pass through the filter, depending on the pore size or molecular weight cutoff of the hollow fiber material. The retained material (retentate) is returned to the bioreactor. Fresh perfused cell culture media is added to the bioreactor, and the permeate is withdrawn from the filter at predetermined intervals or continuously to maintain a desired or constant bioreactor volume. The permeate can be discarded, stored in a holding tank, holding bag, or holding tote, or transferred directly to another unit operation, such as filtration, flocculation, centrifugation, and / or other downstream purification methods.

[0052] In various embodiments, hollow fibers have an inner diameter of about 0.5 mm to about 1 mm and can be any suitable length (e.g., about 30 cm to about 110 cm). Hollow fibers for microfiltration generally have pore sizes in the range of 0.1 μm to 10 μm or a molecular weight cutoff of 500 to 750 kDa or more and can be used to pass proteins into the permeate. Ultrafiltration hollow fibers generally have pore sizes in the range of 0.01 μm to 0.1 μm or a molecular weight cutoff of 300 kDa or less and can be used to retain the desired protein in the retentate and return it to the bioreactor. This can be used, for example, to concentrate recombinant protein products for recovery. Such filters are commercially available, for example, Xampler UFP-750-E-4MA, Xampler UFP-30-E-4MA (GE Healthcare, Pittsburgh, Pa.), Midikros TC Modules T02-E030-10, T02-050-10, T02-E750-05, T02-M10U-06 (Spectrum Laboratories, Inc., Dominguez, Calif.), and XCell ATF (registered trademark), Repligen, Waltham, Mass.

[0053] The cell culture medium can be drawn from the bioreactor into the filter module by a pumping system that passes the cell culture through or along the filter (e.g., through the lumen side of the hollow fibers). Examples of cell pumping systems include peristaltic pumps, double diaphragm pumps, low shear pumps (Levitronix™ pumps, Zurich, Switzerland), and alternating tangential flow systems (ATF™, Repligen, Waltham, MA). The permeate can be drawn from the filter by using a peristaltic pump. In an embodiment, perfusion is achieved by using an alternating tangential flow system.

[0054] The disclosed methods can be used as part of a large-scale production process in which cells are cultured in three or more distinct phases. For example, cells can be cultured in one or more growth phases prior to an N-1 production phase, cultured in an N-1 production phase, and then transferred to an (N) production phase under conditions that maximize protein production. Each phase can be carried out in its own bioreactor vessel or other vessel suitable for cell culture. Alternatively, multiple phases can be carried out in a common vessel. In one such example, the growth and production phases are carried out in the same bioreactor vessel. In commercial processes for the production of proteins by mammalian cells, there are generally multiple, e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10, growth phases occurring in different culture vessels preceding the final production phase.

[0055] The duration of the N-1 stage can range, for example, from 3 to 14 days and can be designed to maintain cells in exponential growth prior to inoculation of the production (N-type) bioreactor.

[0056] Cell culture media are media suitable for the growth of animal cells, such as mammalian cells, in in vitro cell culture. Cell culture medium formulations are well known in the art. Typically, cell culture media contain buffers, salts, carbohydrates, amino acids, vitamins, and trace amounts of essential elements. Cell culture media may or may not contain serum, peptone, and / or protein. Various tissue culture media, including serum-free, chemically defined culture media, are commercially available. For example, any one or combination of the following cell culture media can be used: RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle's Medium (DMEM), Eagle's Minimum Essential Medium, F-12K medium, Ham's F12 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media such as the EX-CELL™ 300 series (JRH Biosciences, Lenexa, KS). Cell culture media may 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., depending on the requirements of the cells being cultured and / or the desired cell culture parameters.

[0057] Cell culture media can be serum-free, protein-free, and / or peptone-free. "Serum-free" refers to cell culture media that do not contain animal serum, such as fetal bovine serum. "Protein-free" refers to cell culture media that do not contain exogenously added proteins, such as transferrin, the protein growth factor IGF-1, or insulin. Protein-free media may or may not contain peptone. "Peptone-free" refers to cell culture media that do not contain exogenous protein hydrolysates, such as animal and / or plant protein hydrolysates. Removing serum and / or hydrolysates from cell culture media has the advantage of reducing lot-to-lot variability and enhancing processing steps such as filtration. However, when serum and / or peptone are removed from cell culture media, cell growth, viability, and / or protein expression may be reduced or suboptimal. Therefore, serum-free and / or peptone-free cell culture media may contain significantly increased concentrations of amino acids, trace elements, and the like. See, for example, U.S. Patent Nos. 5,122,469 and 5,633,162.

[0058] Chemically defined cell culture media formulations are complexes containing amino acids, inorganic salts, carbohydrates, lipids, vitamins, buffers, and trace essential elements. Identifying the components necessary and beneficial for maintaining cell cultures with desired characteristics is an ongoing challenge. Chemically defined basal media formulations that are supplemented or enriched to meet the requirements of specific host cells or to meet desired performance parameters are one approach to developing chemically defined media.

[0059] Induction of the production period Bioreactor process parameters that can be used to switch to non-steady-state conditions are permeate rate and temperature. A combination of permeate rate, temperature, and biomass can be used to prioritize protein production over cell growth, resulting in non-steady-state CM operation. These parameters are empirically determined for each cell line to maximize productivity.

[0060] The cell culture temperature is typically 35°C to about 38°C. Cell culture typically includes at least one exponential growth phase and may include a production phase. The growth phase can occur at a higher temperature than the production phase. For example, the growth phase can occur at a first temperature of about 35°C to about 37°C, and 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. Additionally, chemical inducers of protein production, such as caffeine, sodium butyrate, and hexamethylene bisacetamide (HMBA), can be added simultaneously with, before, and / or after the temperature shift. If the inducer is added after the temperature shift, it can be added 1 hour to 5 days after the temperature shift, optionally 1 to 2 days after the temperature shift. The growth phase can also occur at a higher pH than the production phase.

[0061] To induce a non-steady state, the liquid flow rate can be reduced to about 1.0 to about 2.5 working volumes per day.

[0062] Various media formulations may be used during the culture period, for example, to facilitate the transition from one stage (e.g., growth stage or growth phase) to another (e.g., production stage or growth phase) and / or optimize conditions during cell culture (e.g., concentrated media provided during perfusion culture). Growth media formulations may be used to promote cell growth and minimize protein expression. Production media formulations may be used to promote production of the protein of interest and cell maintenance while minimizing cell growth. Feed media, typically media containing more concentrated components such as nutrients and amino acids consumed during the cell culture process, may be used to supplement and maintain active cultures, particularly cultures operated in fed-batch mode. "Perfusion" medium refers to a feed medium specifically developed for use in cell cultures maintained by perfusion methods and sufficiently complete to support the culture during the process. Perfusion medium formulations are typically richer and more concentrated than basal culture medium and fed-batch feed medium formulations, and are continuously supplied in a higher total volume over the culture period to accommodate higher cell densities and the methods used to remove spent medium. Perfusion media can be used during both the growth and production phases of the culture. Such concentrated feed and perfusion media typically contain depleted components and / or components necessary to maintain the culture, and may be present in concentrated cell culture media in amounts of, for example, about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X or more in the basal medium.

