Cell culture process

Abstract

The present invention belongs to the field of production of recombinant proteins, particularly antibodies. More specifically, this relates to a method for producing recombinant proteins (such as antibodies) in a bioreactor and / or a method for enhancing cell culture ability during production of recombinant proteins in a bioreactor (N-stage) by applying organized operating stress to a seed culture through a specific feeding strategy in a seed bioreactor (N-1 stage), that is, by increasing the feeding rate, the feeding amount, and / or by increasing the power input by an impeller.

Description

Technical Field

[0001] The present invention belongs to the field of production of recombinant proteins, particularly antibodies. More specifically, it relates to a method for producing recombinant proteins (such as antibodies) in a bioreactor and / or a method for enhancing cell culture ability during the production of recombinant proteins in a bioreactor (N-stage) through a specific feeding strategy in a seed bioreactor (N-1 stage).

Background Art

[0002] The development of recombinant proteins as therapeutic proteins such as therapeutic antibodies requires the production of recombinant proteins on an industrial scale. To achieve this, various expression systems such as both prokaryotic and eukaryotic systems can be utilized. However, over the past 20 years, the majority of therapeutic proteins approved as therapeutic agents have been produced by culturing mammalian cells, and such systems still continue to be the preferred expression systems for producing large amounts of recombinant proteins for use in humans.

[0003] Over the past 30 years, while most of the research focus has been directed towards achieving optimal cell growth by changing the composition of the cell culture medium (see, for example, Hecklau C. et al., 2016; Zang Li. et al., 2011) and operating conditions, much effort has been devoted to establishing the basic parameters of cell culture and recombinant protein expression and to the development of large-capacity bioreactors.

[0004] It is known that the production step (N-stage) ability can be greatly affected by the conditions of the N-1 stage. As described by Grammatikos et al (1999), the relative intracellular content of nucleotides and ATP is affected by, for example, the conditions of the bioreactor, particularly the conditions of the seed bioreactor.

[0005] Nevertheless, there is still a continuing need to provide an N-1 stage cell culture method that positively affects the N stages of recombinant protein production. SUMMARY OF THE INVENTION

[0006] In a first aspect, the present invention provides a process for producing a recombinant protein in a production bioreactor, the process comprising: a. inoculating a mammalian cell comprising a gene encoding a recombinant protein into an N-1 bioreactor; b. culturing the mammalian cells in the N-1 bioreactor in fed-batch mode under specific conditions selected from: i. a specific mode and period for adding a feed or at least one of the feeds, ii. controlling the total amount of the feed added, and / or iii. controlling at least one industrial parameter; c. inoculating the cells obtained from step (b) into an N bioreactor at a seeding density of at least 2.00×10 6 viable cells / ml of viable cells; d. culturing the cells in the N bioreactor under conditions that allow production of the recombinant protein; and e. optionally harvesting the recombinant protein, purifying the recombinant protein, and formulating the recombinant protein.

[0007] In a second aspect, the present invention provides a process for improving the growth of mammalian cells in a production bioreactor, the process comprising: a. inoculating a mammalian cell comprising a gene encoding a recombinant protein into an N-1 bioreactor; b. culturing the mammalian cells in the N-1 bioreactor in fed-batch mode under specific conditions selected from: i. a specific mode and period for adding a feed or at least one of the feeds, ii. controlling the total amount of the feed added, and / or iii. controlling at least one industrial parameter; c. inoculating the cells obtained from step (b) into an N bioreactor at a seeding density of at least 2.00×10 6Inoculating an N bioreactor at a seeding density of viable cells per ml of viable cells, and d. culturing the cells in the N bioreactor under conditions that enable the production of the recombinant protein, and e. optionally harvesting the recombinant protein, purifying the recombinant protein, and formulating the recombinant protein.

[0008] In a third aspect, the present invention relates to a process for increasing the production yield of a recombinant protein expressed by mammalian cells in a culture in a production bioreactor, the process comprising: a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding the recombinant protein, and b. culturing the mammalian cells in the N-1 bioreactor in fed-batch mode under specific conditions selected from: i. a specific mode and period of addition of a feed or at least one of the feeds, ii. control of the total amount of the feed added, and / or iii. control of at least one industrial parameter, and c. the cells obtained from step (b) being at least 2.00×10 6 Inoculating an N bioreactor at a seeding density of viable cells per ml of viable cells, and d. culturing the cells in the N bioreactor under conditions that enable the production of the recombinant protein, and e. optionally harvesting the recombinant protein, purifying the recombinant protein, and formulating the recombinant protein.

[0009] Definitions In case of conflict, the present specification including the definitions shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the subject matter of this specification belongs. As used herein, the following definitions are provided to facilitate understanding of the present invention.

[0010] As used in this specification and the claims, the term "and / or" as used in the phrase "A and / or B" herein is intended to include "A and B", "A or B", "A", and "B".

[0011] As used in this specification and the claims, the terms "cell culture" or "culturing" mean the growth and proliferation of cells in vitro, i.e., outside an organ or tissue. Suitable culture conditions for mammalian cells are known in the art, such as those taught by Ozturk & Hu (2005). Mammalian cells may be cultured while in suspension or while attached to a solid substrate.

[0012] The terms "cell culture medium", "culture medium", "medium" and any plural forms thereof refer to any medium in which any type of cell can be cultured. "Basal medium" refers to a cell culture medium that contains all of the essential components useful for cell metabolism. This includes, for example, amino acids, lipids, carbon sources, vitamins and inorganic salts. DMEM (Dulbeccos’ Modified Eagles Medium), RPMI (Roswell Park Memorial Institute Medium) or Medium F12 (Ham's F12 medium) are examples of commercially available basal media. Other suitable media are described, for example, in WO 98 / 08934 and US 2006 / 0148074 (the entire contents of both are incorporated herein by reference in their entirety). Further suitable commercially available media include, but are not limited to, AmpliCHO CD medium, Dynamis™ medium, EX-CELL® Advanced™ CHO fed-batch system, CD FortiCHO™ medium, CP OptiCHO™ medium, Minimum Essential Media (MEM), BalanCD® CHO Growth A medium, ActiPro™ medium, DMEM - Dulbecco's Modified Eagle Medium and RPMI-1640 medium. Alternatively, the basal medium can be a trademarked medium also referred to as a "chemically defined medium" or "chemically defined culture medium", in which all of the components are described by chemical formula and are present at specific concentrations. The medium preferably contains no protein, no serum, and can be supplemented with any additional compounds such as amino acids, salts, sugars, vitamins, hormones, growth factors, etc., as required by the cells in culture.

[0013] The term "feed medium" (and its plural forms) refers to a medium used as a supplement during cultivation in fed-batch mode to replenish nutrients consumed during cultivation. The feed medium can be a commercially available feed medium or a trademarked feed medium. Suitable commercially available feed media include, but are not limited to, Cell Boost™ supplement, EfficientFeed™ supplement, ExpiCHO™ Feed. Alternatively, the feed medium can be a trademarked feed medium also referred to as a "defined feed medium" or a "chemically defined feed medium", where all of the components are described by chemical formula and are present at specific concentrations. The feed medium is typically concentrated so as not to increase the total volume of the culture in the bioreactor to a high level. Such feed medium can contain most of the components, for example, at about 1.5 times, 2 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 12 times, 14 times, 16 times, 20 times, 30 times, 50 times, 100 times, 200 times or even 500 times the normal amount in the basal medium. Trademarked feed media are typically in powder form. Commercially available feeds can be either liquid or powder. When the feeds are already in liquid form, they are typically used as is according to the pamphlet. Powdered feeds need to be solubilized in water, for example, before use.

