Cell culture method

Abstract

The invention belongs to the field of preparation of recombinant proteins, in particular antibodies. More specifically, the present invention relates to methods of producing recombinant proteins, such as antibodies, in bioreactors and / or methods of improving cell culture performance during the production of recombinant proteins in bioreactors (N-stage), by specific feeding strategies in seed bioreactors (N-1-stage), that is, by increasing the feeding speed, feeding amount, and / or increasing the power input via the thruster, ordered operational stresses are applied during seed cultivation.

Description

Technical Field

[0001] The present invention belongs to the field of preparation of recombinant proteins, especially antibodies. More specifically, the present invention relates to a method for producing recombinant proteins (such as antibodies) in a bioreactor and / or a method for improving cell culture performance during production of recombinant proteins in a bioreactor (N stage), which is carried out by a specific feeding strategy in a seed bioreactor (N-1 stage). Background Art

[0002] The development of recombinant proteins into therapeutic proteins such as therapeutic antibodies requires the production of recombinant proteins on an industrial scale. To achieve this goal, different expression systems can be employed, including prokaryotic and eukaryotic systems. However, in the past two decades, most therapeutic proteins approved as therapeutic agents have been produced by mammalian cell culture, and this system remains the preferred expression system for producing large quantities of recombinant proteins for human use.

[0003] Over the past 30 years, a great deal of effort has been invested in establishing the fundamental parameters for cell culture and recombinant protein expression, with much of the research focused on achieving optimal cell growth by varying the composition of cell culture media (see, e.g., Hecklau C. et al., 2016; Zang Li. et al., 2011) and operating conditions, as well as developing large-scale bioreactors.

[0004] It is known that N-1 phase conditions can strongly influence the performance of the production step (N phase). As described by Grammatikos et al. (1999), for example, the relative intracellular content of nucleotides and ATP is affected by bioreactor conditions, especially seed bioreactor conditions.

[0005] However, there remains a need to provide N-1 stage cell culture methods that will positively impact the N stage of recombinant protein production. Summary of the invention

[0006] In a first aspect, the present invention provides a method for producing a recombinant protein in a production bioreactor, wherein the method comprises: a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding the recombinant protein; b. culturing the mammalian cells in an N-1 bioreactor operating in a batch feeding mode under specific conditions selected from the following: i. adding a feed or a specific pattern and duration of at least one feed, ii. controlling the total amount of one or more feeds to be added, and / or iii. controlling at least one engineering parameter; c. using the cells obtained from step (b) at a rate of at least 2.00 x 10 6viable cells / ml; d. culturing the cells in the N bioreactor under conditions that allow production of the recombinant protein, and e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

[0007] In a second aspect, the present invention provides a method for improving the growth of mammalian cells in a production bioreactor, wherein the method comprises: a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding the recombinant protein; b. culturing the mammalian cells in an N-1 bioreactor operating in a batch feeding mode under specific conditions selected from the following: i. adding a feed or a specific mode and duration of at least one feed, ii. controlling the total amount of one or more feeds to be added, and / or iii. controlling at least one engineering parameter; c. using the cells obtained from step (b) at a rate of at least 2.00 x 10 6 viable cells / ml; d. culturing the cells in the N bioreactor under conditions that allow production of the recombinant protein, and e. and 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 method for increasing the yield / production of a recombinant protein expressed by cultured mammalian cells in a production bioreactor, wherein the method comprises: a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding the recombinant protein; b. culturing the mammalian cells in an N-1 bioreactor operating in a batch feeding mode under specific conditions selected from the following: i. adding a feed or a specific pattern and duration of at least one feed, ii. controlling the total amount of one or more feeds to be added, and / or iii. controlling at least one engineering parameter; c. using the cells obtained from step (b) at a rate of at least 2.00 x 10 6 viable cells / ml; d. culturing the cells in the N bioreactor under conditions that allow production of the recombinant protein, and e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

[0009] definition

[0010] In the event of a conflict, the present specification, including definitions, will control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this document belongs. As used herein, the following definitions are provided to facilitate understanding of the present invention.

[0011] As used in the specification and claims, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A", and "B".

[0012] As used in the specification and claims, the term "cell culture" or "culture" refers to the growth and reproduction of cells in vitro, i.e., outside of an organism or tissue. Suitable culture conditions for mammalian cells are known in the art, such as those taught by Ozturk & Hu (2005). Mammalian cells can be cultured in suspension or attached to a solid substrate.

[0013] 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 containing all the necessary components useful for cell metabolism. This includes, for example, amino acids, lipids, carbon sources, vitamins and mineral salts. DMEM (Double's Modified Eagle Medium), RPMI (Roswell Park Memorial Institute medium) or F12 medium (Ham's F12 medium) are examples of commercially available basal culture media. Other suitable culture media are described, for example, in WO98 / 08934 and US2006 / 0148074 (both of which are incorporated herein in their entirety). Additional suitable commercially available culture media include, but are not limited to, AmpliCHO CD medium, Dynamis TM Culture medium, EX- Advanced TM CHO batch feeding system, CD FortiCHO TM Culture medium, CP OptiCHO TM Culture medium, minimum essential medium (MEM), CHO Growth A Medium, ActiPro TM The basal medium may be a proprietary medium, also referred to herein as a "chemically defined medium" or "chemically defined medium", in which all components can be described by chemical formulas and are present at specific concentrations. The medium is preferably protein-free and serum-free, and may be supplemented with any additional compound or compounds, such as amino acids, salts, sugars, vitamins, hormones, growth factors, as required by the cultured cells.

[0014] The term "feed medium" (and its plural form) refers to a medium used as a supplement in a batch feeding mode during the culture to replenish the nutrients consumed during the culture. The feed medium can be a commercially available feed medium or a proprietary feed medium. Suitable commercially available feed media include, but are not limited to, Cell Boost TMSupplements, EfficientFeed TM Supplements, ExpiCHO TM Feeding. Alternatively, the feed medium can be a proprietary feed medium, also referred to herein as a "defined feed medium" or a "chemically defined feed medium", in which all components can be described by chemical formulas and are present in specific concentrations. The feed medium is usually concentrated so as not to increase the total volume of the culture in the bioreactor to a higher level. The amount of most of the components contained in this feed medium can be, for example, 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 of their normal amount in the basal medium. The proprietary feed medium is usually in powder form. Commercial feeds are liquid or powdered. When the feeds are already in liquid form, they are usually used as is according to the instructions. The powdered feed needs to be dissolved before use, for example dissolved in water.

[0015] 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: a feed medium containing most of the nutrients consumed (this feed is also called the main feed medium), a feed medium containing some additional nutrients (this feed is also called the secondary feed), for example, when these nutrients have aggregation / stability problems, and a feed medium containing a carbon source (e.g., glucose) or consisting of it. It is worth noting that the secondary feed (one or more) is not mandatory and will depend on the components contained in the main feed. It may happen that only two feeds (i.e., the main feed and the feed containing the carbon source) are used, or it may also happen that only one main feed containing all the required components (including the carbon source) is used. It should be understood that in the context of the present invention, when only one feed is used, it can be indifferently referred to as "feed" or "main feed".

[0016] The term "bioreactor" refers to any system in which cells can be cultured. It includes but is not limited to flasks, static flasks, rotating bottles, culture tubes, shake tubes, shake bottles, wave bags, bioreactors, fiber bioreactors, and stirred tank bioreactors with or without microcarriers. Alternatively, the term also includes microtiter plates, capillaries, or porous plates. Any size bioreactor can be used, for example, from 1 ml (1 mL, very small scale) to 20000 liters (20000L or 20KL, super large scale), such as 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).