[0063] The culture pH is controlled to a desired pH, typically about 6 to 7.4, preferably 6.85 to 7.2. In one embodiment, the pH is 6.90 to 6.95. The pH can be controlled using sparged CO2 and 1 M sodium carbonate. The dissolved oxygen concentration is preferably 40 to 88 mmHg, more preferably 60 to 70 mmHg. Antifoaming agent may be added at a frequency and volume appropriate for the culture operation, and supplemental bolus antifoaming agent may be added as needed.

[0064] A "cell" or "cells" includes any prokaryotic or eukaryotic cell. Cells include "host cells," also called "cell lines," which have been genetically engineered to express a protein of interest. Host cells are typically derived from lines arising from a primary culture that can be maintained in culture indefinitely. Genetic engineering of host cells involves transfecting, transforming, or transducing the cells with a recombinant polynucleotide molecule and / or otherwise modifying (e.g., by homologous recombination and gene activation, or fusion of recombinant cells with non-recombinant cells) the host cell 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 of skill in the art.

[0065] 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 connection with 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 cell (HKB-11), CEVEC's amniocyte production (CAP) cells, human liver (HuH-7) cells, and any other cells used or suitable for use in clinical and / or commercial manufacturing. The most commonly used cell lines are derived from CHO cells. CHO cells are widely used to produce complex 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 efficient DHFR-selectable and amplifiable gene expression systems enable 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 GS-based methionine sulfoximine (MSX) selection, is also widely used. CHOK1 cells (ATCC CCL61) are also included. Measuring key attributes and performance parameters of cell lines allows for more accurate decisions regarding the performance of each step during manufacturing. These key attributes and parameters can be monitored in real time, near real time, and / or retrospectively. Important parameters such as consumed medium components (such as glucose), metabolic levels of by-products accumulating in the medium (such as lactate and ammonia), and those related to cell maintenance and survival, such as dissolved oxygen content, can be measured during cell culture.Important attributes such as specific productivity, viable cell density, pH, osmolality, appearance, viability, aggregation, cell number, cell volume in solution, product quality, yield, titer, etc. can be monitored at appropriate stages in the manufacturing process. Process and product impurities may also be monitored throughout the manufacturing process.

[0066] Monitoring and measurement can be performed using known techniques and commercially available equipment. Detection of product quality attributes can be achieved using mass spectrometry, liquid chromatography with UV and / or mass spectrometry detection, capillary electrophoresis, etc. Amino acid processing and post-translational modifications such as glycosylation can be characterized, for example, using a polyhydroxyethyl aspartamide column operated in size exclusion mode and coupled with ESI-MS (Brady et al., (2008) J Am Soc Mass Spectro, 19:502-509). Real-time monitoring of the eluate from ion exchange chromatography can be performed by monitoring the normalized LS / UV ratio for each fraction using a laser light scattering detector and UV absorbance (see, e.g., U.S. Patent Application Publication No. 2013 / 0303732).

[0067] For example, viable cell density (VCD) and viability (%) can be measured using a Cedex HiRes (Roche, Basel, Switzerland). Glucose, lactate, and NH4+ concentrations can be measured using a Cedex BioHT (Roche, Basel, Switzerland). Titer can be measured by high-performance liquid chromatography (HPLC) via affinity chromatography (Protein A, Waters, Milford, MA). The percentage of impurities can be measured using reduced capillary electrophoresis with sodium dodecyl sulfate (rCE-SDS), non-reduced capillary electrophoresis with sodium dodecyl sulfate (nrCE-SDS), ultra-high performance liquid chromatography (SE-UHPLC), and acidic and basic charge variant species using cation exchange chromatography (CEX-HPLC).

[0068] The viscosity of the cell culture, including the media components and the cells themselves, can be about 1 to about 6 centipoise (e.g., about 2 to about 6 centipoise). The viscosity of the cell culture can be measured using any suitable viscometer, such as a "cone-and-plate" viscometer. The density of the cell culture, including the media components and the cells themselves, can be about 1 g / L to about 1.5 g / L. The density of the cell culture can be measured, for example, by using an automated cell counter, such as the Roche Cedex HiRes, using the trypan blue exclusion method.

[0069] harvest The methods provided herein may further include recovering the protein product from the cell culture. In the harvesting step, the protein product is separated from the cell debris and producer 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 lowered to below 12°C (but above 0°C). Harvesting can be performed by any suitable method, including accelerated sedimentation such as acid precipitation or flocculation, gravity separation, centrifugation, sonication, filtration including membrane filtration using ultrafilters, microfilters, tangential flow filtration including the use of tangential flow filtration in alternative tangential flow and recirculating tangential flow, depth filters, and synovial filters. Depth filtration can be part of the harvesting process to provide further removal of 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 generally composed of cellulose or polypropylene fibers, diatomaceous earth or perlite, and charged resins. The depth filter may optionally include filter membrane layers of various pore sizes, such as 0.22 μm.

[0070] downstream purification The harvested protein can then be further purified from impurities such as residual cell culture medium, cell extracts, host cell proteins, DNA, viruses, improperly expressed proteins, product-related impurities, etc. through one or more downstream purification steps. Downstream process operations can be performed in batch, semi-continuous, and / or continuous modes. Two or more operations may have a direct connection, for example, with a surge tank, holding tank, bag, or other suitable vessel adapted to receive the feed from at least one operation to another.

[0071] Capture chromatography is often used as the first purification step, followed by one or more intermediate and / or polish chromatography steps.

[0072] Affinity chromatography is commonly used as the initial capture step for harvested recombinant proteins because it is suitable for purifying crude or clarified material. Affinity chromatography media can include, for example, substrate-binding capture mechanisms, aptamer-binding capture mechanisms, and cofactor-binding capture mechanisms. Proteins containing Fc components can use substrate-binding capture mechanisms such as Protein A, Protein G, Protein A / G, and Protein L. Numerous resins and materials for Protein A affinity chromatography are commercially available, including, but not limited to, GE Healthcare's MabSelect™, Millipore's PROSEP® Ultra Plus, and Purolite's Praesto® APc+. An exemplary resin is MabSelect™ SuRe resin (GE Healthcare Life Sciences), which exhibits improved clearance of low molecular weight species (LMWS).

[0073] One or more intermediate and / or polish chromatography steps remove remaining contaminants and / or impurities. The intermediate and / or polish chromatography steps involve the use of a chromatographic medium, such as a resin, monolith, and / or membrane, that can be used in a bind-and-elute mode (where the protein of interest binds to the chromatographic medium and is eluted after contaminants and impurities have passed through or been washed away from the chromatographic medium), a frontal or overload mode (where a solution containing the protein of interest is loaded onto the column until the adsorption sites on the column are occupied and the species with the lowest affinity for stationary phase (the protein of interest) begins to elute), a flow-through mode (where the protein of interest flows through the chromatographic material unbound, while contaminants and impurities bind to the chromatographic medium), or any other suitable mode or combination of modes. The most common chromatography modalities used in intermediate and / or purification steps in biologics manufacturing 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 multimode anion exchange chromatography (MMC), and hydroxyapatite chromatography (HA). The operations of each polishing chromatography unit can be performed in the same or different configurations and / or in different modes.