[0014] Different feed media of different compositions can be added throughout the culture process. For example, three different feed media can be used in the same process: one feed media contains most of the nutrients consumed (this feed is also called the main feed media), one feed media contains some additional nutrients when these nutrients present problems such as aggregation / stability (this feed is also called the secondary feed), and one feed media contains or consists of a carbon source (e.g., glucose). Note that the secondary feed is not essential and varies depending on the components contained in the main feed. There may be cases where only two feeds are used (i.e., the main feed and the feed containing the carbon source), or there may be cases where only one main feed containing all the required components (including the carbon source) is used. It should be understood that if only one feed is used in the context of the present invention, this can be referred to without distinction as "feed" or "main feed".

[0015] The term "bioreactor" refers to any system capable of culturing cells. This includes, but is not limited to, flasks, stationary flasks, spinner flasks, test tubes, shaking tubes, shaking bottles, wave bags, bioreactors, stirred tank bioreactors with or without microcarriers, and also includes, alternatively, microtiter plates, capillaries or multi-well plates. Bioreactors of any size can be used, for example, 1 mL, 5 mL, 0.01 L, 0.1 L, 1 L, 2 L, 5 L, 10 L, 50 L, 100 L, 500 L, 1000 L (or 1 KL), 2000 L (or 2 KL), 5000 L (or 5 KL), 10000 L (or 10 KL), 15000 L (or 15 KL) or 20000 L (20 KL), from 1 milliliter (1 mL, a very small scale) to 20000 liters (20000 L or 20 KL, a very large scale).

[0016] The term "fed-batch culture" refers to a method of culturing cells, where there is a bolus (typically several boluses) or continuous feed medium (or feed media) supplementation to replenish the nutrients consumed without removing any of the medium already present in the bioreactor. The feed can be added according to a predetermined schedule, for example, daily, once every other day, once every three days, etc. Alternatively, the feeding must be continuous and the feeding rate can be varied throughout the culture. This cell culture technique has the potential to obtain high cell densities on the order of 8×10 6 ~30×10 6 cells / ml or higher, depending on the medium formulation, cell line, and other cell growth conditions. The two-phase culture conditions can be generated and maintained by various feeding strategies and medium formulations.

[0017] The term "N-1 stage" according to the present invention corresponds to the stage just before the "production stage" where cells are expected to grow and increase rapidly (in a so-called seed bioreactor) in order to have sufficient material to inoculate an N-stage bioreactor. Alternatively, this stage is called the seed stage.

[0018] The terms "production period", "production stage", or "N stage" according to the present invention correspond to the cell culture stage during the process for manufacturing a recombinant protein when the cells express (i.e., produce) a recombinant polypeptide. The production period starts when the titer of the desired product increases and ends with the harvest of the cells or cell culture fluid or supernatant. Typically, at the start of the production period, the cell culture is transferred from the seed bioreactor to the production bioreactor. Harvesting is the step of removing the cell culture fluid from the production bioreactor for the purpose of recovering and purifying the recombinant protein (e.g., recombinant antibody) in subsequent steps.

[0019] As used herein, for a given volume, "cell concentration" (also known as "cell density") refers to the number of cells in a given volume of medium.

[0020] The term "viable cell concentration" (or "VCC") refers to the number of viable cells in a given volume of medium. This is determined by a standard viability assay. It is to be understood that those skilled in the art know how to determine the maximum VCC for each specific cell line. This is typically carried out by one or more initial experiments. Alternatively, this may refer to "VCD" or "viable cell density".

[0021] The term "IVCC" refers to the integrated viable cell number, which can be determined by finding the area under the cell culture growth curve (IVCC =

Number

[0022] The term "viability" or "cell viability" refers to the ratio of the total number of viable cells to the total number of cells in the culture. Viability is typically acceptable as long as it is not less than 60% of the threshold value compared to the start of the culture, although the acceptable threshold can be determined on a case-by-case basis. Viability is often used to determine the harvest time (this determination is made once throughout the preliminary experiment). For example, in fed-batch culture, harvest can be carried out when the viability in the culture reaches at least 60% or after approximately 14 days (typically 14 days + / - 1 day). Standard methods can be used to determine cell viability (alternatively VCC or VCD), such as through the use of a VI-CELL® XR automated cell counter (Beckman-Coulter Inc.).

[0023] The expression "titer" refers to the concentration of the protein of interest in a given volume of solution. This is determined by standard titer assays such as serial dilution in combination with a detection method (e.g., colorimetry, chromatography), using CEDEX or Protein A high-pressure liquid chromatography (HPLC), Biacore C® or ForteBIO Octet® methods as used in the Examples section.

[0024] The term "specific production rate", also known as "qp", refers to the amount of the protein of interest produced per cell per day.

[0025] The terms "higher titer" or "higher production rate", and their equivalents, mean that the titer or production rate is increased by at least 10% when compared to control culture conditions. The titer or specific production rate is considered to be maintained if it is in the range of -10% to 10% compared to control culture conditions. The terms "lower titer" or "lower production rate", and their equivalents, mean that the titer or production rate is decreased by at least 10% when compared to control culture conditions.

[0026] The term "lag phase" refers to the period of slow growth when the cells are adapting to the culture environment and preparing for rapid growth.

[0027] The term "specific power input" describes the ratio of the power input (P) to the volume (V) of the working fluid. The power input describes the amount of energy delivered per second by the impeller to the bulk of the bioreactor. The specific power input is as follows:

Number

[0028] As used herein, terms such as "protein" include peptides, polypeptides, and proteins, and refer to compounds containing two or more amino acid residues. Proteins according to the present invention include, but are not limited to, cytokines, growth factors, hormones, fusion proteins, antibodies or fragments thereof. A therapeutic protein refers to a protein that can be used for treatment or that is being used for treatment.

[0029] The term "recombinant protein" means a protein produced by recombinant technology. Recombinant technology is well within the knowledge of those skilled in the art (see, for example, Sambrook et al., 1989 and updates).

[0030] Preferably, the proteins by this method, use, and process of the present invention are antibodies, or antigen-binding fragments thereof, or fusion proteins.