[0017] The term "fed-batch culture" refers to a method of culturing cells in which a bolus (usually several boluses) or continuous feed medium (or feed media) is added to replace the nutrients consumed without removing any medium already in the bioreactor. One or more feeds can be added according to a predetermined schedule, for example, once a day, once every other day, once every three days, etc. Alternatively, if the feeding is continuous, the feed rate can vary throughout the culture. Depending on the medium formulation, cell line, and other cell growth conditions, this cell culture technique has the potential to achieve greater than 8 x 10 6 Up to 30x 10 6 High cell densities of the order of 10 cells / ml can be achieved. Biphasic culture conditions can be created and maintained through a variety of feeding strategies and media formulations.

[0018] According to the present invention, the term "N-1 phase" corresponds to the phase just before the "production phase", during which the cells are thought to grow and proliferate rapidly (in the so-called seed bioreactor) so that there is enough material to inoculate the N phase bioreactor. Alternatively, this phase is also called the seed phase.

[0019] According to the present invention, the term "production phase", "production stage" or "N phase" corresponds to the cell culture phase during the preparation of a recombinant protein when the cells express (i.e., produce) one or more recombinant polypeptides. The production phase begins when the titer of the desired product increases and ends with the harvesting of cells or cell culture fluid or supernatant. Typically, at the beginning of the production phase, 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 during the process in order to recover and purify the recombinant protein, such as a recombinant antibody, in a subsequent step.

[0020] As used herein, "cell concentration" (also referred to as "cell density") refers to the number of cells in a given volume of culture medium.

[0021] The term "viable cell concentration" (or "VCC") refers to the number of viable cells in a given volume of culture medium. This is determined by standard viability assays. It will be appreciated that the skilled person knows how to determine the maximum VCC for each specific cell line: this is usually achieved by one or more preliminary experiments. Alternatively, it may also be referred to as "VCD" or "viable cell density".

[0022] The term "IVCC" refers to the integrated viable cell count and can be determined by calculating the area under the cell culture growth curve.

[0023] The term "viability" or "cell viability" refers to the ratio between the total number of viable cells in the culture and the total number of cells. Although viability is generally acceptable as long as it does not fall below a threshold of 60% compared to the start of the culture, the acceptable threshold can be determined on a case-by-case basis. Viability is generally used to determine the time of harvest (this determination is done once during a preliminary experiment). For example, in a fed-batch culture, harvesting can be performed once the viability reaches at least 60% or after about 14 days of culture (usually 14 days + / - 1 day). Cell viability (alternatively VCC or VCD) can be determined using standard methods, for example by using VI- XR automated cell counting apparatus (Beckman-Coulter Inc.).

[0024] The term "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 combined with assays (colorimetric, chromatographic, etc.), combined with CEDEX or Protein A high pressure liquid chromatography (HPLC), Biacore or ForteBIO Methods as used in the Examples section.

[0025] The term "specific productivity", also referred to as "qp", refers to the amount of a protein of interest produced per cell per day.

[0026] The term "higher titer" or "higher yield" and their equivalents means that the titer or yield is increased by at least 10% compared to the control culture condition. If the titer or specific productivity ranges from -10% to 10% compared to the control culture condition, it is considered to remain unchanged. The term "lower titer" or "lower yield" and their equivalents means that the titer or yield is reduced by at least 10% compared to the control culture condition.

[0027] The term "lag phase" refers to a period of slow growth as cells adapt to the culture environment and prepare for rapid growth.

[0028] The term "specific power input" describes the ratio between the power input (P) and the volume of the working fluid (V). The power input describes the amount of energy delivered by the mover to the bioreactor body per second. The specific power input is expressed as follows:

[0029]

[0030] in:

[0031] Np is the power number, several examples of which are reported in the literature (Roustan, 2005);

[0032] ρ is the density of the liquid (kg / m 3 );

[0033] N is the mixing speed (rpm);

[0034] d is the diameter of the impeller (m);

[0035] V is the working volume (m 3 ).

[0036] The term "protein" as used herein includes peptides, polypeptides and proteins, and refers to a compound comprising 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. Therapeutic proteins refer to proteins that can be used or used for treatment.

[0037] The term "recombinant protein" means a protein produced by recombinant techniques. Recombinant techniques are well within the knowledge of those skilled in the art (see, e.g., Sambrook et al., 1989, and updates).

[0038] Preferably, the protein according to the methods, uses and processes of the invention is an antibody or an antigen binding fragment thereof or a fusion protein.

[0039] The term "antibody" as used herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies produced by recombinant techniques known in the art. "Antibodies" include antibodies of any species, particularly mammalian species; such as human antibodies of any isotype, including IgG1, IgG2a, IgG2b, IgG3, IgG4, IgE, IgD, and antibodies produced in the form of dimers (including IgGA1, IgGA2) or pentamers (such as IgM and its modified variants) of this basic structure; non-human primate antibodies, such as antibodies from chimpanzees, baboons, rhesus monkeys or cynomolgus monkeys; rodent antibodies, such as antibodies from mice or rats; rabbit, goat or horse antibodies; camelid antibodies (e.g. antibodies from camels or llamas, such as Nanobodies TM) 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 the first portion of at least one heavy chain and / or light chain antibody sequence is from a first species, and the second portion of the heavy chain and / or light chain antibody sequence is from a second species. Chimeric antibodies of interest herein include "primatized" antibodies, which contain variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, such as baboons, rhesus monkeys, or cynomolgus monkeys) and human constant region sequences. "Humanized" antibodies are chimeric antibodies containing sequences derived from non-human antibodies. In most cases, humanized antibodies are human antibodies (acceptor antibodies) in which residues from the hypervariable region of the acceptor are replaced with residues from the hypervariable region [or complementary determining region (CDR)] of a non-human species (donor antibody) such as mouse, rat, rabbit, chicken, or non-human primate, which have the desired specificity, affinity, and activity. In most cases, the residues of the human (acceptor) antibody outside the CDR, i.e., the residues in the framework region (FR), are additionally replaced with corresponding non-human residues. In addition, humanized antibodies may also contain residues that are not present in the acceptor antibody or the donor antibody. These modifications are made to further improve the antibody properties. Humanization reduces the immunogenicity of non-human antibodies in humans, thereby promoting the use of antibodies in the treatment of human diseases. Humanized antibodies and several different techniques for producing them are well known in the art. The term "antibody" also refers to human antibodies, which can be produced as an alternative to humanization. For example, transgenic animals (e.g., mice) can be produced that are able to produce a complete reservoir of human antibodies after immunization without producing endogenous murine antibodies. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies, such as phage display or ribosome display technologies, in which a recombinant DNA library is used, at least a portion of which is artificially generated, or a reservoir of immunoglobulin variable (V) domain genes from a donor. Phage and ribosome display technologies for producing human antibodies are well known in the art. Human antibodies can also be generated from isolated human B cells, which are immunized in vitro with an antigen of interest and subsequently fused to produce hybridomas, which can then be screened to obtain the best human antibodies. The term "antibody" refers to both glycosylated and non-glycosylated antibodies. Moreover, the term "antibody" as used herein refers not only to full-length antibodies, but also to antibody fragments thereof, more specifically to antigen-binding fragments thereof. The fragment of an antibody comprises at least one heavy chain or light chain immunoglobulin domain known in the art, and binds to one or more antigens. 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.The fragment may also be a diabody, a triabody, a triabody, a tetrabody, a minibody, a single domain antibody (dAb) such as an sdAb, a VL, VH, VHH or a camelid antibody (e.g. an antibody from a camel or llama, such as a Nanobody. TM ) and VNAR fragments. Antigen-binding fragments according to the invention may also comprise a Fab linked to one or two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binds a therapeutic target and one scFv or dsscFv increases half-life by binding to, for example, albumin). Examples of such antibody fragments are FabdsscFv (also known as ) or Fab-(dsscFv)2 (also known as See, e.g., WO2015197772). Antibody fragments as defined above are known in the art. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present disclosure relates to a process (or method) for producing recombinant proteins. More specifically, the present disclosure describes a process (or method) for producing recombinant proteins in mammalian cells and improving cell culture performance (such as cell growth, specific productivity and / or titer / yield). In particular, the present invention is based on the following research results of the inventors, that is, by applying specific conditions during the N-1 stage, such as controlling the feeding strategy of the N-1 stage (i.e., in the seed bioreactor), cell growth can be improved during the production phase (such as increasing the growth rate, increasing cell viability, reducing the stagnation period that may occur at the beginning of the N stage) and the yield of recombinant proteins produced by cells can be increased, more specifically in the high seeding density (HSD) process (see also Fig. 9 Surprisingly, the inventors have found that stressing cells (e.g., by osmotic stress) during the N-1 phase (i.e., in a seed bioreactor) run in a batch feeding mode can improve cell growth and increase the yield of recombinant protein production in the subsequent N phase (i.e., in a production bioreactor), particularly during an intensification process such as an HSD process. For some expression cells, the stagnation period observed at the beginning of the production phase (in the N phase) can also be reduced or even avoided. 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 performance). The inventors refer to this stress as "organized stress."