[0074] Each chromatographic unit can be operated as a single uncoupled unit, multiple coupled units, and / or a composite unit. Single chromatographic columns can be operated, for example, as a staggered cycling system, countercurrent loading (cyclic countercurrent chromatography), or a multi-column countercurrent solvent gradient purification process (MCSGP).

[0075] Chromatography media are well known in the art, are common, 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 sulfonates, carboxylates, carboxymethylsulfonates, sulfoisobutyls, sulfoethyls, carboxyl salts, sulfopropyls, sulfonyls, sulfoxyethyls, orthophosphates, etc. CEX resins include, but are not limited to, Mustang S, Sartobind S, SO3 Monolith, S Ceramic HyperD, Poros XS, Poros HS50, Poros HS20, SPSFF, SP-Sepharose XL (SPXL), CM Sepharose Fast Flow, SP Sepharose Fast Flow XL™, SP-Sepharose High Performance™, Capto S, Capto SP ImpRes™, TOYOPEARL® HS, TOYOPEARL® XS, UNOsphere™, FractoPrep™, Fractogel Se HiCap, Fractogel SO3, or Fractogel COO. Anion exchange media include, but are not limited to, Source 15Q, Capto™ Q, Q-sepharose Fast Flow™, Fractogel EDM TMEAT™, Fractogel EDM DEAE, TOYOPEARL Super Q®, Poros HQ™, and POROS XQ™. Mixed mode or multimode media include Capto™ Adhere.Hydrophobic interaction chromatography materials include, but are not limited to, Fractogel™ EMD Propyl or Fractogel™ EMD Phenyl columns (Merck), Octyl Sepharose™ High Performance columns (Pharmacia LKB Biotechnology), low- or high-substituted Phenyl Sepharose™ 6 Fast Flow columns (Pharmacia LKB Biotechnology), Phenyl Sepharose™ High Performance columns (Pharmacia LKB Biotechnology), Macro-Prep™ Methyl or Macro-Prep™ t-Butyl Supports (Bio-Rad), WP HI-Propyl (C3)™ columns (JT Baker), and Toyopearl™ ether, phenyl, or butyl columns (TosoHaas).

[0076] Viral inactivation and viral filtration Viral suppression measures are crucial to ensuring the safety of protein therapeutics. Viral contaminants can arise from a variety of sources, including the use of animal-derived reagents, adventitious viral contaminants in host cell lines, or system failures in GMP manufacturing sites. Viruses are classified as enveloped and non-enveloped. Enveloped viruses have capsids surrounded by a lipoprotein membrane or "envelope" composed of host cell proteins and phospholipids, along with viral glycoproteins that coat the virus upon release from the host cell. This envelope allows the virus to identify, bind, invade, and infect target host cells. Therefore, enveloped viruses are more susceptible to inactivation methods. Non-enveloped viruses are more difficult to inactivate without risk to the manufactured protein and are removed by filtration. Viral suppression strategies can be implemented one or more times through downstream purification.

[0077] Various methods can be employed for virus inactivation, including heat inactivation / pasteurization, UV and gamma irradiation, the use of high-intensity broad-spectrum white light, chemical inactivation agents, the addition of detergents, and solvent / detergent treatment. Low pH and solvent / detergent treatment are the most common virus inactivation methods in the manufacturing process of protein therapeutics. Viral inactivation is typically performed after purification of the harvested solution using affinity chromatography (particularly affinity chromatography utilizing substrate-binding ligands derived from Staphylococcus aureus, such as protein A chromatography), because elution from such chromatographic materials is usually performed at low pH. The acidified eluate is held for a determined time to inactivate the virus concentration by the required log number. Following this, the inactivated material is neutralized. Exemplary low-pH virus inactivation methods are described in U.S. Patent Application Nos. 63 / 168,608 and 63 / 159,217. Exemplary surfactant deactivation is described in WO 2020 / 190985.

[0078] Non-enveloped viruses are difficult to inactivate without risk to 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 remove certain contaminants in the product pool or eluate stream before applying the pool or eluate to the virus filter to maintain continuous flow during the virus filtration operation. An exemplary process is described in WO 2020 / 159838. Virus filtration can be performed using microfilters or nanofilters, such as those available from PLAVONA® (Asahi Kasei, Chicago, IL), VIROSART® (Sartorius, Goettingen, Germany), VIRESOLVE® Pro (MilliporeSigma, Burlington, MA), Pegasus™ Prime (Pall Biotech, Port Washington, NY), and CUNO Zeta Plus VR (3M, St. Paul, MN). Viral filtration can occur at one or more steps in downstream operations of a biomanufacturing process. Typically, viral filtration precedes the UFDF operation, but it can also occur after UFDF.

[0079] UF / DF The method optionally further comprises concentrating the protein product using ultrafiltration and diafiltration (UFDF). The purified protein is subjected to an ultrafiltration and diafiltration operation that includes concentrating or diluting the purified protein by ultrafiltration; buffer exchanging the purified, concentrated / diluted protein into the desired formulation by diafiltration; and further diluting or concentrating the formulated purified protein by a second ultrafiltration until the target protein concentration is reached. One or more stability-enhancing excipients can be added directly to the UFDF retentate feed tank containing the formulated purified protein, which results in the formulated drug substance, or to the UFDF eluate pool. Filters for use in UF / DF operations are well known and common in the art and are commercially available from a number of sources. There are many types of materials available, such as regenerated cellulose Pellicon (MilliporeSigma, Danvers, MA), stabilized cellulose, Sartocon® Slice, Sartocon® ECO Hydrosart® (Sartorius, Goettingen, Germany), polyethersulfone (PES) membrane, Omega (Pall Corporation, Port Washington, NY), etc. Multiple filters can be used up to the capacity that the holder, skid, or physical setup of the UFDF system will allow or that is necessary to achieve the desired objectives of the production process.

[0080] Biopharmaceuticals The methods provided herein can be used to produce a protein product, such as a recombinant protein. The recombinant protein can be a eukaryotic protein, such as a mammalian protein.

[0081] The mammalian protein can be an antigen-binding protein, such as an antibody, antibody fragment, antibody derivative, antibody analog, antibody construct, fusion protein, mutein, multispecific protein, bispecific protein, bispecific T cell engager, or peptidobody. The antibody can be a whole antibody, a single-chain variable fragment, Fv, Fab, Fab', F(ab')2, diabody, triabody, tetrabody, Fd, dAb, minibody, or maxibody.

[0082] "Multispecific proteins" and "multispecific antibodies" refer to proteins that have been 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 agents to tumors or infectious agents. These multispecific proteins have been found to be useful in a variety of applications, such as redirecting immune effector cells to tumor cells, blocking signaling pathways to alter cell signaling, targeting tumor angiogenesis, and cancer immunotherapy by blocking cytokines, as well as for the delivery of chemotherapeutic agents, radiolabeling (to improve detection sensitivity), and as pretargeting delivery vehicles for drugs such as nanoparticles (targeted to specific cells / tissues, such as cancer cells).

[0083] The most common and diverse types of multispecific proteins are those that bind to two antigens, and are referred to interchangeably herein as "bispecific proteins" and "bispecific antibodies." Multispecific proteins also include triabodies, tetravalent bispecific antibodies, multispecific proteins without antibody components such as diabodies, triabodies, or tetrabodies that can bind to multiple targets, minibodies, and single-chain proteins. Coloma, MJ, et al., Nature Biotech. 15 (1997) 159-163.