[0031] As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies produced by recombinant techniques known in the art. "Antibody" includes antibodies of any species, particularly mammalian species; for example, antibodies produced as dimers of this basic structure including IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, IgD, as well as IgGA1, IgGA2, or pentamers such as IgM, and any isotype of human antibodies including modified variants thereof; for example, non-human primate antibodies derived from chimpanzee, baboon, rhesus monkey, or cynomolgus monkey; for example, rodent antibodies derived from mouse or rat; rabbit, goat, or horse antibodies; camelid antibodies (e.g., derived from camel or llama such as Nanobodies™) and derivatives thereof; avian antibodies such as chicken antibodies; or fish antibodies such as shark antibodies. The term "antibody" also refers to "chimeric" antibodies in which at least a first portion of the heavy chain and / or light chain antibody sequences is derived from a first species and a second portion of the heavy chain and / or light chain antibody sequences is derived from a second species. Chimeric antibodies for the purposes herein include "primateized" antibodies that include variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys such as baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences. A "humanized" antibody is a chimeric antibody that contains sequences derived from a non-human antibody. In most cases, a humanized antibody is a human antibody (recipient antibody) in which residues from the hypervariable regions of the recipient have been replaced with residues from the hypervariable regions [or complementarity-determining regions (CDRs)] of a non-human species such as mouse, rat, rabbit, chicken, or non-human primate that have the desired specificity, affinity, and activity. In most cases, residues of the human (recipient) antibody are further replaced by the corresponding non-human residues outside of the CDRs, i.e., within the framework regions (FRs). Furthermore, a humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody properties. Humanization reduces the immunogenicity of non-human antibodies in humans and thus facilitates the application of antibodies for the treatment of human diseases.Humanized antibodies and several different techniques for making them are well known in the art. The term "antibody" also refers to human antibodies that can be made as an alternative to humanization. For example, it is possible to generate transgenic animals (e.g., mice) that, when immunized, can produce a complete repertoire of human antibodies in the absence of production of endogenous mouse antibodies. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display techniques such as phage display or ribosome display techniques, and use recombinant DNA libraries that are made at least in part artificially or from a donor immunoglobulin variable (V) domain gene repertoire. Phage and ribosome display techniques for making human antibodies are well known in the art. Human antibodies can also be made from isolated human B cells that are immunized ex vivo with an antigen of interest and then fused to generate hybridomas, which are then screened for optimal human antibodies. The term "antibody" refers to both glycosylated and non-glycosylated antibodies. Further, as used herein, the term "antibody" refers not only to full-length antibodies, but also to antibody fragments, more specifically antigen-binding fragments thereof. Antibody fragments contain at least one heavy or light chain immunoglobulin domain and bind to one or more antigens, as is known in the art. Examples of antibody fragments according to the present invention include Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv and Bis-scFv fragments. Said fragments can also be diabodies, tribodies, triabodies, tetra-bodies, minibodies, single domain antibodies (dAb), e.g., sdAb, VL, VH, VHH or camelid antibodies (e.g., from camel or llama such as Nanobody™) and VNAR fragments.The antigen-binding fragment according to the present invention can also comprise a Fab linked to one or two scFvs or dsscFvs, where each scFv or dsscFv binds to the same or a different target (e.g., one scFv or dsscFv that binds to a therapeutic target and one scFv or dsscFv that increases the half-life by binding to, e.g., albumin). Examples of such antibody fragments are FabdsscFv (also called BYbe®), or Fab-(dsscFv)2 (also called TrYbe®, see, e.g., WO 2015197772). The antibody fragments defined above are known in the art.

Mode for Carrying Out the Invention

[0032] The present disclosure relates to a process (or method) for the production of recombinant proteins. More specifically, it describes a process (or method) for producing recombinant proteins in mammalian cells and improving cell culture performance (cell growth, specific production rate and / or titer / yield). In particular, the present invention improves cell growth (e.g., increases growth rate, increases cell viability, reduces the possible lag phase at the start of the N stage) by applying specific conditions during the N-1 stage (i.e., in the seed bioreactor), such as controlling the feeding strategy, and is based on the inventors' finding that it was possible to increase the yield of recombinant protein production by cells during the production phase, more specifically during a high seeding density (HSD) process (see also Figure 9). Surprisingly, the inventors found that subjecting the cells to stress (such as osmotic stress) during the N-1 stage (i.e., in the seed bioreactor) operating in fed-batch mode improved cell growth and was subsequently able to increase the yield of recombinant protein production in the N stage (i.e., the production bioreactor), particularly in enhanced processes such as the HSD process. For some expressing cells, it was also possible to reduce or even avoid the lag phase observed at the start of the production phase (during the N stage). As shown herein, the timing and / or duration of stress, such as osmotic stress, are important factors (e.g., osmotic stress one day before inoculation may be sufficient to improve production capacity). The inventors refer to this stress as "tissue stress".

[0033] Accordingly, the process described herein relies on specific conditions (such as specific stress conditions alternatively named "tissue stress" conditions), i.e., during the N-1 stage, particularly in fed-batch implemented during the N-1 stage such as the control of feeding mode and / or industrial parameters when integrated into an enhanced process, for the production of the recombinant protein of interest during the subsequent N stage (i.e., the production stage).

[0034] The present disclosure particularly describes a method for controlling feeding conditions / industrial parameters during the N-1 stage in order to maximize the yield of a production bioreactor and to maximize cell growth. The present disclosure provides specific examples of fed-batch processes, such as the HSD fed-batch process, for a seed bioreactor (N-1 stage) in which these parameters are controlled within the claimed scope, and details specific examples of possible addition modes of the feed (including period and timing for bolus, continuous / semi-continuous, etc.).

[0035] In one embodiment, the present invention provides a process for producing a recombinant protein in a production bioreactor, the process comprising a. inoculating a mammalian cell containing a gene encoding a recombinant protein into an N-1 bioreactor; b. culturing the mammalian cells in an N-1 bioreactor operated in fed-batch mode under specific conditions selected from i. a specific mode and period for adding a feed or at least one of the feeds, ii. control of the total amount of the feed added, and / or iii. control of at least one industrial parameter; and c. inoculating the cells obtained from step (b) into an N bioreactor at a seeding density of at least 2.00×10 6 viable cells / ml of viable cells; d. culturing the cells in the N bioreactor under conditions that allow production of the recombinant protein; and e. optionally harvesting, purifying and formulating the recombinant protein.

[0036] In this process for producing a recombinant protein, it has been shown that the yield of the recombinant protein is increased and / or the cell growth in the production bioreactor is improved. Thus, in an alternative embodiment, the present invention provides a process for producing a recombinant protein in a production bioreactor, the process comprising: a. inoculating a mammalian cell comprising a gene encoding a recombinant protein into an N-1 bioreactor; b. culturing the mammalian cells in the N-1 bioreactor in fed-batch mode under specific conditions selected from: i. a specific mode and period for adding a feed or at least one of the feeds, ii. controlling the total amount of the feed added, and / or iii. controlling at least one industrial parameter; c. inoculating the cells obtained from step (b) into an N bioreactor at a seeding density of viable cells of at least 2.00×10 6 viable cells / ml; d. culturing the cells in the N bioreactor under conditions enabling the production of the recombinant protein; and e. optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein, wherein the yield of the recombinant protein and / or the cell growth in the production bioreactor are also improved. It is understood that the yield of the recombinant protein is increased and / or the cell growth in the production bioreactor is improved as compared to a process not comprising the specific conditions of step b) (i.e., the cell growth is increased as compared to cells that have not grown under "tissue stress" during the "N-1 stage").

[0037] In another embodiment, the present invention provides a process for improving the growth of mammalian cells in a production bioreactor, the process comprising a. inoculating a mammalian cell comprising a gene encoding a recombinant protein into an N-1 bioreactor; b. i. a specific mode and period for adding a feed or at least one of the feeds, ii. controlling the total amount of the feed added, and / or iii. controlling at least one industrial parameter, culturing mammalian cells in an N-1 bioreactor operated in fed-batch mode under specific conditions selected from 6 seeding the N bioreactor with viable cells at a seeding density of at least 2.00×10 d. culturing the cells in an N bioreactor under conditions that allow for the production of a recombinant protein; e. optionally harvesting the recombinant protein, purifying the recombinant protein, and formulating the recombinant protein,

[0038] One skilled in the art will understand that an improvement in cell growth (i.e., cell growth is increased compared to when the cells do not grow under "tissue stress" during the N-1 stage) is observed compared to processes where the cells do not grow according to the specific conditions of step b).

[0039] In this process for improving mammalian cell growth in a production bioreactor, it has also been shown that the yield of the recombinant protein is also increased. Thus, in an alternative embodiment, the present invention provides a process for improving the growth of mammalian cells in a production bioreactor, the process comprising: a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding a recombinant protein; b. i. a specific mode and period of adding a feed or at least one of the feeds, ii. controlling the total amount of the added feed, and / or iii. controlling at least one industrial parameter, culturing the mammalian cells in an N-1 bioreactor operated in fed-batch mode under specific conditions selected from 6Inoculating an N bioreactor with a seeding density of viable cells in viable cells / ml, and d. culturing the cells in the N bioreactor under conditions enabling the production of the recombinant protein, and e. optionally harvesting the recombinant protein, purifying the recombinant protein, and formulating the recombinant protein, wherein cell growth in the bioreactor is improved compared to cells not growing under the conditions of step b), and the yield of the recombinant protein is also increased.