[0042] Therefore, the process described in this article relies on an N-1 stage carried out in batch feeding under specific conditions (such as specific stress conditions, or so-called "sequential stress" conditions), that is, during the N-1 stage, the feeding pattern and / or engineering parameters are controlled, especially when integrated with the intensification process, so as to produce the recombinant protein of interest during the subsequent N stage (i.e., the production stage).

[0043] The present disclosure particularly describes how to control feeding conditions / engineering parameters during the N-1 stage to maximize the yield of the production bioreactor and maximize cell growth. The present disclosure provides specific examples of batch feeding processes such as HSD batch feeding processes for seed bioreactors (N-1 stage), wherein these parameters are controlled within the claimed ranges, and specific examples of possible modes of adding one or more feeds (bolus mode, including duration and timing, relative to continuous / semi-continuous mode, etc.) are described in detail.

[0044] In one embodiment, the present invention provides a method for producing a recombinant protein in a production bioreactor, wherein the method comprises:

[0045] a. inoculating an N-1 bioreactor with mammalian cells containing a gene encoding the recombinant protein,

[0046] b. Cultivating mammalian cells in an N-1 bioreactor operated in a fed-batch mode, wherein the fed-batch is carried out under specific conditions selected from the group consisting of:

[0047] i. adding a feed or a specific mode and duration of at least one feed,

[0048] ii. controlling the total amount of one or more feedstocks to be added, and / or

[0049] iii. Control at least one engineering parameter,

[0050] c. Using the cells obtained from step (b) at a concentration of at least 2.00 x 10 6 The N bioreactor was inoculated at an inoculation density of viable cells / ml;

[0051] d. culturing the cells in an N bioreactor under conditions that allow production of the recombinant protein,

[0052] e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

[0053] The results show that in the method for producing recombinant protein, the yield of recombinant protein is increased and / or the cell growth in the production bioreactor is improved. Therefore, in an alternative embodiment, the present invention provides a method for producing recombinant protein in a production bioreactor, wherein the method comprises: a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding the recombinant protein, b. culturing mammalian cells in an N-1 bioreactor operated in a batch feeding mode, wherein the batch feeding is performed under specific conditions selected from the following: i. adding a specific mode and duration of feed or at least one feed, ii. controlling the total amount of one or more feeds to be added, and / or iii. controlling at least one engineering parameter, c. using the cells obtained from step (b) at a rate of at least 2.00 x 10 6 viable cells / ml; d. culturing the cells in the N bioreactor under conditions that allow the production of the recombinant protein, e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein, wherein the yield of the recombinant protein is also increased and / or the cell growth in the production bioreactor is improved. It should be understood that the yield of the recombinant protein is increased and / or the cell growth in the production bioreactor is improved (i.e., wherein the cell growth is increased compared to cells that were not grown under "ordered stress" during the N-1 stage) compared to a method that does not include the specific conditions of step b).

[0054] In another embodiment, the present invention provides a method for improving growth of mammalian cells in a production bioreactor, wherein the method comprises:

[0055] a. inoculating an N-1 bioreactor with mammalian cells containing a gene encoding the recombinant protein,

[0056] b. Cultivating mammalian cells in an N-1 bioreactor operated in fed-batch mode under specific conditions selected from:

[0057] i. adding a feed or a specific mode and duration of at least one feed,

[0058] ii. controlling the total amount of one or more feedstocks to be added, and / or

[0059] iii. Control at least one engineering parameter,

[0060] c. Using the cells obtained from step (b) at a concentration of at least 2.00 x 10 6 The N bioreactor was inoculated at an inoculation density of viable cells / ml;

[0061] d. culturing the cells in an N bioreactor under conditions that allow production of the recombinant protein,

[0062] e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

[0063] Those skilled in the art will appreciate that an improvement in cell growth is observed compared to a method during which the cells are not grown under the specific conditions of step b) (i.e., wherein cell growth is increased compared to cells not grown under "ordered stress" during the N-1 stage).

[0064] The results show that in the method for improving the growth of mammalian cells in a production bioreactor, the yield of the recombinant protein is also improved. Therefore, in an alternative embodiment, the present invention provides a method for improving the growth of mammalian cells in a production bioreactor, wherein the method comprises: a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding the recombinant protein, b. culturing the mammalian cells in an N-1 bioreactor operating in a batch feeding mode under specific conditions selected from the following: i. adding a feed or a specific mode and duration of at least one feed, ii. controlling the total amount of one or more feeds to be added, and / or iii. controlling at least one engineering parameter; c. using the cells obtained from step (b) at a rate of at least 2.00 x 10 6 viable cells / ml; d. culturing the cells in the N bioreactor under conditions that allow production of the recombinant protein, and e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein, wherein growth of the cells in the bioreactor is improved compared to cells not grown under the conditions of step b), and wherein the yield of the recombinant protein is also improved.

[0065] In yet another embodiment, the present invention relates to a method for increasing the yield of a recombinant protein expressed by cultured mammalian cells in a production bioreactor, wherein the method comprises:

[0066] a. inoculating an N-1 bioreactor with mammalian cells containing a gene encoding the recombinant protein,

[0067] b. Cultivating mammalian cells in an N-1 bioreactor operated in fed-batch mode under specific conditions selected from:

[0068] i. adding a feed or a specific mode and duration of at least one feed,

[0069] ii. controlling the total amount of one or more feedstocks to be added, and / or

[0070] iii. Control at least one engineering parameter,

[0071] c. Using the cells obtained from step (b) at a concentration of at least 2.00 x 10 6The N bioreactor was inoculated at an inoculation density of viable cells / ml;

[0072] d. culturing the cells in an N bioreactor under conditions that allow production of the recombinant protein,

[0073] e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

[0074] Those skilled in the art will appreciate that an increase in the yield of recombinant protein production is observed compared to a method during which the cells are not grown under the specific conditions of step b) (i.e., wherein the yield of recombinant protein production is increased compared to cells not grown under "ordered stress" during the N-1 stage).