[0084] Bispecific proteins can be classified into two broad 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), and the Fc region improves solubility and stability and helps facilitate some purification procedures. Non-IgG-like molecules are smaller, enhancing tissue penetration. See 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, pages 837-855. Bispecific proteins have binding specificities for different antigens or several epitopes and may be used as a framework for additional components that increase the binding specificity of the molecule.

[0085] Bispecific proteins come in a variety of formats, including knob-in-hole, crossmab, dual variable domain IgG (DVD-IgG), IgG-single chain Fv (scFv), scFv-CH3 KIH, dual action Fab (DAF), half molecule exchange, κλ-body, tandem scFv, scFv-Fc, diabody, single chain diabody (scDiabodies), scDiabodies-CH3, triple body, miniantibody, minibody, TriBi minibody, tandem diabody, scDiabody-HAS, tandem scFv-toxin, dual affinity retargeting molecules (DARTs), nanobody, nanobody. Body-HSA, Dock & Lock (DNL), Strand Exchange Modification Domain SEEDbody, Triomab, Leucine Zipper (LUZ-Y), XmAb®; Fab-Arm Exchange, DutaMab, DT-IgG, Charge Pair, Fcab, Orthogonal Fab, IgG(H)-scFv, scFV-(H)IgG, IgG(L)-scFV, IgG(L1H1)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-VV(L)-IgG, KIH IgG-scFab, 2scFV-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-Ig4 (Four-in-One), Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, intrabody, ImmTAC, 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 (BiTE®), half-life extended bispecific T cell engager (HLE BITE) (Fan supra; Spiess supra; Sedykh supra; Seimetz et al., Cancer Treat Rev 36(6)458-67,2010;Examples of suitable antibodies include, but are not limited to, Shulka and Norman, Chapter 26 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody-Drug Conjugates, in Process Scale Purification of Antibodies Second Edition, Uwe Gottswchalk editor, pp. 559-594, John Wiley & Sons, 2017; Moore et al., MAbs 3:6, 546-557, 2011);

[0086] 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 hormone, growth hormone receptor, T cell receptor; neurotrophic factors, neurotrophins, relaxin, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, or immunoadhesins. The growth factor can be nerve growth factor, fibroblast growth factor, transforming growth factor, or insulin-like growth factor. The colony-stimulating factor can be granulocyte colony-stimulating factor (G-CSF). Such G-CSF molecules include, but are not limited to, Neupogen® (filgrastim) and Neulasta® (pegfilgrastim). Also listed are Epogen® (epoetin alfa), Aranesp® (darbepoetin alfa), Dynepo® (epoetin delta), Mircera® (methoxypolyethylene glycol-epoetin beta), Hematide®, MRK-2578, INS-22, Retacrit® (epoetin zeta), Neorecormon® (epoetin beta), Silapo® (epoetin zeta), Binocrit® (epoetin alfa), epoetin alfa Hexal, Abse Also included are erythropoiesis-stimulating agents (ESAs) such as amed® (epoetin alfa), Ratioepo® (epoetin theta), Eporatio® (epoetin theta), Biopoin® (epoetin theta), epoetin alfa, epoetin beta, epoetin zeta, epoetin theta, and epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activator, GLP-1 receptor agonists, and molecules or variants or analogs thereof and biosimilars of any of the foregoing.

[0087] 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 and coagulation-related proteins, colony-stimulating factors (CSFs), other blood and serum proteins, blood group antigens; receptors, receptor-related 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, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0088] The methods provided herein involve the detection of 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, CD174, and the like; HER receptor family proteins, including but not limited to, HER2, HER3, HER4, and the EGF receptor EGFRvIII; LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, αv / β3 integrin, and the like. cell adhesion molecules, 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 inhibitory substance, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factor (NGF-β, etc.), platelet-derived growth factor (PDGF), fibroblast growth factor (including, but not limited to, aFGF and bFGF), epidermal growth factor (EGF), Cryptogen, transforming Growth factors (TGF) (especially TGF-α and TGF-β, including TGF-α and TGF-α, particularly TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5), insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone morphogenetic factors; insulin and insulin-related proteins, including, but not limited to, insulin, insulin A chain, insulin B chain, proinsulin, insulin-like growth factor binding proteins, and the like; e.g., factor VIII, tissue coagulation and coagulation-related proteins, including, but not limited to, colony-stimulating factors (CSFs), such as M-CSF, GM-CSF, and G-CSF; other blood and serum proteins, including, but not limited to, albumin, IgE, blood group antigens, and the like;Receptors and receptor-associated proteins, including, but not limited to, flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptor, T cell receptor, and the like; neurotrophic factors, including, but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, -6 (NT-3, NT-4, NT-5, or NT-6); relaxin A chain, relaxin B chain, and prorelaxin; interferons, including, for example, interferon-α, -β, and -γ; IL-1 through IL-10, IL-12, IL-15, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59 ... Interleukins (ILs) including, but not limited to, 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 factors α and β; enkephalinase; BCMA; IgKappa; RO R-1; ERBB2; mesothelin; RANTES (activation regulator typically expressed and secreted by T cells); mouse gonadotropin-related peptide; Dnase; FRα; 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; transport protein; homing receptor; MIC (MIC-a, MIC-B); ULBP1-6; EPCAM; PSA; addressin; regulatory protein; Immunoadhesin; antigen-binding protein; somatropin; 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; 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 conjugates; Legionella pneumophila (ly); gpA33; B7H3; IFNγ; interferon-γ-inducible protein 10 (IP10); IFNAR; TALL-1; thymic stromal lymphopoietin (TSLP); protein convertase subtilisin / kexin type 9 (PCSK9); stem cell factor; Flt-3; calcitonin gene-related peptide (CGRP); OX40L; α4β7; platelet-specific (platelet glycoprotein Iib / IIIb (PAC-1); transforming growth factor β (TFGβ); zona pellucida sperm-binding protein 3 (ZP-3); TWEAK; platelet-derived growth factor receptor α (PDGFRα); sclerostin; and biologically active fragments or variants of any of the foregoing.

[0089] The methods provided herein include the use of any of the following: abciximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, bioozumab, brentuximab vedotin, brodalumab, cantuzumab mertansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, lerdelimumab, lumivumab, and thiazol-111. Riximab, ixekizumab, mapatumumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelvekin, palivizumab, panitumumab, pembrolizumab, pertuzumab, pexelizumab, ranibizumab, rilotumumab, rituximab, romiplostim, romosozumab, sargamostim, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, visilizumab, volociximab, zanolimumab, zalutumumab, and variants or analogs thereof, and can be used to produce biosimilars of any of the foregoing.