[0040] In a further embodiment, the present invention relates to a process for increasing the production yield of a recombinant protein expressed by mammalian cells in a culture in a production bioreactor, the process comprising a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding the recombinant protein, b. i. culturing the mammalian cells in the N-1 bioreactor in fed-batch mode under specific conditions selected from a specific mode and period of adding a feed or at least one of the feeds, ii. controlling the total amount of the feed added, and / or iii. controlling at least one industrial parameter, and c. inoculating the N bioreactor with the cells obtained from step (b) at a seeding density of viable cells in viable cells / ml of at least 2.00×10 and d. culturing the cells in the N bioreactor under conditions enabling the production of the recombinant protein, and e. optionally harvesting the recombinant protein, purifying the recombinant protein, and formulating the recombinant protein. 6 Inoculating an N bioreactor with a seeding density of viable cells in viable cells / ml, d. culturing the cells in the N bioreactor under conditions enabling the production of the recombinant protein, and e. optionally harvesting the recombinant protein, purifying the recombinant protein, and formulating the recombinant protein.

[0041] One skilled in the art will understand that an improvement in the production yield of the recombinant protein (i.e., the production yield of the recombinant protein is increased compared to the cells not growing under "tissue stress" during the N-1 stage) is observed compared to a process in which the cells do not grow according to the specific conditions of step b).

[0042] In this process for increasing the production yield of recombinant proteins, improved cell growth in the bioreactor was demonstrated. Thus, in an alternative embodiment, the present invention relates to a process for increasing the production yield of a recombinant protein expressed by mammalian cells in a culture in a production bioreactor, the process comprising: a. inoculating a mammalian cell containing a gene encoding the recombinant protein into an N-1 bioreactor; b. culturing the mammalian cells in the N-1 bioreactor in fed-batch mode under specific conditions selected from: i. a specific mode and period for adding a feed or at least one of the feeds, ii. controlling the total amount of the feed added, and / or iii. controlling at least one industrial parameter; c. inoculating the cells obtained from step (b) into an N bioreactor at a seeding density of at least 2.00×10 6 viable cells / ml of viable cells; d. culturing the cells in the N bioreactor under conditions that allow production of the recombinant protein; and e. optionally harvesting, purifying, and formulating the recombinant protein, wherein the production yield is increased compared to cells not grown under the conditions of step b), and cell growth in the bioreactor is also improved.

[0043] Those skilled in the art will understand that a certain level of viable cell density must be obtained at the end of step (b) in order to perform step (c) using the cells obtained from step (b). Thus, also encompassed by the present invention is a process for producing a recombinant protein in a production bioreactor, a process for improving the growth of mammalian cells in a production bioreactor, and / or a process for increasing the production yield of a recombinant protein expressed by mammalian cells in a culture in a production bioreactor, the process comprising: a. inoculating a mammalian cell containing a gene encoding the recombinant protein into an N-1 bioreactor; b. at least 8×10 6Culturing mammalian cells in an N-1 bioreactor operated in fed-batch mode under specific conditions until viable cells / ml are obtained, wherein the specific conditions are i. a specific mode and period for adding a feed or at least one of the feeds, ii. control of the total amount of the added feed, and / or iii. control of at least one industrial parameter, selected from c. inoculating the N bioreactor with the cells obtained from step (b) at a seeding density of at least 2.00×10 6 viable cells / ml, d. culturing the cells in the N bioreactor under conditions that allow production of the recombinant protein, e. optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

[0044] According to the present invention, it is important to operate the N-1 stage in fed-batch mode, but the N stage can be operated according to any mode such as perfusion, batch or fed-batch. Preferably, the N stage is operated in perfusion mode or fed-batch mode. Those skilled in the art know how to operate the production stage in perfusion mode, batch mode or fed-batch mode.

[0045] Overall, in the context of the present invention, the improvement of cell growth in the production bioreactor can be, for example, an increase in cell growth, as determined by an increase in VCC compared to cells that do not grow under "tissue stress" during the N-1 stage and / or a decrease in the induction period that may occur at the start of the production period compared to cells that do not grow under "tissue stress" during the N-1 stage. By increasing the VCC or decreasing the induction period, when an induction period exists, the production of the recombinant protein can be prevented from being delayed.

[0046] Overall, in the context of the present invention, the specific conditions applied during the fed-batch N-1 stage are considered stress conditions for the cells. Since these conditions (alternatively referred to as stress conditions) are controlled, they can alternatively be "tissue stress" conditions. In all of the processes described herein, step b) can alternatively be: - b) culturing mammalian cells in an N-1 bioreactor operated in fed-batch mode under specific stress conditions, wherein the specific stress conditions are: i. a specific mode and period for adding a feed or at least one of the feeds, ii. control of the total amount of the feed added, and / or iii. control of at least one industrial parameter, or - b) culturing mammalian cells in an N-1 bioreactor operated in fed-batch mode under specific tissue stress conditions, which can be described as: i. a specific mode and period for adding a feed or at least one of the feeds, ii. control of the total amount of the feed added, and / or iii. control of at least one industrial parameter.

[0047] In the context of the present invention as a whole, mammalian cells containing a gene encoding a recombinant protein (in other words, mammalian cells expressing a recombinant protein) are preferably cultured in fed-batch mode for at least 4 days, preferably 7 days or less, more preferably at least 5 days such as 5 days, 6 days or 7 days, but 7 days or less, during the N-1 stage. Those skilled in the art can carry out a longer N-1 stage, but there are associated risks such as a decrease in cell viability or the start of production that must be avoided during the N-1 stage. The feed (at least the main feed and / or the secondary feed if present) is preferably added daily. The feed is added at the start, on day 0, 1, 2, 3 or 4 after the start, and over a period of at least 2 days, at least 3 days (3 days, 4 days, 5 days or 6 days) in the case of the N-1 stage (i.e., day 0 is the inoculation day). Typically, the final feed is added until the day before the end of the culture. Thus, as a non-limiting example, if the N-1 stage has a period of 7 days and the feed is added at the 3rd day during a period of 4 days, the final feed is added on the 6th day. In another non-limiting example, if the N-1 stage has a period of 6 days and the feed is added at the 0th day during a period of 6 days, the final feed is added on the 5th day.

[0048] Overall, in the context of the present invention, one of the conditions for implementing "tissue stress" is the period during which at least one of the feeds or feeds is added. Preferably, such feed in step (b) is added daily as a bolus. Preferably, i) at least the main feed in step (b) is added daily as a bolus, and / or ii) at least one (if any) of the secondary feeds in step (b) is added daily as a bolus. The daily bolus is preferably added in about 3 hours or less, about 2 hours or less, or about 1 hour or less. If there are two or more feeds added (such as one main feed and one secondary feed), preferably all feeds are added daily as a bolus in step (b). In such a case, the daily bolus is preferably added in about 3 hours or less, about 2 hours or less, or about 1 hour or less. The addition periods of different feeds (when different feeds exist) need not be the same. In a non-limiting example, the main feed can be added daily as a bolus over 3 hours, and the secondary feed can be added daily as a bolus over 1 hour. In another non-limiting example, the main feed can be added daily as a bolus over 1 hour, and the secondary feed can be added daily as a bolus over 30 minutes. Those skilled in the art will understand that, if desired, at least one feed can alternatively be a feed containing a carbon source, as long as it is sufficient to cause the stress conditions.