[0075] The results show that in the method for increasing the yield of recombinant protein production, cell growth in the bioreactor is improved. Therefore, in an alternative embodiment, the present invention relates to a method for increasing the yield of recombinant protein production expressed by cultured mammalian cells in a production bioreactor, wherein the method comprises: a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding the recombinant protein, b. culturing mammalian cells in an N-1 bioreactor operating in a batch feeding mode under specific conditions selected from the following: i. adding a feed or a specific mode and duration of at least one feed, ii. controlling the total amount of one or more feeds to be added, and / or iii. controlling at least one engineering parameter, c. using the cells obtained from step (b) at a rate of at least 2.00 x 10 6 viable cells / ml; d. culturing the cells in the N bioreactor under conditions that allow production of the recombinant protein, e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein, wherein the yield produced is increased compared to cells not grown under the conditions of step b), and wherein cell growth in the bioreactor is also improved.

[0076] The skilled person will appreciate that in order to carry out step (c) with the cells obtained from step (b), a certain level of viable cell density should be obtained at the end of step (b). Therefore, the present invention also encompasses a method for producing a recombinant protein in a production bioreactor, a method for improving the growth of mammalian cells in a production bioreactor and / or a method for increasing the production yield of a recombinant protein expressed by cultured mammalian cells in a production bioreactor, wherein the method comprises:

[0077] a. inoculating an N-1 bioreactor with mammalian cells containing a gene encoding the recombinant protein,

[0078] b. Cultivate mammalian cells in an N-1 bioreactor operating in fed-batch mode under specified conditions until at least 8 x 10 6 viable cells / ml, and wherein the specific conditions are selected from:

[0079] i. adding a feed or a specific mode and duration of at least one feed,

[0080] ii. controlling the total amount of one or more feedstocks to be added, and / or

[0081] iii. Control at least one engineering parameter,

[0082] c. Using the cells obtained from step (b) at a concentration of at least 2.00 x 10 6 The N bioreactor was inoculated at an inoculation density of viable cells / ml;

[0083] d. culturing the cells in an N bioreactor under conditions that allow production of the recombinant protein,

[0084] e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

[0085] Although it is important to run the N-1 stage in batch feeding mode according to the present invention, the N stage can be run in any mode, such as perfusion mode, batch mode or batch feeding mode. Preferably, the N stage is run in perfusion or batch feeding mode. Those skilled in the art know how to run the production stage in perfusion, batch or batch feeding mode.

[0086] For the purposes of the present invention as a whole, an improvement in cell growth in a production bioreactor can be, for example, an increase in cell growth as determined by an increase in VCC compared to cells not grown under "ordered stress" during the N-1 phase and / or a reduction in the lag phase that may occur at the beginning of the production phase compared to cells not grown under "ordered stress" during the N-1 phase. If a lag phase exists, increasing the VCC or reducing the lag phase will allow the production of the recombinant protein to be undelayed.

[0087] For the present invention as a whole, the specific conditions applied during the batch feeding N-1 phase are considered stress conditions for the cells. Since these conditions (also called stress conditions) are controlled, they can also be called "ordered stress" conditions. In all methods described herein, step b) can alternatively be described as:

[0088] -b) culturing mammalian cells in an N-1 bioreactor operating in a fed-batch mode under specific stress conditions, wherein the specific stress conditions are selected from: i. adding a feed or a specific pattern and duration of at least one feed, ii. controlling the total amount of one or more feeds to be added, and / or iii. controlling at least one engineering parameter, or can also be described as

[0089] -b) culturing mammalian cells in an N-1 bioreactor operated in a fed-batch mode under applied specific ordered stress conditions, and wherein the specific ordered stress conditions are selected from: i. adding feeds or a specific pattern and duration of at least one feed, ii. controlling the total amount of one or more feeds to be added, and / or iii. controlling at least one engineering parameter.

[0090] For the present invention as a whole, mammalian cells containing genes encoding recombinant proteins (in other words, mammalian cells expressing recombinant proteins) are preferably cultured in batch feeding mode for at least 4 days during the N-1 stage, but preferably no more than 7 days, more preferably at least 5 days and no more than 7 days, for example 5 days, 6 days or 7 days. Those skilled in the art can perform longer N-1 stages, but there are associated risks, such as cell viability decline or start of production, which are all to be avoided in the N-1 stage. It is preferred to add feed every day (at least the main feed and / or secondary feed, if any). Feed can be added from the 0th day, the 1st day, the 2nd day, the 3rd day or the 4th day after the start of the N-1 stage (i.e., the 0th day is the day of inoculation), and continue for at least 2 days, at least 3 days, for example 3 days, 4 days, 5 days or 6 days. Typically, the last feed is added until the day before the last day of cultivation. Therefore, as a non-limiting example, if the N-1 stage lasts for 7 days and feed is added from the 3rd day for 4 days, the last feed addition will be on the 6th day. In another non-limiting example, if the N-1 phase lasts 6 days and feed is added starting on day 0 and continuing for 6 days, the last feed addition would be on day 5.

[0091] For the present invention as a whole, one of the conditions for performing "sequential stress" is the duration of adding a feed or at least one feed. Preferably, this feed of step (b) is added in a bolus mode every day. Preferably, i) at least the main feed of step (b) is added in a bolus mode every day, and / or ii) at least one secondary feed of step (b) (if any) is added in a bolus mode every day. The daily bolus feed is preferably added within about 3 hours or less, within about 2 hours or less, or within about 1 hour or less. If more than one feed (e.g., one main feed and one secondary feed) is to be added, preferably in step (b), all feeds are added in a bolus mode every day. In this case, the addition of the daily bolus feed is preferably within about 3 hours or less, within about 2 hours or less, or within about 1 hour or less. The duration of adding different feeds (when different feeds are present) does not need to be similar. In one non-limiting example, the main feed can be added in a bolus mode every day within 3 hours, and one or more secondary feeds can be added in a bolus mode every day within 1 hour. In another non-limiting example, the main feed can be added in a bolus mode every day within 1 hour, and one or more secondary feeds can be added in a bolus mode every day within 30 minutes. Those skilled in the art will understand that if they wish, at least one feed can alternatively be a feed comprising a carbon source, as long as it is sufficient to induce stress conditions.

[0092] Another condition for performing "sequential stress" in the context of the present invention as a whole is the total amount of feed added during step (b). The total amount of feed is important and should preferably account for at least about 3.5% of the culture starting volume, but preferably not more than 30% per culture starting volume. Therefore, in the context of the present invention as a whole, the total amount of feed added during step (b) preferably accounts for at least about 3.5% of the culture starting volume, but preferably not more than 30% per culture starting volume. Alternatively, the total amount of feed added during step (b) preferably accounts for at least about 3.5% of the culture starting volume, but preferably does not exceed 25% per culture starting volume (in other words, the total amount of feed added during step (b) preferably accounts for about 3.5% to about 30% of the culture starting volume), such as about 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 15.0%, 20.0%, 25.0% or 25.0% of the culture starting volume. As a non-limiting example, if the total amount of feed added during step (b) accounts for 3.5% of the culture starting volume, and the culture starting volume is 100 L, this means that a total of 3.5 L of feed will be 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) accounts for 4.2% of the culture starting volume, and the culture starting volume is 500 L, this means that a total of 21 L of feed will be 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) accounts for 15% of the culture starting volume, and the culture starting volume is 200 L, this means that a total of 30 L of feed will be added during the N-1 stage, resulting in a final culture volume of 230 L.