[0090] The methods provided herein include the use of blinatumomab, catumaxomab, ertumaxomab, solitomab, targomR, rutikizumab (ABT981), vanucizumab (RG7221), lemtolumab (ABT122), ozoralixumab (ATN103), floteusumab (MGD006), pasotuximab (AMG112, MT112), lymphomunin (FBTA05), (ATN-103), AMG200, and thiazolinone (TH100). 11 (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 IMP-205, xm734, LY3 164530, OMP-305BB3, REGN1979, COV322, ABT112, ABT165, RG-6013(ACE910), RG7597(MEDH7945A), RG7802, R G7813 (RO6895882), RG7386, BITS7201A (RG7990), RG7716, BFKF8488A (RG7992), MCLA-128, MM-111, MM141, M OR209 / ES414, MSB0010841, ALX-0061, ALX0761, ALX0141; BII034020, AFM13, AFM11, SAR156597, FBTA05, PF06671008, GSK2434735, MEDI3902, MEDI0700, MEDI7352, and variants or analogs thereof, as well as biosimilars of any of the foregoing.

[0091] Other terms Various embodiments herein are referred to using the term "comprising" under various circumstances; however, related embodiments may also be described using "consisting of" or "consisting essentially of." The present disclosure contemplates that embodiments described as "comprising" a feature include embodiments "consisting of" or "consisting essentially of" that feature. The terms "a" or "an" mean one or more; the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The term "or" should be understood to include items alternatively or together unless the context clearly dictates otherwise. The term "and / or" should be understood to include each item in a list (individually), combinations of items in a list, and all items in a list taken together.

[0092] When "about" is used in connection with a numerical value, the present disclosure contemplates a range around the numerical value. For example, "about" can mean ±1%, ±2%, ±5%, ±10%, etc. Also, when referring to units such as temperature or pH, it can mean ±0.1 units, ±0.2 units, ±0.3 units, ±0.4 units, ±0.5 units, or ±1 unit.

[0093] When describing a range of values, the present disclosure contemplates each individual value found within that range. For example, a "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. In any range described herein, the endpoints of the range are included within the range. However, the present disclosure also contemplates the same range with the lower and / or upper limits excluded. When a value is described as "about," the exact value is also intended, whether as an individual value or as an endpoint of a range.

[0094] As used herein, "may be," "may," "can be," or "can" refers to something envisioned by the inventors to function and be available as part of the subject matter provided.

[0095] Further features and variations of the present invention will be apparent to those skilled in the art from the entirety of this application, including the drawings and detailed description. It should be understood that this entire document is intended to be related as an integrated disclosure, and that all combinations of features described herein are contemplated, even if the combinations of features are not together in the same sentence, paragraph, or section of this document (even if described in separate sections). [Example]

[0096] While the following examples illustrate specific embodiments, variations and modifications will occur to those skilled in the art, and therefore only limitations set forth in the appended claims should be placed on this invention.

[0097] Example 1 Experiment 1 The first experiment shows a non-steady state cell culture in the first condition. In the first experiment, there were process conditions to induce a non-steady state cell culture.

[0098] In a 100 L single-use bioreactor (Xcellerex, Marlborough, MA), CHO cells expressing the antigen-binding protein were cultured at 45 × 10 in 60 L of serum-free, chemically defined basal medium. 5 After inoculation, the target volume in the bioreactor was adjusted to 100 L of basal medium at a setpoint of pH 6.90 and an initial temperature of 36.0°C, and maintained for 24 hours.

[0099] The bioreactor was equipped with two ATF 6 (0.2 μm) alternating tangential flow devices (Repligen, Waltham, MA). On day 1, the culture was continuously perfused with serum-free, chemically defined perfusion medium at pH 6.90 and 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). Cells were left in the retentate and returned to the bioreactor to build and / or maintain cell density. Any recombinant product was passed through the permeate stream and discharged to waste during the growth phase (days 0–6). The biomass-specific permeate velocity was 0.0211 cm / pF·day at the end of the growth phase.

[0100] Cell count, viability, cell diameter, pH, pCO2, pO2, dielectric constant, glucose, lactate, ammonia, osmolality, cell volume in solution, bioreactor titer, flow-through titer, and harvest titer were measured daily. Bioreactor bulk supernatant and harvest samples were taken for PQ. Antifoam was added directly to the bioreactor to control foaming.

[0101] [Table 1]

[0102] [Table 2]

[0103] Cultures were maintained until a target dielectric constant of 95 pF / cm was achieved (day 6), at which point a temperature shift from 36.0 °C to 34.0 °C was implemented to control cell growth and increase protein production (Table 3). A capacitance probe was used to monitor cell biomass and induce a temperature shift at the target dielectric constant value. The capacitance probe was also used to increase and maintain biomass during the growth and biomass expansion phases by controlling cell bleed and removing cells from the bioreactor. The minimum cell-in-solution volume was ≥ 25%.

[0104] Biomass Increase: Starting on day 6, after the temperature shift signaling the start of the production phase, the biomass increased to a maximum of 105 pF / cm. This high cell density at the start of the production phase sustains a high protein production rate throughout production. The biomass-to-flow rate was 0.0190 pF / cm at this maximum biomass.

[0105] Non-steady state CM: The combination of process parameters in this run, namely temperature, biomass, and permeate velocity (or biomass-specific permeate velocity), resulted in a non-steady state CM where the dielectric constant target and cell bleed were no longer maintained.

[0106] Experiment 1, Condition 1: This condition was run in the production phase, so the recombinant product in the permeate stream was continuously harvested into sterile harvest bags. From day 8 to day 20, the temperature setpoint was maintained at 34.0°C, and the permeate flow rate was maintained at 2.0–2.2 wv / day. Because the cell culture was run in a non-steady-state condition, VCD and viability decreased, while cell diameter and PCV increased. Initially, the dielectric constant was maintained at a target of 95 pF / cm while allowing cell bleed. After approximately one week, the dielectric constant decreased below this maximum of 95 pF / cm, as expected, due to biomass loss during non-steady-state operation. The benefits of reducing VCD, viability, PCV, dielectric constant, bleed, and cell growth rate were stable and high protein production rates.

[0107] Experiment 1, Condition 2: On day 21, the culture temperature was increased to 36.0°C, and the dielectric constant target was set to 65 pF / cm and maintained by cell bleed. The cell culture was also operated in a non-steady-state environment; however, increasing the temperature reduced the rate of biomass loss in the culture. The permeate flow rate was maintained at 2.2 WV / day. The biomass-specific permeate flow rate was 0.0338 pF / cm·day. The recombinant product in the permeate stream was continuously harvested into sterile harvest bags. The culture was continued under these conditions until day 28, at which point it was terminated. While the protein production rate remained as high as in the initial Condition 1, it declined over the last few days. This indicates that relaxing the process parameters in Condition 1 reduces productivity.

[0108] Example 1: Results The first experiment showed biomass growth and non-steady-state operation, resulting in high protein production rates. Thus, biomass growth and the effects of Condition 1 appear to determine the production rate for the majority of the run. Condition 2, which had a higher temperature and reduced the rate of biomass loss, resulted in a decline in production rates during the final few days of culture.

[0109] Experiment 2 The second experiment begins with steady-state conditions followed by unsteady-state conditions where viability declines.

[0110] In the second experiment, the process design space was explored to differentiate between steady-state and unsteady-state CM runs and the magnitude of each run. Several conditions involving successively decreasing temperature and increasing dielectric constant were tested during the production phase. The objective was to determine which combination of temperature and biomass ratio (flow-through rate to biomass during flow-through rate culture) could sustain cell growth and result in high viability (steady-state culture) as opposed to conditions where viability began to decline (unsteady-state culture). Biomass expansion steps were not examined in this experiment.