[0049] Overall, in the context of the present invention, another condition for implementing "tissue stress" is the total amount of feed added during step (b). The total amount of feed is important and is preferably at least about 3.5% of the starting culture volume, but preferably should not exceed 30% of the starting culture volume. Thus, overall, in the context of the present invention, the total amount of feed added during step (b) is preferably at least about 3.5% of the starting culture volume, but preferably exhibits no more than 30% of the starting culture volume. Alternatively, the total amount of feed added during step (b) is preferably about 3.5% of the starting culture volume, but preferably exhibits no more than 25% of the starting culture volume (in other words, the total amount of feed added during step (b) is preferably at least about 3.5% to about 30% of the starting culture volume), which is about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 15.0%, about 20.0%, about 25.0% or 25.0% of the starting culture volume. As a non-limiting example, if the total amount of feed added during step (b) exhibits 3.5% of the starting culture volume and the starting culture volume is 100 L, this means that a total of 3.5 L of feed is added during the N-1 stage, resulting in a final culture volume of 103.5 L. As another non-limiting example, if the total amount of feed added during step (b) exhibits 4.2% of the starting culture volume and the starting culture volume is 500 L, this means that a total of 21 L of feed is added during the N-1 stage, resulting in a final culture volume of 521 L. In yet another non-limiting example, if the total amount of feed added during step (b) exhibits 15% of the starting culture volume and the final culture volume is 200 L, this means that a total of 30 L of feed is added during the N-1 stage, resulting in a final culture volume of 230 L.

[0050] Overall, in the context of the present invention, another of the conditions for implementing "tissue stress" is the control of at least one industrial parameter during step (b). Preferably, at least one industrial parameter controlled during step (b) is the specific power input. More preferably, the specific power input in step (b) is controlled daily, and the said specific power input in step (b) preferably reaches at least 100 W / m 3 for all or part of the period of step (b). Preferably, the specific power input in step (b) reaches at least 100 W / m 3 , at least 110 W / m 3 , at least 120 W / m 3 , at least 130 W / m 3 , at least 140 W / m 3 , at least 150 W / m 3 , at least 160 W / m 3 or at least 170 W / m 3 . In a non-limiting example, if the period of step (b) is 6 days, the specific power input may be controlled at about 120 W / m 3 for the entire period of this step (i.e., from day 0 to day 6). In another non-limiting example, if the period of step (b) is 7 days, for example, for only 5 days (i.e., part of the period of step (b)), such as from day 1 to day 5, the specific power input may be controlled at about 140 W / m 3 .

[0051] This is mainly (alternatively, solely) the P / V applied to the seed bioreactor rather than the production bioreactor, but it has been shown by the inventors that it had a significant impact on the cell culture. Alternatively, the difference (or ratio) between the P / V in the seed bioreactor and the production bioreactor is still considered a potential factor that greatly affects the production capacity of the cell culture. In other words, the difference in mechanical stress caused by the power input of the seed bioreactor and the production bioreactor is still a potential factor that affects the production capacity of the cell culture: Conditions with a high P / V in the seed bioreactor were associated with a lower P / V (lower mechanical stress) in the production step. As shown in the example section, the conditions that result in the best cell culture ability during the production phase were those with a high P / V in the seed bioreactor associated with a lower P / V in the production step. Thus, alternatively, at least one industrial parameter controlled during step (b) is not only the specific power input but also an additional industrial parameter controlled during step (d), namely, the specific power input of the production bioreactor. As a result, a specific ratio between the power input of the seed bioreactor (P / V SBR) and the power input of the production bioreactor (P / V PBR) is considered. Preferably, the ratio of P / V SBR:P / V PBR is at least 1.2:1, at least 1.25:1, at least 1.30:1, at least 1.35:1, at least 1.40:1, at least 1.45:1, at least 1.50:1, at least 1.55:1, at least 1.60:1, finally 1.65:1, at least 1.70:1, at least 1.80:1, at least 1.90:1 or even at least 2:1. During the initial experiments, the best ratio can be determined. Thus, the process according to the present invention as a whole leads to an improvement in the growth of mammalian cells in the production bioreactor and / or an improvement in the production yield of recombinant proteins expressed by mammalian cells in the culture of the production bioreactor, compared to a standard process, and further includes a preliminary step of performing at least a series of initial experiments to determine the ratio of P / V SBR:P / V PBR.It should be understood that at least a series of initial experiments for a set need not be repeated each time the process according to the invention is carried out. In other words, once the conditions are determined, for at least a series of initial experiments for one particular clone under a given condition, it is not necessary to control this each time the process according to the invention is carried out.

[0052] Accordingly, the present invention also encompasses a process for producing a recombinant protein in a production bioreactor, a process for improving the growth of mammalian cells in a production bioreactor, and / or a process for increasing the production yield of a recombinant protein expressed by mammalian cells in a culture in a production bioreactor, and this process comprises 0. Optionally, carrying out at least a series of initial experiments for determining the ratio of P / V SBR:P / V PBR preferably maintained at the production stage, and a. inoculating mammalian cells containing a gene encoding a recombinant protein into an N-1 bioreactor; b. culturing mammalian cells in an N-1 bioreactor operated in fed-batch mode under "tissue stress" conditions until at least 8×10 6 viable cells / ml are obtained, wherein the "tissue stress" conditions are i. a specific mode and period of adding a feed or at least one of the feeds, ii. controlling the total amount of the added feed, and / or iii. controlling at least one industrial parameter selected from the group consisting of controlling at least one industrial parameter, wherein the at least one industrial parameter is the specific power input, c. inoculating the cells obtained from step (b) into an N bioreactor at a seeding density of at least 2.00×10 6 viable cells / ml, and d. Culturing cells in an N-bioreactor under conditions enabling the production of a recombinant protein, comprising the step of controlling at least one industrial parameter, wherein the at least one industrial parameter is the specific power input such that, at any step (0), or such that the ratio of P / V SBR:P / V PBR determined in the aforementioned initial experiment is maintained, and e. Optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

[0053] The best results for producing a recombinant protein in a production bioreactor and improving the growth of mammalian cells in the production bioreactor, and / or the production yield of the recombinant protein expressed by mammalian cells in the culture of the production bioreactor, result from a combination of three conditions that reach tissue stress. However, it is already sufficient for an acceptable result to occur with one of these, and it may be sufficient depending on the VCC obtained at the end of the N-1 steps. Implementing one, two or three of the "tissue stress" conditions is variable depending on the producing cells. The best conditions can be determined during the initial experiment. Thus, the process according to the invention as a whole includes a preliminary step of performing at least one initial experiment to determine the VCC at the end of the N-1 steps, and to determine whether to apply during the N-1 steps such that preferably at least 8×10 6 viable cells / ml are reached, and such that it is possible to produce a recombinant protein in the production bioreactor. Compared to a standard process, it is possible to improve the growth of mammalian cells in the production bioreactor and / or the production yield of the recombinant protein expressed by mammalian cells in the culture of the production bioreactor. It should be understood that this initial experiment need not be repeated every time the process according to the invention is carried out. In other words, once the conditions are determined, for at least one initial experiment for one particular clone under a given condition, it is not necessary to control this every time the process according to the invention is carried out.