[0093] In the context of the present invention as a whole, another condition for performing "sequential stress" is to control at least one engineering parameter during step (b). Preferably, at least one engineering parameter controlled during step (b) is the unit power input. More preferably, the unit power input in step (b) is controlled every day, and during the entire or partial duration of step (b), the unit power input in step (b) should preferably reach at least 100 W / m 3 Preferably, during the whole or part of the duration of step (b), the unit power input in step (b) reaches at least 100 W / m 3 , at least 110W / m 3 , at least 120W / m 3 , at least 130W / m 3 , at least 140W / m 3 , at least 150W / m 3 , at least 160W / m3 or at least 170W / m 3 In a non-limiting example, if the duration of step (b) is 6 days, the unit power input may be controlled to be approximately 120 W / m throughout the duration of the step (i.e., from day 0 to day 6). 3 In another non-limiting example, if the duration of step (b) is 7 days, the unit power input may be controlled at about 140 W / m only for 5 days (i.e., part of the duration of step (b)), for example, from day 1 to day 5. 3 .

[0094] The inventors' research shows that it is mainly (or only) the power input (P / V) applied in the seed bioreactor, rather than the P / V applied in the production bioreactor, that has a significant impact on cell culture production. Alternatively, it seems that the difference (or ratio) between the P / V in the seed bioreactor and the production bioreactor is still a potential factor that significantly affects cell culture production performance. In other words, the difference between the mechanical stress caused by the power input in the seed bioreactor and the production bioreactor is still a potential factor affecting cell culture production performance: the condition of high P / V in the seed bioreactor is associated with lower P / V (lower mechanical stress) in the production step. As shown in the Example section, the conditions for achieving optimal cell culture performance during the production stage are the conditions of high P / V in the seed bioreactor associated with lower P / V in the production step. Thus, in an alternative, not only is the at least one engineering parameter controlled during step (b) the specific power input, but there is also an additional engineering parameter controlled during step (d), namely the specific power input in the production bioreactor, such that a certain ratio is followed between the power input in the seed bioreactor (P / V SBR) and the power input in the production bioreactor (P / V PBR). 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, at least 1.65:1, at least 1.70:1, at least 1.80:1, at least 1.90:1 or even at least 2:1. The optimal ratio can be determined during preliminary experiments. Therefore, the method according to the invention as a whole may further comprise the preliminary step of carrying out at least one set of preliminary experiments to determine the P / V SBR:P / V PBR ratio, which ratio results in an improvement in the growth of mammalian cells in the production bioreactor and / or an increase in the yield of recombinant protein expressed by mammalian cells cultured in the production bioreactor compared to the standard method. It must be understood that the at least one set of preliminary experiments need not be repeated each time the method according to the invention is performed. In other words, once the conditions have been determined in at least one set of preliminary experiments, it is not necessary to control the conditions for a particular clone each time the method according to the invention is performed under given conditions.

[0095] Thus, the present invention also encompasses a method for producing a recombinant protein in a production bioreactor, a method for improving the growth of mammalian cells in a production bioreactor and / or a method for increasing the yield of the production of a recombinant protein expressed by cultured mammalian cells in a production bioreactor, wherein the method comprises:

[0096] 0. optionally performing at least one set of preliminary experiments to determine the P / V SBR:P / VPBR ratio preferably to be maintained during the production phase,

[0097] a. inoculating an N-1 bioreactor with mammalian cells containing a gene encoding the recombinant protein,

[0098] b. Cultivating the mammalian cells in an N-1 bioreactor operating in a fed-batch mode under "ordered stress" conditions until at least 8 x 10 6 viable cells / ml, and wherein the "ordered stress" condition is selected from:

[0099] i. adding a feed or a specific mode and duration of at least one feed,

[0100] ii. controlling the total amount of one or more feedstocks to be added, and / or

[0101] iii. controlling at least one engineering parameter, wherein the at least one engineering parameter is a specific power input,

[0102] c. Using the cells obtained from step (b) at a concentration of at least 2.00 x 10 6 The N bioreactor was inoculated at an inoculation density of viable cells / ml;

[0103] d. culturing the cells in an N bioreactor under conditions that allow production of the recombinant protein, comprising the step of controlling at least one engineering parameter, wherein the at least one engineering parameter is unit power input, so as to maintain the P / V SBR:P / V PBR ratio determined in optional step (0) or in previous preliminary experiments,

[0104] e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

[0105] Although the combination of the three conditions to achieve ordered stress has yielded the best results for the production of recombinant proteins in a production bioreactor, for improving the growth of mammalian cells in a production bioreactor and / or for increasing the yield of recombinant proteins expressed by mammalian cells cultured in a production bioreactor, one of the conditions has been sufficient to achieve acceptable results and may be sufficient, depending on the VCC obtained at the end of the N-1 stage. The implementation of one, two or three of the "ordered stress" conditions will depend on the production cell. One or more optimal conditions can be determined during preliminary experiments. Therefore, the method according to the present invention as a whole can further include the preliminary step of performing at least one preliminary experiment to determine the VCC at the end of the N-1 stage and to determine which conditions to apply during the N-1 stage so that the cells obtained from step (b) reach preferably at least 8 x 10 6viable cells / ml and capable of producing recombinant proteins in a production bioreactor, the growth of mammalian cells in a production bioreactor can be improved and / or the yield of recombinant proteins expressed by mammalian cells cultured in a production bioreactor can be increased compared to standard methods. It must be understood that this preliminary experiment does not need to be repeated each time the method according to the invention is performed. In other words, once the conditions have been determined in at least one preliminary experiment, it is not necessary to control it for a particular clone under given conditions each time the method according to the invention is performed.

[0106] Thus, the present invention also encompasses a method for producing a recombinant protein in a production bioreactor, a method for improving the growth of mammalian cells in a production bioreactor and / or a method for increasing the yield of the production of a recombinant protein expressed by cultured mammalian cells in a production bioreactor, wherein the method comprises:

[0107] 0. optionally carrying out at least one preliminary experiment to determine the VCC at the end of the N-1 stage and / or optionally carrying out at least one preliminary experiment to determine the duration of feeding during step (b), the total amount of feed to be added during step (b) and / or the specific power input to be used during step (b),

[0108] a. inoculating an N-1 bioreactor with mammalian cells containing a gene encoding the recombinant protein,

[0109] b. Cultivate mammalian cells in an N-1 bioreactor operating in fed-batch mode under “ordered stress” conditions until at least 8 x 10 6 viable cells / ml, and wherein the "ordered stress" condition is selected from:

[0110] i. adding a feed or a specific mode and duration of at least one feed,

[0111] ii. controlling the total amount of one or more feedstocks to be added, and / or

[0112] iii. Control at least one engineering parameter,

[0113] c. Using the cells obtained from step (b) at a concentration of at least 2.00 x 10 6 The N bioreactor was inoculated at an inoculation density of viable cells / ml;

[0114] d. culturing the cells in an N bioreactor under conditions that allow production of the recombinant protein,

[0115] e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

[0116] Although the inoculation of the N bioreactor can be performed at a lower inoculation density (e.g., less than 2.00 x 10 6 viable cells / ml, less than 1.50 x 10 6 viable cells / ml, less than 1.00 x 10 6 viable cells / ml or even less than 0.50 x 10 6 viable cells / ml), but when the density is at least about 2.00 x 10 6 When the inoculation density is higher than about 2.00×10 viable cells / ml (i.e., high inoculation density), the results are better in terms of the production of recombinant protein, the increase of cell growth and / or the yield of recombinant protein production. Therefore, in the context of the present invention as a whole, the inoculation of the N bioreactor (step c) is preferably carried out with cells obtained from the N-1 bioreactor at a density of at least about 2.00×10 6 Alternatively, the bioreactor of step (c) is inoculated at an inoculation density of at least 3.00 x 10 6 viable cells / ml, at least 4.00 x 10 6 viable cells / ml, at least 5.00 x 10 6 viable cells / ml, at least 6.00 x 10 6 viable cells / ml, at least 7.00 x 10 6 viable cells / ml, at least 8.00 x 10 6 viable cells / ml, at least 9.00 x 10 6 viable cells / ml or at least 10.00 x 10 6 viable cells / ml. A person skilled in the art will understand that “10.00 x 10 6 "viable cells / ml" is not a limit, and higher inoculation densities are envisioned with respect to the cell density obtained at the end of step (b), depending on the output of the N-1 bioreactor.