[0111] CHO cells were grown at 45 × 10 in 60 L of serum-free synthetic basal medium. 5 A 100 L bioreactor was inoculated at a density of 100 cells / ml. After inoculation, the target volume in the bioreactor was adjusted to 100 L of basal medium at a setpoint of pH 6.90 and an initial temperature of 36.0°C and maintained for 24 hours. The permeate flow rate was adjusted daily to reach a maximum daily working volume (WV) of 2.0 on day 6 (see Table 3).

[0112] [Table 3]

[0113] The culture was maintained until a dielectric constant target of 75 pF / cm was achieved (day 6), as shown in Table 3. The minimum cell volume in solution was 25% or greater. The biomass-specific permeability rate was 0.0293 pF / cm·day.

[0114] Experiment 2, Condition 1: The culture temperature was 36.0°C, the target dielectric constant was 75 pF / cm, and the permeate rate was 2.2 wv / day, maintained until day 8. The biomass-specific permeate rate remained at 0.0293 pF / cm·day when the target dielectric constant was reached.

[0115] Experiment 2, Condition 2: On day 8, a temperature shift from 36.0°C to 35.5°C was implemented to control cell growth and promote protein production. The dielectric constant target (75 pF / cm) and permeate velocity (2.2 wv / day) were maintained until day 12. The biomass-specific permeate velocity remained at 0.0293 pF / cm·day when the dielectric constant target was reached.

[0116] 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 permeate flow rate was maintained at 2.2 wv / day. The biomass-specific permeate flow rate increased to 0.0259 pF / cm·day once the dielectric constant target was reached.

[0117] 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 permeate flow rate was maintained at 2.2 wv / day. The biomass-specific permeate flow rate increased to 0.0232 pF / cm·day once the dielectric constant target was reached.

[0118] 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 permeate flow rate was maintained at 2.2 wv / day. The biomass-specific permeate flow rate was increased to 0.0191 pF / cm·day once the dielectric constant target was reached.

[0119] In Experiment 2, conditions 1–3, cell culture parameters remained steady, and production rates were low. In Experiment 2, conditions 4–5, as temperature and biomass increased (decreased biomass-to-permeate rate), the cultures began to exhibit characteristics of non-steady-state CM (i.e., decreased viability). Because cultures in conditions 4–5 still exhibited some cell growth (as evidenced by cell bleed), 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 indicate the first signs of non-steady-state characteristics in conditions 4–5 are decreased viability and decreased cell bleed rate. Production rates also increased in conditions 4–5 relative to steady-state conditions 1–3. Due to the steady-state nature (high cell growth rates) of many of the conditions tested, production rates in Experiment 2 were approximately half of those in Experiment 1.

[0120] Experiment 3 The third experiment illustrates non-steady state conditions without a biomass growth phase.

[0121] Experiment 3 repeated the growth phase of Experiment 2 and Condition 1 without the biomass expansion phase. The objective was to determine the effect of the biomass expansion phase on the performance parameters of the cell culture.

[0122] CHO cells were cultured as in Experiment 1, except that biomass expansion was not 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 permeate velocity (2.0). The biomass-specific permeate velocity for both the growth phase and Condition 1 was 0.0210 pF / cm.

[0123] Although Run 3 was an unsteady-state CM operation, the rate of viability decline was not as rapid, and the cell growth rate (indicated by the bleed rate) exceeded that of Run 1. This run had lower productivity compared to Run 1. This phenomenon may be due to the lack of a biomass growth phase.

[0124] [Table 4]

[0125] Experiment 4 The fourth experiment is a repeat of the first, demonstrating the reproducibility of cell culture in non-steady state conditions.

[0126] Experiment 4 was conducted as a non-steady-state CM operation with different dielectric constant targets and permeate velocities. The biomass specific permeate velocity was lower than in Experiment 1, while the dielectric constant target was higher and the permeate velocity was lower. Similar to Experiment 1, this experiment also demonstrated high productivity in non-steady-state CM culture.

[0127] CHO cells were grown at a lower permeate velocity (1.8 wv / day) and a higher dielectric constant (115 pF / cm) during the growth phase as described in Experiment 1 (see Table 5). The biomass specific permeate velocity was 0.0157 pF / cm.

[0128] [Table 5]

[0129] On day 7, the temperature was reduced to 34.0°C, and the dielectric constant and permeate velocity remained the same with increasing biomass. The biomass specific permeate velocity was 0.0157 pF / cm.

[0130] On the 9th day, the dielectric constant was reduced to 105 pF / cm, and the biomass specific liquid flow rate was set to 0.0171 pF / cm.

[0131] Example 1: Results In Experiment 1, high protein production was demonstrated throughout the low-temperature production phase. Production rates were similar throughout the process despite changes in setpoint (see Figure 1A-F). Therefore, the biomass expansion phase and Condition 1 appeared to determine the high protein production rate (shown in total production). In contrast, the effect of Condition 2 was only seen in the last few days because it takes several days for the effects of the conditions to appear in cell culture. Therefore, the biomass expansion phase should optimally occur immediately after the growth phase, as this step largely determines the protein production rate for the majority of the culture period.

[0132] Two examples of non-steady-state CM with biomass expansion phases (Experiments 1 and 4) were validated for product quality in both lots of each run and compared to a single lot of a discontinuous perfusion process of the same cell line / molecule. Samples were taken from each run to determine the product quality attributes of the recombinant protein produced. These PQAs were compared to a discontinuous perfusion culture producing an antigen-binding protein. PQAs were also similar between lots of non-steady-state CM. There appeared to be no adverse effects on PQAs between different CM runs or between CM and the discontinuous perfusion process.

[0133] [Table 6]

[0134] Two production lots were obtained from each CM bioreactor experimental run. The two lots within each experimental run were compared for product quality. Product quality attributes were similar between the two CM lots in Experiment 1 and between the two CM lots in Experiment 2. Product quality was also fairly similar between the two different CM runs for most attributes. A discontinuous perfusion process was performed to produce the same protein from the same cell line. This process was also operated at unsteady state, but the culture conditions were different (e.g., temperature, perfusion rate). Product was maintained in the reactor and perfused for the final three days. Product quality attributes from the perfusion study were similar to those from the two CM studies, despite differences in process conditions and format.

[0135] Experiment 3 had the same growth phase and initial conditions as Experiment 1, but without biomass expansion. The production volume was lower under the conditions of Experiment 3 compared to Experiment 1 (see Figures 1A-F). This may be due to the absence of a biomass expansion phase in Experiment 3. Therefore, the presence of a biomass expansion phase is important for ensuring high productivity.

[0136] Run 4 had both a growth and biomass expansion phase, followed by a production phase at the same temperature as runs 1 and 3. The dielectric constant and permeate setpoints were adjusted to achieve a lower biomass-to-permeate velocity. Productivities in runs 1 and 4 were similar because the growth, biomass expansion, and production phases were similar (see Figures 1A–F). This indicates that all three stages are important for establishing high productivity in unsteady-state CM cultures.