[0054] Accordingly, the present invention also encompasses a process for producing a recombinant protein in a production bioreactor, a process for improving the growth of mammalian cells in a production bioreactor, and / or a process for increasing the production yield of a recombinant protein expressed by mammalian cells in a culture in a production bioreactor, the process comprising: 0. Optionally, performing at least one initial experiment to determine VCC at the end of the N-1 stage, and / or optionally, performing at least one initial experiment to determine the feed period during step (b), the total amount of feed added during step (b) and / or the specific power input used during step (b); a. inoculating a mammalian cell containing a gene encoding a recombinant protein into an N-1 bioreactor; b. culturing the mammalian cells in an N-1 bioreactor operated in fed-batch mode under "tissue stress" conditions until at least 8×10 6 viable cells / ml are obtained, wherein the "tissue stress" conditions are selected from: i. a specific mode and period for adding a feed or at least one of the feeds; ii. control of the total amount of feed added; and / or iii. control of at least one industrial parameter; c. inoculating the cells obtained from step (b) into an N bioreactor at a seeding density of at least 2.00×10 6 viable cells / ml; d. culturing the cells in an N bioreactor under conditions that allow production of the recombinant protein; e. optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

[0055] Inoculation of the N bioreactor is at a lower seeding density (e.g., less than 2.00×10 6 viable cells / ml, 1.50×10 6less than viable cells / ml, 1.00×10 6 less than viable cells / ml or even 0.50×10 6 less than viable cells / ml), and the results regarding the production of the recombinant protein, the increase in cell growth, and / or the production yield of the recombinant protein are at least about 2.00×10 6 The density of viable cells / ml (i.e., high seeding density) is good. Thus, overall in the context of the present invention, the inoculation of the N bioreactor (step c) is preferably carried out using the cells obtained from the N-1 bioreactor at least at about 2.00×10 6 seeding density of viable cells / ml. Alternatively, the N bioreactor of step (c) is at least 3.00×10 6 viable cells / ml, at least 4.00×10 6 viable cells / ml, at least 5.00×10 6 viable cells / ml, at least 6.00×10 6 viable cells / ml, at least 7.00×10 6 viable cells / ml, at least 8.00×10 6 viable cells / ml, at least 9.00×10 6 viable cells / ml or at least 10.00×10 6 seeding density of viable cells / ml. One skilled in the art will understand that with respect to the cell density obtained at the end of step (b), depending on the yield of the N-1 bioreactor, "10.00×10 6 viable cells / ml" is not a limit and higher seeding densities can be expected.

[0056] In order to be able to inoculate the N bioreactor under a high seeding density strategy, at least about 8.00×10 6It is preferable to reach a (viable) cell density of viable cells / ml. It is also preferable not to reach an overly high cell density at the end of this stage. This may mean that the cells have been cultured for an overly long period, which may affect their quality and production rate during the N stages. Thus, overall in the context of the present invention, the (viable) cell density obtained at the end of the N-1 stage (end of step b) is at least about 8.00×10 6 viable cells / ml and at most about 30.00×10 6 viable cells / ml. Alternatively, the (viable) cell density obtained at the end of the N-1 stage (end of step b) is at least 9.00×10 6 viable cells / ml, at least 10.00×10 6 viable cells / ml, at least 15.00×10 6 viable cells / ml, at least 20.00×10 6 viable cells / ml, at least 25.00×10 6 viable cells / ml, although preferably it is at most 30.00×10 6 viable cells / ml.

[0057] Overall in the context of the present invention, the medium at the start of the culture (alternatively, herein named the basal medium and used for both the N-1 stage-medium in step (a) and the N stage-medium in step (c)) is preferably a protein- and serum-free medium. Such protein- and serum-free medium can be a commercially available medium or a (self-made or commercially available) chemically defined medium. Such a medium can thus be used at the start of the N-1 stage (initial medium for step (a)) and at the start of the N stage (initial medium for step (c)).

[0058] Overall, in the context of the present invention, the main feed medium can be any main feed medium. In one example, this main feed medium does not contain Cys (cysteine and cystine), and Trp and Tyr, and these components are provided by at least one additional feed (such an additional feed is one of the secondary feeds or secondary feeds).

[0059] Overall, in the context of the present invention, the process is preferably carried out on a large scale in a bioreactor having a volume of, for example, preferably 50 L or more, 100 L or more, 500 L or more, 1000 L or more, 2,000 L or more, 55,000 L or more, 10,000 L or more, or 20,000 L or more. In other words, mammalian cells producing the recombinant protein are preferably cultured in a bioreactor (e.g., a production bioreactor) having a volume of 50 L or more, 100 L or more, 500 L or more, 1000 L or more, 2,000 L or more, 5,000 L or more, 10,000 L or more, or 20,000 L or more.

[0060] Overall, in the context of the present invention, suitable mammalian host cells (also referred to as mammalian cells) include Chinese Hamster Ovary (CHO cells), lymphocyte cell lines (e.g., NSO myeloma cells and SP2 cells, COS cells, myeloma or hybridoma cells). In a preferred embodiment, the mammalian cells are CHO cells. Suitable types of CHO cells can include, for example, CHO-K1, CHOK1-SV, dhfr-CHO, or even CHO-S cells such as CHO-DG44, CHO-DXB11, CHO-DXB1. The host cells are preferably stably transformed or transfected with an expression vector encoding the recombinant protein of interest.

[0061] In the context of the present invention as a whole, a recombinant protein is a protein such as a cytokine, a growth factor, a hormone, a fusion protein or an antibody. When the protein is an antibody, this can be, for example, a chimeric antibody, a humanized antibody or a fully human antibody, preferably an IgG such as IgG1, IgG2, IgG3 or IgG4. Alternatively, this can be any kind of protein according to the definitions given herein.

[0062] The process according to the invention can further comprise the step of recovering a cell culture fluid (CCF) containing a recombinant protein (collection step), in other words the step of collecting the recombinant protein. Following recovery, the recombinant protein can be purified, for example, using protein A chromatography and other chromatography / filtration steps when the protein is an antibody. This process can optionally further comprise the step of formulating the purified recombinant protein into a formulation with a protein concentration, for example, of 50 mg / ml or more, such as 10 mg / ml or more, for example, 100 mg / ml or more, 150 mg / ml or more or even 200 mg / mL or more. Without limitation, the formulation can be a liquid formulation, a lyophilized formulation or a spray-dried formulation.

Brief Description of the Drawings

[0063]

Figure 1A

Figure 1B

Figure 2

Figure 3A

Figure 3B

Figure 4

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 5A

Figure 5B

Figure 6A

Figure 6B

Figure 7A

Figure 7B

Figure 8A

Figure 8B

Figure 9

Figure 10

[0064] Example Cell Lines, Cell Culture, and Experimental Procedures Two different production CHO-DG44 cell lines were used to produce mAb-1 (a full-length IgG4 antibody with a pI of 5.7 - 5.9) and mAb-2 (a trispecific antibody with a pI of 8.9 - 9.2), respectively.

[0065] Two sets of media were used: - The first source (media from Cytiva): ActiPro™ as the basal medium, Cell Boost™ 7a as the main feed (alternatively referred to herein as CB7a), and Cell Boost™ 7b as the secondary feed (alternatively referred to herein as CB7b) that supplies additional elements to that from the main feed. - The second source (modified CHO Pro medium): "BM1" as the basal medium, "FM1" as the main feed, and "FM2" as the secondary feed that supplies additional elements to that from the main feed.

[0066] Cells were cultured in a 2L stirred tank glass bioreactor (STR) equipped with a supply tower (C-DCUII, Sartorius Stedim Biotech) controlled by a multi-fermentation control system (MFCS, Sartorius Stedim Biotech). The bioreactor was equipped with a 3-segment blade impeller. The starting culture volume was adapted so that the final culture volume would surely be optimal. The production bioreactor was seeded at the target seeding density (TSD) in the basal medium. The pH control of the production STR was set to 7.0 with a dead band of ±0.2. The pO2 target was set to 40% air saturation. To control pO2, air, nitrogen, and oxygen were sparged into the culture vessel based on a cascade controller using a predetermined mixing profile. The temperature was controlled at approximately 36.8 °C. The seed bioreactor (N-1 stage) was operated in fed-batch mode for 5 or 6 days (by the addition of two different feeds). Then production (N stage) was operated in supply experiment mode for 14 days (by the addition of two different feeds). During this stage, monoclonal antibody (mAb) was secreted into the medium. Samples were taken daily (-80 °C storage) to determine VCD, viability, offline pH, pCO2, osmolarity, glucose-lactate concentration, amino acid concentration, and mAb concentration. To control the increase in foam, an antifoaming agent was added manually daily as needed. 72 hours after inoculation, continuous feeding of nutrients was started at a predetermined rate. When the glucose concentration dropped below a predetermined threshold, a glucose bolus feed was added to the culture. The glucose concentration was measured daily. Samples for amino acid analysis were taken before the addition of the feed. The extracellular concentration after feeding was calculated based on the composition of the feed and the concentration of nutrients measured before the addition of the feed. At the end of the production period, the cell culture supernatant sample (i.e., the recovered cell culture fluid) was purified by protein A purification of the TECAN automated system after recovery.