[0117] In order to be able to inoculate the N bioreactor under the high inoculation density strategy, it is preferred to reach at least about 8.00 x 10 6 It is also preferred not to reach a (viable) cell density of at least about 8.00 x 10 viable cells / ml at the end of this stage, since this may mean that the cells are cultured for too long, which may affect their quality and productivity during the N stage. Thus, in the context of the present invention as a whole, at the end of the N-1 stage (at the end of step (b)), the (viable) cell density achieved is preferably at least about 8.00 x 10 6 viable cells / ml, and up to about 30.00x10 6Alternatively, the (viable) cell density obtained at the end of the N-1 stage (at the end of step (b)) is preferably at least 9.00 x 10 6 viable cells / ml, at least 10.00 x 10 6 viable cells / ml, at least 15.00 x 10 6 viable cells / ml, at least 20.00 x 10 6 viable cells / ml, at least 25.00 x 10 6 viable cells / ml, but preferably not more than 30.00 x 10 6 viable cells / ml.

[0118] In the context of the present invention as a whole, the culture medium at the beginning of the culture (also referred to herein as the basal medium; the culture medium used for both the N-1 stage (i.e. the culture medium used in step (a)) and the N stage (i.e. the culture medium used in step (c))) is preferably a protein-free and serum-free culture medium. The protein-free and serum-free culture medium may be a commercially available culture medium or a (homemade or commercially available) chemically defined culture medium. Thus, such a culture medium may be used at the beginning of the N-1 stage (the initial culture medium of step (a)) and at the beginning of the N stage (the initial culture medium of step (c)).

[0119] In the context of the present invention as a whole, the main feed medium can be any main feed medium. In one example, this main feed medium does not contain Cys (neither cysteine ​​nor cystine), Trp and Tyr, and these components are brought in by at least one additional feed (this additional feed will be a secondary feed or one of the secondary feeds).

[0120] In the context of the present invention as a whole, the method is preferably performed on a large scale, for example in a bioreactor, the volume of which is preferably equal to or greater than 50 L, equal to or greater than 100 L, equal to or greater than 500 L, equal to or greater than 1000 L, equal to or greater than 2,000 L, equal to or greater than 55,000 L, equal to or greater than 10,000 L or equal to or greater than 20,000 L. In other words, the mammalian cells producing the recombinant protein are cultured in a bioreactor, such as a production bioreactor, the volume of which is preferably equal to or greater than 50 L, equal to or greater than 100 L, equal to or greater than 500 L, equal to or greater than 1000 L, equal to or greater than 2,000 L, equal to or greater than 5,000 L, equal to or greater than 10,000 L or equal to or greater than 20,000 L.

[0121] In the context of the present invention as a whole, suitable mammalian host cells (also referred to as mammalian cells) include Chinese hamster ovary cells (CHO cells), lymphocyte cell lines, such as NSO myeloma cells and SP2 cells, COS cells, myeloma or hybridoma cells. In a preferred embodiment, the mammalian cell is a CHO cell. Suitable types of CHO cells may include CHO-K1, CHOK1-SV, dhfr-CHO, such as CHO-DG44, CHO-DXB11, CHO-DXB1 or CHO-S cells. The host cell is preferably stably transformed or transfected with an expression vector encoding a recombinant protein of interest.

[0122] 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. If the protein is an antibody, it can be, for example, a chimeric antibody, a humanized antibody or a fully human antibody, and is preferably an IgG, such as IgG1, IgG2, IgG3 or IgG4. Alternatively, it can be any kind of protein according to the definition given herein.

[0123] The method according to the present invention may further include the step (harvest step) of reclaiming the cell culture fluid (CCF) comprising the recombinant protein, in other words the step of harvesting the recombinant protein. After harvesting, the recombinant protein can be purified, for example, if the protein is an antibody, protein A chromatography and other chromatography / filtration steps are used. The method further optionally includes the step of preparing the purified recombinant protein, for example, it is formulated into a preparation with a high protein concentration, such as a concentration of 10 mg / ml or higher, for example 50 mg / ml or higher, such as 100 mg / ml or higher, 150 mg / ml or higher or even 200 mg / mL or higher. Without any restriction, the preparation can be a liquid preparation, a lyophilized preparation or a spray-dried preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1 : A) Cell growth of cells expressing mAbl in N-1 bioreactor. B) Cumulative IVCC of cells expressing mAbl in N bioreactor.

[0125] Figure 2 : Summary of multiple linear regression fits for cumulative IVCC in N bioreactors.

[0126] Figure 3 : A) Cell growth curve of cells expressing mAb1 in N bioreactor. B) Titer of cells expressing mAb1 in N bioreactor.

[0127] Figure 4: Data for cells expressing mAb1 in N bioreactor. A) Cell viability; B) Viable cell count. C) Titer (normalized). D) Figure 4 Illustration of the A-4C.

[0128] Figure 5 : A) Cell growth of cells expressing mAbl in N-1 bioreactor. B) Cumulative IVCC of cells expressing mAbl in N bioreactor.

[0129] Figure 6 : A) Cell growth curve of cells expressing mAb1. B) Titer of cells expressing mAb1 in N bioreactor.

[0130] Figure 7 : A) Cell growth curve of cells expressing mAb2 in N bioreactor. B) Titer of cells expressing mAb2 in N bioreactor.

[0131] Figure 8 : A) Cell growth curve of cells expressing mAb2 in N bioreactor. B) Titer of cells expressing mAb2 in N bioreactor.

[0132] Fig. 9 : Design of a long lag phase scale-down model screen from a seed bioreactor (N-1) to a production bioreactor (N) in a glass stirred vessel bioreactor. "Feed 1" corresponds to the primary feed and "Feed 2" corresponds to the secondary feed.

[0133] Fig.10 : The influence of unit power input on the cell culture performance of the enhanced process of cells expressing mAb2 in the seed bioreactor. In order to verify the small-scale data, the "ordered stress" concept was applied to two 2000L cell culture production bioreactors. The unit power input in the seed bioreactor (400L bioreactor) is variable (Table 3). The unit production rate curves of viable cell density, cell viability, glucose and lactic acid in the enhanced production bioreactor are given. Example

[0134] Cell lines, cell culture, and experimental procedures

[0135] Two different production CHO-DG44 cell lines were used to produce: mAb-1 (whole IgG4 antibody with a pI of 5.7-5.9) and mAb-2 (trispecific antibody with a pI of 8.9-9.2).