[0137] The objective of Experiment 2 was to determine which temperature and biomass-specific permeate flow rate setpoints could sustain cell growth and result in high viability (steady-state culture), and at which temperature viability began to decline (unsteady-state culture). Experiment 2 demonstrated that there is a process design space that favors cell growth and another that favors protein production. Steady-state conditions were observed in the first three conditions, and unsteady-state conditions were present in the last two conditions, as indicated by the decline in viability. See Figures 1A-F.

[0138] Overall Results of Example 1 There are several examples of non-steady-state CM conditions mentioned above. There were various levels of biomass reduction and corresponding differences in production rates, which were the result of testing various combinations of process parameters. The importance of the biomass expansion phase was demonstrated in Experiment 3 by removing this phase from the culture and observing the effect on production rate. The transition between steady-state and non-steady-state CM was demonstrated by gradually increasing the biomass and decreasing the temperature.

[0139] Experiments 1 and 4 show non-steady-state CM with a phase of biomass growth.

[0140] Viable cell density-VCD increased during the growth phase (days 0-6) and biomass expansion phase (days 6-8), peaking on day 8, and then steadily decreased during the remainder of the production phase (due to non-steady-state operation).

[0141] Survival rates - Survival rates also began to decline, similar to VCD.

[0142] Permittivity - Permittivity decreased in the transient state, but in contrast to VCD and viability, which often showed a delay in exhibiting transient characteristics, this decreased quickly.

[0143] The slope of Bleed Sum - Bleed Sum is a measure of cell proliferation. A horizontal slope indicates no cell proliferation. A sharp decline in viability corresponds to a slower bleed rate and a slower cell proliferation rate.

[0144] Cell volume in solution - PCV increases even though VCD decreases in non-steady-state cultures, indicating that cell diameter (and therefore cell volume) increases in non-steady-state cultures.

[0145] The slope of total production minus total production represents the protein production rate, which is steeper in non-steady-state CM than in steady-state CM cultures.

[0146] Example 1: Conclusion Experiments 1–4 helped define the non-steady-state production phase. We investigated the process parameters that influence non-steady-state behavior and their impact on the culture. Furthermore, we clarified the role of the biomass expansion phase in improving productivity. In Experiment 1, a biomass expansion phase was performed; the first condition resulted in non-steady-state operation, and the second condition attempted to return the process to steady-state operation. In Experiment 2, the culture was gradually shifted from steady-state to non-steady-state by manipulating the biomass level and temperature. In Experiment 3, the process parameters that induced the non-steady-state condition were repeated (i.e., Experiment 1, Condition 1 without the biomass expansion phase). This resulted in a decrease in productivity, which is attributed to the absence of the biomass expansion phase. In Experiment 4, the non-steady-state process (i.e., Experiment 1, Condition 1 with the biomass expansion phase) was repeated with the same process parameters as Experiment 1. This resulted in a similar productivity to Experiment 1, confirming non-steady-state operation.

[0147] Cell cultures operating under non-steady-state conditions (VCD, viability loss) yielded much higher productivity than those operating under steady-state conditions. This may shorten the culture period, improve plant utilization, and therefore improve the cost of the process. The reduction in bleed volume increased the yield in the culture. Higher productivity and permeate velocity led to even higher yields. Thus, operating under non-steady-state conditions resulted in an economically advantageous process. Product quality was similar across the non-steady-state CMs of both lots, indicating that the process was in control.

[0148] Example 2 Example 2 shows cell culture under non-steady state conditions using different cell lines and antigen-binding proteins, demonstrating the reproducibility of these methods. The conditions in this example were cell culture under non-steady state conditions.

[0149] In a 100 L single-use bioreactor (Xcellerex, Marlborough, MA), CHO cells expressing the second bispecific T cell engager were cultured at 45 × 10 in 60 L of serum-free, chemically defined basal medium. 5 After inoculation, the target volume in the bioreactor was adjusted to 100 L of basal medium as described in Example 1, with a setpoint pH of 6.95 and an initial temperature of 36.0°C, and maintained for 24 hours.

[0150] 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 serum-free, chemically defined perfusion medium at pH 6.90 and 36.0°C. The permeate flow rate was adjusted daily to reach a maximum working volume (WV) of 1.8 per day by day 5 (see Table 7). The process parameters for the experiment are listed in Table 8. Cells were left in the retentate and returned to the bioreactor to build and / or maintain biomass. Any recombinant product was passed through the permeate stream and discharged to waste during the growth phase (days 0–6). The biomass dielectric constant was 0.0157 pF / cm·day.

[0151] Cell count, viability, cell diameter, pH, pCO2, pO2, dielectric constant, glucose, lactate, ammonia, osmolality, cell volume in solution, bioreactor titer, flow-through titer, and harvest titer were measured daily. Bioreactor bulk supernatant and harvest samples were taken for PQ.

[0152] [Table 7]

[0153] [Table 8]

[0154] The purpose of this experiment was to demonstrate unsteady-state operation for different molecules and to test the effect of process conditions on different molecules. The growth phase, biomass expansion phase, and condition 1 in this example were the same as experiment 4 in Example 1. Condition 2 involved increasing the temperature to 35°C and manually bleeding the cells by 5% to examine the effect on cell culture performance. In the third condition, the manual bleeding was stopped and the effect on the culture was observed.

[0155] Cultures were maintained until a dielectric constant target of 115 pF / cm was achieved (day 6), Table 8. This dielectric constant was chosen to achieve a high VCD (>1E8 cells / mL) during the biomass expansion phase. The minimum cell volume in solution was ≥25%. The biomass specific flow rate was 0.0157 pF / cm·day. No manual cell bleed was used to maintain the desired dielectric constant. The biomass specific dielectric constant was 0.0157 pF / cm·day.

[0156] Condition 1: The culture temperature was reduced to 34.0°C and the dielectric constant was reduced to 105 pF / cm. The reduction in dielectric constant was performed to reduce the biomass in the culture and reduce the risk of ATF failure. The permeate flow rate was maintained at 18 wv / day until day 14. No manual cell bleeding was used to achieve or maintain the desired dielectric constant. The biomass relative dielectric constant was 0.0171 pF / cm·day.

[0157] Condition 2: The culture temperature was increased to 35.0°C. The dielectric constant was maintained at 105 pF / cm by manual 5% cell bleeding. The permeate flow rate was maintained at 18 wv / day until day 20. The biomass relative dielectric constant was 0.0171 pF / cm·day.

[0158] Condition 3: The incubation temperature, dielectric constant, and permeate velocity were all the same as in Condition 2, but manual cell bleed was not used. The biomass relative dielectric constant was 0.0171 pF / cm·day.

[0159] The recombinant product in the flow-through was continuously harvested into sterile harvest bags during conditions 1 to 3. The culture was terminated on day 28.

[0160] Example 2: Results Condition 1, as expected, resulted in non-steady-state CM, which was characterized by VCD, reduced viability, and high protein production rates.

[0161] Bleeding in Condition 2 served to remove some of the cells and cell debris during culture, resulting in a lower VCD in Condition 2 despite the higher temperature. This cell line showed a dramatic increase in PCV (and cell diameter) compared to the cell line in Example 1. Experience has shown that a PCV of 30-35% increases the risk of ATF failure in the settings used. Cell bleeding was necessary in Condition 2 to help maintain or reduce PCV, reduce the risk of ATF failure, and extend the cell culture period.