[0067] Analysis method Cells were counted by using a VI-CELL® XR (Beckman-Coulter, Inc.) automated cell counter operated based on the trypan blue exclusion method. The amounts of glucose and lactate in the medium were determined using a NOVA 400 BioProfile automated analyzer (Nova Biomedical) or Cedex Bio HT (Roche). A Model 2020 freezing point osmometer (Advanced Instruments, Inc.) was used for osmotic pressure determination. Off-line gas and pH measurements were performed with a Model BioProfile pHOx® blood gas analyzer (Nova Biomedical Corp.). Metabolite concentrations were determined daily using the CedexBioHT system (Roche). The titer analysis of the product was performed by protein A high performance liquid chromatography (HPLC) using cell culture supernatant samples stored at -80 °C before analysis. The relative percentages of the major acidic (APG, i.e., acidic peak group) and basic (BPG, i.e., basic peak group) isoforms of the purified mAb were determined by imaging capillary electrophoresis (ProteinSimple iCE3). The amounts of aggregates (HMWS), monomer, and fragments (LMWS) of the purified mAb were determined by size exclusion chromatography (SE-UPLC) or by a protein A HPLC gradient.

[0068] Example 1 High feeding management system, mode of feed addition, and specific power input of the seed bioreactor affect the cell culture ability of the production bioreactor (mAb1) For this experiment, five 2-L N-1 seed bioreactors (SBRs) were used at 0.35×10 6CHO cells producing mAb-1 were inoculated under various conditions of seeding density in cells / mL and feeding mode, and the daily feed addition period and specific power input were tested over 5 days (see Table 1). Then, 15 production bioreactors (PBRs) (see Table 1 and Figure 9) were inoculated with 3.0×10 6 cells / mL from the different N-1 seed bioreactors described above. The purpose was to evaluate the effect of various parameters of the seed bioreactor, namely the amount of feed added, addition mode (bolus or continuous), and specific power input, on cell growth in the production bioreactor. The specific power input was also varied in the production bioreactor for some conditions.

Table 1

[0069] For all of the conditions tested, comparable cell growth with a maximum VCC of approximately 14×10 6 cells / mL was observed during the N-1 stage (Figure 1A). The results shown in Figure 1B (i.e., cumulative IVCC) and Figure 3A (VCC) highlight that cell growth in the production bioreactor (N stage) was lower for conditions having - a lower amount of feed added in the seed bioreactor (N-1 stage), - a lower specific power input in the seed bioreactor, and / or - a continuous addition mode for the main feed in the seed bioreactor.

[0070] In contrast, as shown in Figure 2, the specific power input in the subsequent production bioreactor had no significant effect on cell growth in the production bioreactor.

[0071] The effect on titer was similar to the effect on cell growth. That is, the titer was - a lower amount of feed added in the seed bioreactor (N-1 stage), - Lower specific power input of the seed bioreactor, and / or - For conditions having a continuous addition mode of the main feed of the seed bioreactor (see Figure 3B), it was low.

[0072] This example shows that higher cell growth and an increased final production rate can be obtained in an N - bioreactor by culturing mammalian cells in a fed - batch mode in an N - 1 bioreactor operated under specific conditions selected from i) a specific mode and period of adding at least one of the feeds, ii) control of the total amount of the feeds added, and / or iii) control of at least one industrial parameter such as the specific power input (see Figures 4A - 4C). In other words, by controlling the feeding mode and period, the total amount of the feeds added during the N - 1 stage, and / or the specific power input, it was possible to improve the growth of mammalian cells and increase the production yield of recombinant proteins expressed by mammalian cells in the N - bioreactor.

[0073] Regarding the specific power input (P / V), considering the results of this example, the P / V applied in the seed bioreactor was considered to have a great impact on cell culture production. More specifically, the difference in P / V between the seed bioreactor and the production bioreactor (P / V in SBR is higher than that in PBR compared with P / V in PBR) is still a potential factor affecting the production capacity of the cell culture. Without limitation, this result indicated that a higher cell culture capacity was associated with a P / V(SBR):P / V PBR ratio of at least 1.20:1.

[0074] Example 2 The mode of feed addition in the seed bioreactor affects the cell culture capacity of the production bioreactor (mAb1) For this experiment, 4 × 10 L N - 1 stage bioreactors (SBR) were filled with 0.35 × 10 over 6 days under various conditions (see Table 2). 6CHO cells producing mAb1 at cells / mL were inoculated. Then, 8 × 2 L production bioreactors (PBRs) were inoculated from the N-1 bioreactor at a seeding density of 3.0 × 10 6 cells / mL as described in the materials and methods. In this experiment, multiple conditions were evaluated, including the daily feed addition periods of Cell Boost™ 7a and Cell Boost™ 7b (see Table 2).

Table 2

[0075] In Figure 5A, the inventors were able to observe the comparative cell growth profiles in the seed bioreactor for the different conditions tested, while Figure 6A shows that for conditions with a longer feed addition period in the seed bioreactor, cell growth in the production bioreactor was lower. The cumulative IVCC in the N bioreactor is represented in Figure 5B. The results show that for conditions with a longer feed addition period in the seed bioreactor, cell growth in the production bioreactor was lower. The inventors were able to observe that the effects of CB7a and CB7b were cumulative. Conditions with a longer feed addition period for both cell boosts 7a and 7b showed a lower final titer at the end of 14 days of production (Figure 6B): This example confirms the results obtained in Example 1. That is, by controlling at least one feeding mode and period of the feeds during the N-1 stage, it was possible to improve the growth of mammalian cells in the N bioreactor and increase the production yield of the recombinant protein expressed by mammalian cells in the N bioreactor, thus confirming this.

[0076] Example 3 - The feed addition mode in the seed bioreactor affects the cell culture ability in the production bioreactor (mAb2) For this experiment, 4 × 10 L N-1 stage bioreactors (SBRs) were inoculated with 0.35 × 10 over 6 days under various conditions (see Table 3).6 CHO cells producing mAb2 were inoculated at [X] cells / mL. In 13 × 2 L production bioreactors (PBR), CHO cells producing mAb2 were inoculated at a seeding density of [X] cells / mL in a fed-batch process as described in the materials and methods. 6 CHO cells producing mAb2 were inoculated at a seeding density of [X] cells / mL. In this experiment, multiple conditions including various addition modes of FM1 were tested in the seed bioreactor.

Table 3

[0077] The cell growth profile is shown in Figure 7. The results shown in Figure 7A indicate that for conditions with a longer feed addition period in the seed bioreactor, cell growth in the production bioreactor is lower.

[0078] The final productivity titer at the end of 14 days of production (Figure 7B) indicates that for conditions with a longer feed addition period in the seed bioreactor, the final production rate in the production bioreactor is lower. It can be observed that a short addition period of FM1 in the seed bioreactor has a significant impact on cell growth and the final production rate in the production bioreactor. This confirms that the mode and period of feed addition in the seed bioreactor (preferably 3 hours or less) have a significant positive impact on increasing cell growth and productivity titer in the production bioreactor, and although in Examples 1 and 2, it also confirms the results with different cell clones (producing different antibodies) and different media platforms.