[0136] Two sets of culture media were used:

[0137] -First source (culture medium from Cytiva): ActiPro as basal culture mediumTM , Cell Boost as the main feed TM 7a (also referred to herein as CB7a) and Cell Boost as secondary feed TM 7b (also referred to herein as CB7b) which supplements the elements from the main feed with additional elements.

[0138] - Second source (Modified CHO Pro Medium): "BM1" as basal medium, "FM1" as main feed and "FM 2" as secondary feed which supplements the elements from the main feed with additional elements.

[0139] The cells were cultured in a 2 L stirred tank glass bioreactor (STR) 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 three-stage paddle impeller. The culture start volume was adjusted to ensure that the culture end volume was optimal. The production bioreactor was inoculated at a target inoculation density (TSD) in the basal medium. The pH control of the production STR was set to 7.0 with a deadband 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 using a predefined mixing curve based on a cascade controller. The temperature was controlled at approximately 36.8°C. The seed bioreactor (N-1 stage) was operated in batch feeding mode for 5 or 6 days (by adding two different feeds). Then, the production bioreactor (N stage) was operated in feeding experiment mode for 14 days (by adding two different feeds). During this phase, monoclonal antibodies (mAbs) are secreted into the culture medium. Samples are taken daily to determine VCD, viability, offline pH, pCO2, osmotic pressure, glucose-lactic acid concentration, amino acid concentration, and mAb concentration (stored at -80°C). Defoamers are added manually as needed every day to control the accumulation of foam. 72 hours after inoculation, continuous nutrient feeding at a predetermined rate begins. When the glucose concentration drops below a given threshold, a glucose bolus feed is added to the culture. Glucose concentration is measured every day. Samples for amino acid analysis are collected before adding the feed. The extracellular concentration after feeding is calculated based on the feed composition and the nutrient concentration measured before adding the feed. At the end of the production period, after harvesting, cell culture supernatant samples (i.e., harvested cell culture fluid) are purified using protein A purification on the TECAN automation system.

[0140] Analytical methods

[0141] By using VI- Cells were counted using an XR (Beckman-Coulter, Inc.) automated cell counter that operates based on the trypan blue exclusion method. Glucose and lactate levels in the culture medium were determined using a NOVA400 BioProfile automated analyzer (Nova Biomedical) or a Cedex Bio HT (Roche). Osmolarity was determined using a 2020 freezing point osmometer (Advanced Instruments, Inc.). Offline gas and pH measurements were performed using a blood gas analyzer (Nova Biomedical Corp.). Metabolite concentrations were determined daily using a CedexBioHT system (Roche). Product titer analysis was performed on cell culture supernatant samples using protein A high pressure liquid chromatography (HPLC), which were stored at -80°C prior to analysis. The relative percentages of the major, acidic (APG for acidic peak group) and basic (BPG for basic peak group) isoforms of purified mAbs were determined by imaging capillary electrophoresis (ProteinSimple iCE3). The levels of aggregates (HMWS), monomers, and fragments (LMWS) of purified mAbs were determined by size exclusion chromatography (SE-UPLC) or protein A HPLC gradient.

[0142] Example 1 - High feed regimen, feed addition pattern and specific power input in seed bioreactors affect production Cell culture performance in bioreactor (mAb1)

[0143] In this experiment, 5 N-1 2L seed bioreactors (SBR) were fed with 0.35x10 6 CHO cells producing mAb-1 were inoculated at an inoculum density of 10 cells / mL and various conditions of feeding pattern, daily feeding addition duration and specific power input were tested (see Table 1) for 5 days. 6 Fifteen production bioreactors (PBRs) were inoculated at a density of 1 cell / mL (see Table 1 and Fig. 9 The aim was to evaluate the effect of different parameters in the seed bioreactor, namely the amount of feed added, the mode of addition (bolus or continuous) and the specific power input on the growth of cells in the production bioreactor. For some conditions, the specific power input in the production bioreactor was also varied.

[0144] Table 1 - Experimental conditions of Example 1

[0145]

[0146]

[0147] Under all conditions tested, comparable cell growth was observed during the N-1 phase, with a maximum VCC of approximately 14x10 6 cells / mL( Figure 1 A). Figure 1 B (i.e., cumulative IVCC) and Figure 3 The results shown in A(VCC) highlight that cell growth in the production bioreactor (N phase) was lower under the following conditions:

[0148] - the feed added in the seed bioreactor (N-1 stage) is lower,

[0149] - lower specific power input in the seed bioreactor and / or

[0150] -Continuous mode of adding main feed in seed bioreactor.

[0151] On the contrary, Figure 2 As shown, the unit power input in the subsequent production bioreactor has no significant effect on the cell growth in the production bioreactor.

[0152] The effect on titer is similar to the effect on cell growth, i.e., titer is lower under the following conditions:

[0153] - the feed added in the seed bioreactor (N-1 stage) is lower,

[0154] - lower specific power input in the seed bioreactor and / or

[0155] -Continuous mode of adding main feed to seed bioreactor (see Figure 3 B).

[0156] This example demonstrates that by culturing mammalian cells in an N-1 bioreactor operating in fed-batch mode under specific conditions selected from i) a specific pattern and duration of addition of at least one feed, ii) controlling the total amount of one or more feeds to be added, and / or iii) controlling at least one engineering parameter, such as specific power input, higher cell growth and increased ultimate productivity can be obtained in an N bioreactor (see Figure 4 A-4C). In other words, by controlling the feed pattern and duration, the total amount of feed added and / or the specific power input during the N-1 phase, the growth of mammalian cells in the N bioreactor can be improved and the production yield of recombinant proteins expressed by the mammalian cells can be increased.

[0157] For unit power input (P / V), given the results in this example, the P / V applied in the seed bioreactor appears to have a significant impact on cell culture production. More specifically, the difference between the P / V in the seed bioreactor and the production bioreactor (the P / V in the SBR is higher than the P / V in the PBR) remains a potential factor affecting cell culture production performance. Although not restrictive, the results show that a ratio of P / V (SBR): P / V PBR of at least 1.20:1 can achieve higher cell culture performance.

[0158] Example 2 - Feed addition pattern in the seed bioreactor affects cell culturing performance in the production bioreactor Can (mAb1)

[0159] In this experiment, 0.35x10 6 CHO cells producing mAb1 were inoculated at a density of 10 cells / mL and continued for 6 days under different conditions (see Table 2). Then, the fed-batch process described in Materials and Methods was followed at a density of 3.0×10 6 From these N-1 bioreactors, 8x2 L production bioreactors (PBRs) were inoculated at an inoculum density of 10 cells / mL. TM 7a and Cell Boost TM 7b duration under various conditions (see Table 2).

[0160] Table 2 - Experimental conditions in the seed bioreactor (N-1 stage) of Example 2

[0161]

[0162]

[0163] exist Figure 5 In A, we can observe comparable cell growth curves in the seed bioreactors under different test conditions, while Figure 6 A shows that the cell growth in the production bioreactor was lower under the condition of longer feed addition in the seed bioreactor. The cumulative IVCC in the N bioreactor is shown in Figure 5 B. The results show that the longer the feed addition duration in the seed bioreactor, the lower the cell growth in the production bioreactor. We can observe that the effects of CB7a and CB7b are cumulative. At the end of 14 days of production, both Cell boost 7a and 7b showed lower final titers with longer feed addition duration ( Figure 6 B).

[0164] This example confirms the results obtained in Example 1, namely that by controlling the feeding pattern and duration of at least one feed during the N-1 phase, the growth of mammalian cells in the N bioreactor can be improved and the production yield of recombinant proteins expressed by the mammalian cells can be increased.