[0162] The increase in temperature from day 15 and the lack of manual cell bleeding in condition 3 starting on day 21 resulted in a gradual and sustained increase in VCD without a decrease in viability. This condition demonstrated that the increase in temperature transitioned the culture from an active, unsteady-state culture promoting protein production to a steady-state culture with cell growth. The protein production rate in condition 3 was lower than that of condition 1, as expected, due to the increased temperature favoring cell growth. PQ was similar for the two lots of this CM run (see Table 9), indicating that the process was in control and that the tested process conditions were suitable for cell culture.

[0163] Growth, biomass growth, and Condition 1 in this Example 2 were the same as in Experiment 4 in Example 1. Similar to Example 1, Example 2 showed decreased VCD and viability, and increased PCV. Because different cell lines / molecules respond differently to process setpoints, the rate of change in viability, VCD, and PCV differed from that observed in Example 1. Production rates were very similar between the two bispecific T cell engagers. In Example 2, viability was decreased and PCV was increased compared to Experiment 4 in Example 1 due to the differences in the cell lines and bispecific T cell engagers used.

[0164] [Table 9]

[0165] The quality of the products from both lots in the CM run was nearly identical, indicating that the process was in control.

[0166] Example 2: Conclusion In this experiment, transient CM, along with a biomass expansion phase, is demonstrated for different cell lines producing different molecules. A transition to steady-state CM operation is also demonstrated. This indicates that this cell line also exhibits distinctive cell culture performance characteristics in these two process design spaces: promoting protein production over cell growth in the transient CM space and promoting cell growth over protein production in the steady-state CM space. The process can be modulated from one type of operation to another by changing the process temperature.

[0167] Example 3 Example 3 illustrates non-steady-state cell culture on a third different cell line and antigen-binding protein, demonstrating the reproducibility of these methods. Cell density and dielectric constant in this example were lower than in the first two examples.

[0168] In a 50 L single-use bioreactor (Hyclone), 38 x 10 cells were cultured in 25 L of serum-free, chemically defined basal medium. 5 CHO cells expressing a second bispecific T cell engager were inoculated at a density of 1000 cells / ml. After inoculation, the target volume in the bioreactor was adjusted to 45 L of basal medium at a setpoint of pH 6.90 and an initial temperature of 36.0°C and maintained for 144 hours.

[0169] The bioreactor was equipped with one ATF 6 (0.2μ) alternating tangential flow device (Refine, Pine Brook, NJ). On day 1, the culture was continuously perfused with serum-free, chemically defined perfusion medium. The permeate flow rate was adjusted daily to reach a maximum daily working volume (WV) of 1.8 on day 6 (see Table 10). The process parameters for the experiment are shown in Table 10. Cells were left in the retentate and returned to the bioreactor to build and / or maintain biomass. Any recombinant product was passed through the permeate stream and discharged to waste during the growth phase (days 0-6).

[0170] Cell count, viability, cell diameter, pH, pCO2, pO2, dielectric constant, glucose, lactate, ammonia, osmolality, cell volume in solution, bioreactor titer, flow-through titer, and harvest titer were measured daily. Bioreactor bulk supernatant and harvest samples were taken for PQ.

[0171] [Table 10]

[0172] [Table 11]

[0173] Cultures were maintained until a dielectric constant target of 70 pF / cm was achieved (day 6).

[0174] 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 permeate rate was maintained at 1.8 wv / day until the 15th day.

[0175] Example 3, Condition 2: The culture temperature was increased to 34.0°C, and the dielectric constant and permeate velocity were decreased to 60 pF / cm. Under both Conditions 1 and 2, the recombinant product in the permeate was continuously harvested into a sterile harvest bag. The culture was continued under these conditions until completion.

[0176] Condition 3: The culture temperature was increased to 36.0°C, and the dielectric constant and flow rate were maintained the same as in Condition 1. In both Conditions 1 and 2, the recombinant product in the flow-through was continuously harvested into a sterile harvest bag. The culture was continued under these conditions until completion.

[0177] Example 3: Results This experiment demonstrated that early-onset biomass expansion and unsteady-state cell culture at low temperatures resulted in high protein production rates. Lowering the dielectric constant in condition 2 further increased protein production, although viability decreased. Increasing the temperature and dielectric constant in condition 3 allowed the culture to maintain viability.

[0178] Example 3: Conclusion In conclusion, dielectric constant and temperature are tools that can be utilized to increase protein production.

[0179] Summary of process parameters in the examples Tables 12 and 13 below summarize exemplary process parameter values ​​contemplated herein as useful for improved protein production.

[0180] [Table 12]

[0181] [Table 13]

[0182] All references cited herein are incorporated by reference in their entirety.

Claims

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

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

3. 10. The method of claim 1, wherein one or more cell bleeds are performed to prevent the culture from exceeding the biomass set point.

4. 4. The method of claim 3, wherein bleeding occurs when the cells increase above the second biomass set point and the bleeding rate is decreased or reduced to zero.

5. 5. The method of any one of claims 1 to 4, wherein the high cell density in (a) is from about 200,000 cells / mL to about 5,000,000 cells / mL.

6. 6. The method of any one of claims 1 to 5, wherein the first biomass set point is between about 50 million cells / mL and about 100 million cells / mL.

7. 7. The method of any one of claims 1 to 6, wherein the second biomass set point is between about 100 million cells / mL and about 150 million cells / mL.

8. 8. The method of any one of claims 1 to 7, wherein the liquid flow rate of (b) is from 0 to about 4.1 working volumes per day.

9. 9. The method of claim 1, wherein the liquid flow rate of (d) is from about 1.0 to about 4.2 working volumes per day.

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

11. 11. The method of any one of claims 1 to 10, wherein the set temperature in (b) is about 35.5 to about 36.5°C.

12. The method of any one of claims 1 to 11, wherein the temperature shift in (c) is to a temperature of about 32.5 to about 35.5°C.

13. 13. The method of any one of claims 1 to 12, wherein the cell bleed during the growth phase is between about 0% and about 3%.

14. The method of any one of claims 1 to 13, wherein the cell viability during the proliferation phase is between about 90% and about 99%.

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

16. 16. The method of any one of claims 1 to 15, wherein the PCV in the growth phase is increased from about 2% to about 24%.

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

18. 18. The method of any one of claims 1 to 17, wherein the duration of the growth phase is about 4 to 12 days.

19. 19. The method of any one of claims 1 to 18, wherein the duration of the production phase before cell growth stops is about 10 days.

20. 20. The method of any one of claims 1 to 19, wherein the duration of the production phase after cell growth arrest is from about 9 days to about 41 days.

21. 21. The method of any one of claims 1 to 20, further comprising subjecting the harvested protein product to downstream capture chromatography, viral inactivation and / or polishing steps.

22. The method of any one of claims 1 to 21, wherein the cell is a mammalian cell.

23. The method of any one of claims 1 to 22, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

24. 24. The method of any one of claims 1 to 23, wherein the protein product is an antigen-binding protein.

25. 25. The method of claim 24, wherein the antigen binding protein is an antibody, an antibody fragment, an antibody derivative, an antibody analogue, 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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