[0079] Example 4 The amount of feed added in the seed bioreactor affects the cell culture ability of the production bioreactor (mAb2) For this experiment, in a 4 × 10 L seed bioreactor (SBR), under various conditions (see Table 4) over 6 days, [X] 6 Please note that the numbers marked as [X] in the translation are the original numbers in the text which are not fully provided in the question. You may need to fill in the correct values according to the original text.CHO cells producing mAb2 were inoculated at cells / mL. In 4 × 2 L production bioreactors (PBRs), CHO cells producing mAb2 were inoculated at a seeding density of 3.75×10 6 cells / mL in a fed-batch process as described in Materials and Methods. In this experiment, multiple conditions with varying amounts of feed added in the seed bioreactor were evaluated. Additionally, the specific power input in the seed bioreactor was set to a low value such that it was possible to evaluate whether it was balanced with the fact that a high feed amount in the seed bioreactor reduces the specific power input. For the same purpose, the feed was added to the seed bioreactor in continuous mode.

Table 4

[0080] (Cell growth profile in the production bioreactor) is shown in Figure 8A, which shows that the specific power input in the SBR is less than 90 W / m 3 due to a higher feeding amount added in the seed bioreactor compared to the control condition (about 5.85% compared to about 4.20% of the total feed added per unit volume of cell culture start in the seed bioreactor w / w), and that even when FM1 is added in continuous mode in the seed bioreactor, it causes an increase in cell growth in the production bioreactor. Additionally, the final product titer at the end of 14 days of production (Figure 8B) shows a higher final production rate in the production bioreactor for conditions with a high feeding amount added in the seed bioreactor compared to the target condition (5.85% compared to 4.20%). This confirms the conclusion drawn in the previous example that by controlling the total feed amount per unit volume of cell culture start in the seed bioreactor (e.g., to be higher than about 3.50%), an increase in higher cell growth and final production rate occurs.

[0081] Example 5 - Industrial parameters affect the cell culture ability in the production bioreactor (mAb1) As shown in Example 1, the difference in P / V between the seed bioreactor and the production bioreactor is a potential factor affecting cell culture productivity: conditions with high P / V in the seed bioreactor associated with low P / V in the production step result in better cell culture performance.

[0082] To verify this finding, one 400 L seed bioreactor (SBR) was inoculated with CHO cells producing mAb1 at 0.35×10 6 cells / mL over 6 days under two conditions (see Table 5). Subsequently, one 2000 L production bioreactor (PBR) was inoculated with cells at a seeding density of 3.0×10 6 cells / mL during the fed-batch process as described in the Materials and Methods. In this experiment, two conditions were tested (see Table 5).

Table 5

[0083] These new experiments confirmed the findings of Example 1. Indeed, as shown in Figure 10 (representing cell culture performance in the PBR), cell growth and product formation decreased in the production bioreactor when cultured at a P / V of 32.8 W / m 3 associated with a higher P / V in the SBR compared to the PBR. The lactate production and glucose consumption rates were higher under these conditions compared to those carried out at a P / V of 134.2 W / cm3 associated with a lower P / V in the SBR compared to the PBR.

[0084] Without being bound by any theory, moving cells from a bioreactor with stronger mechanical stress to an environment within the production bioreactor with lower mechanical stress can still be one of the most important factors. If cells are subjected to stronger mechanical stress in the production bioreactor, they are likely to exhibit a state where their growth is delayed or no growth is observed at all.

[0085] References JPEG2025520392000008.jpg50134

Claims

1. A process for producing recombinant protein in a production bioreactor, wherein the process comprises: a. Inoculating mammalian cells containing the gene encoding the recombinant protein into the N-1 bioreactor, b. i. A feed or at least one specific mode and period of a feed, ii. Control of the total amount of feed added, and / or iii. Control of at least one industrial parameter, The mammalian cells are cultured in the N-1 bioreactor, which is operated in fed-batch mode under specific conditions selected from the above, c. The cells obtained from step (b) should be at least 2.00 × 10⁻⁶ ６ Inoculate the N bioreactor at a seeding density of viable cells / ml, d. Culturing the cells in the N bioreactor under conditions that enable the production of the recombinant protein, e. A process comprising: optionally collecting the recombinant protein, purifying the recombinant protein, and formulating the recombinant protein.

2. A process for improving the growth of mammalian cells in a production bioreactor, wherein the process comprises: a. Inoculating mammalian cells containing genes encoding recombinant proteins into an N-1 bioreactor, b. i. A specific mode and duration for adding a feed or at least one of the feeds, ii. Control of the total amount of feed added, and / or iii. Control of at least one industrial parameter, The mammalian cells are cultured in the N-1 bioreactor, which is operated in fed-batch mode under specific conditions selected from the above, c. The cells obtained from step (b) should be at least 2.00 × 10⁻⁶ ６ Inoculate the N bioreactor at a seeding density of viable cells / ml, d. Culturing the cells in the N bioreactor under conditions that enable the production of the recombinant protein, e. A process comprising: optionally collecting the recombinant protein, purifying the recombinant protein, and formulating the recombinant protein.

3. A process for increasing the production yield of recombinant proteins expressed by mammalian cells in a culture of a production bioreactor, wherein the process comprises: a. Inoculating mammalian cells containing the gene encoding the recombinant protein into the N-1 bioreactor, b. i. A feed or at least one specific mode and period of a feed, ii. Control of the total amount of feed added, and / or iii. Control of at least one industrial parameter, The mammalian cells are cultured in the N-1 bioreactor, which is operated in fed-batch mode under specific conditions selected from the above, c. The cells obtained from step (b) should be at least 2.00 × 10⁻⁶ ６ Inoculate the N bioreactor at a seeding density of viable cells / ml, d. Culturing the cells in the N bioreactor under conditions that enable the production of the recombinant protein, e. A process comprising: optionally collecting the recombinant protein, purifying the recombinant protein, and formulating the recombinant protein.

4. The process according to any one of claims 1 to 3, wherein the feed of step (b) is added daily, and the feed is added on day 0 of culture or thereafter.

5. i) at least the main feed of step (b) is added daily as a bolus, and / or ii) at least one of the secondary feeds of step (b) is added daily as a bolus, if present, the process according to any one of claims 1 to 3.

6. i) at least the main feed of step (b) is added daily as a bolus within about 3 hours or less, about 2 hours or less, or about 1 hour or less, and / or ii) at least one of the secondary feeds of step (b), if present, is added daily as a bolus within about 3 hours or less, about 2 hours or less, or about 1 hour or less.

7. The process according to any one of claims 1 to 3, wherein all of the feed in step (b) is added daily as a bolus.

8. The process according to claim 7, wherein all of the feeds in step (b) are added daily as a bolus within approximately 3 hours or less, approximately 2 hours or less, or approximately 1 hour or less.

9. The process according to any one of claims 1 to 3, wherein the at least one industrial parameter controlled during step (b) is a specific power input.

10. The specific power input is controlled daily, and the specific power input in step (b) is at least 100 W / m for all or part of the period of step (b). ３ The process according to claim 9, which must reach

11. The process according to any one of claims 1 to 3, wherein the total amount of feed added during step (b) is more than about 3.5%, but preferably 30% or less per culture start volume.

12. The process according to any one of claims 1 to 3, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

13. The process according to any one of claims 1 to 3, wherein the recombinant protein is a cytokine, growth factor, hormone, antibody, or fusion protein.

14. The process according to any one of claims 1 to 3, wherein the process is carried out on a large scale.

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