[0165] Example 3 - Feed addition pattern in the seed bioreactor affects cell culturing performance in the production bioreactor Can (mAb2)

[0166] In this experiment, 0.35x10 6 CHO cells producing mAb2 were inoculated at a density of 10 cells / mL and continued for 6 days under different conditions (see Table 3). Following the fed-batch process described in Materials and Methods, 3.75×10 6 CHO cells producing mAb2 were inoculated into 13x2 L production bioreactors (PBR) at an inoculation density of 10 cells / mL. In this experiment, multiple conditions with different modes of FM1 addition were tested in the seed bioreactor.

[0167] Table 3 - Experimental conditions in the seed bioreactor (N-1 stage) of Example 3

[0168]

[0169] Cell growth curve Figure 7 shown. Figure 7 The results shown in A show that cell growth in the production bioreactor was lower under conditions where feed addition duration was longer in the seed bioreactor.

[0170] Under the condition of longer feed addition duration in the seed bioreactor, the final product titer at the end of 14 days of production ( Figure 7 B) shows a lower final productivity of the production bioreactor. It can be observed that a shorter duration of FM1 addition in the seed bioreactor significantly affects cell growth and final productivity in the production bioreactor. This confirms that the mode and duration of feed addition in the seed bioreactor (preferably within ≤ 3 hours) has a significant positive impact on increasing cell growth and product titer in the production bioreactor, thus validating the results of Examples 1 and 2, but using another cell clone (producing a different antibody) and another medium platform.

[0171] Example 4 - Feeding amount added in the seed bioreactor affects cell culture in the production bioreactor Performance (mAb2)

[0172] In this experiment, 0.35x10 6CHO cells producing mAb2 were inoculated at a density of 10 cells / mL and continued for 6 days under different conditions (see Table 4). Following the fed-batch process described in Materials and Methods, 3.75×10 6 CHO cells producing mAb2 were inoculated into 4x2 L production bioreactors (PBRs) at an inoculation density of 10 cells / mL. In this experiment, various conditions were evaluated with different amounts of feed added to the seed bioreactor. Moreover, the specific power input in the seed bioreactor was set to a low value so that it could be evaluated whether a large amount of feed in the seed bioreactor could balance the fact that the specific power input was reduced. For the same purpose, feed was added in a continuous mode in the seed bioreactor.

[0173] Table 4 - Experimental conditions in the seed bioreactor (N-1 stage) of Example 4

[0174]

[0175] Cell growth curve (in production bioreactor) such as Figure 8 Figure 1 is a graph showing that a higher amount of feed added in the seed bioreactor compared to the control condition (about 5.85% vs. about 4.20% total amount of feed added per cell culture starting volume in the seed bioreactor, w / w) resulted in increased cell growth in the production bioreactor, even though the specific power input in the SBR was less than 90 W / m 3 And FM1 was added to the seed bioreactor in continuous mode. In addition, at the end of 14 days of production, the final product titer ( Figure 8 B) shows that the final productivity in the production bioreactor is higher under conditions of higher feed added to the seed bioreactor compared to the control condition (5.85% vs. 4.20%). This confirms the conclusion drawn in the previous example that controlling the total amount of feed per culture starting volume in the seed bioreactor (e.g., to above about 3.50%) leads to higher cell growth and an increase in final productivity.

[0176] Example 5 - Engineering parameters affect cell culture performance in a production bioreactor (mAb1)

[0177] As shown in Example 1, the difference between P / V in the seed bioreactor and the production bioreactor is a potential factor affecting cell culture production performance: conditions with high P / V in the seed bioreactor coupled with lower P / V in the production step lead to better cell culture performance.

[0178] To verify this finding, a 400 L seed bioreactor (SBR) was set up with 0.35 x 10 6CHO cells producing mAb1 were inoculated at a density of 10 cells / mL for 6 days under two conditions (see Table 5). Then, the cells were fed in batches at 3.0×10 6 The cells were inoculated into a 2000 L production bioreactor (PBR) at an inoculation density of 10 cells / mL. In this experiment, two conditions were tested (see Table 5).

[0179] Table 5 - Experimental conditions in the bioreactor of Example 5

[0180]

[0181] These new experiments confirm the findings of Example 1. Fig.10 As shown (depicting cell culture performance in a PBR), when the SBR was operated at 32.8 W / m associated with a higher P / V in the PBR 3 The cell growth and product formation in the production bioreactor were lower when the P / V was lower than that in the SBR. 3 Compared with the conditions with a P / V of 40,000, the lactate production and glucose consumption rates were higher under these conditions.

[0182] Without being bound by any theory, the environment of transferring cells from a bioreactor with higher mechanical stress to a production bioreactor with lower mechanical stress may still be one of the most critical factors. If cells are subjected to greater mechanical stress in the production bioreactor, they are more likely to show growth arrest or not grow at all.

[0183] References

[0184] 1) Hecklau C et al. (2016) J Biotech, 218: 53-63

[0185] 2) Zang Li et al. (2011) Anal. Chem, 83: 5422-5430

[0186] 3) Grammatikos et al. (1999) Biotechnol. Bioeng., 5; 64(3): 357-67

[0187] 4)Ozturk&Hu(2005)Cell Culture Technology for Pharmaceutical and Cell-Based Therapies,ed.CRC Press

[0188] 5)Roustan M.(2005)Engineering Techniques.Article No.J3802

[0189] 6)WO98 / 08934

[0190] 7)US2006 / 0148074

[0191] 8)WO2015 / 197772

Claims

1. A method for producing a recombinant protein in a production bioreactor, wherein the method comprises: a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding the recombinant protein, b. culturing the mammalian cells in the N-1 bioreactor operated in a fed-batch mode under specific conditions selected from: i. dosing or at least a specific mode and duration of dosing, ii. controlling the total amount of one or more feedstocks to be added, and / or iii. Control at least one engineering parameter, c. Using the cells obtained from step (b) at a concentration of at least 2.00 x 10 6 The N bioreactor was inoculated at an inoculation density of viable cells / ml; d. culturing the cells in the N bioreactor under conditions allowing production of the recombinant protein, e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

2. A method for improving the growth of mammalian cells in a production bioreactor, wherein the method comprises: a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding the recombinant protein, b. culturing the mammalian cells in the N-1 bioreactor operated in a fed-batch mode under specific conditions selected from: i. adding a feed or a specific mode and duration of at least one feed, ii. controlling the total amount of one or more feedstocks to be added, and / or iii. Control at least one engineering parameter, c. Using the cells obtained from step (b) at a concentration of at least 2.00 x 10 6 The N bioreactor was inoculated at an inoculation density of viable cells / ml; d. culturing the cells in the N bioreactor under conditions allowing production of the recombinant protein, e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

3. A method for increasing the yield of a recombinant protein expressed by cultured mammalian cells in a production bioreactor, wherein the method comprises: a. inoculating an N-1 bioreactor with mammalian cells comprising a gene encoding the recombinant protein, b. culturing the mammalian cells in the N-1 bioreactor operated in a fed-batch mode under specific conditions selected from: i. dosing or at least a specific mode and duration of dosing, ii. controlling the total amount of one or more feedstocks to be added, and / or iii. Control at least one engineering parameter, c. Using the cells obtained from step (b) at a concentration of at least 2.00 x 10 6 The N bioreactor was inoculated at an inoculation density of viable cells / ml; d. culturing the cells in the N bioreactor under conditions allowing production of the recombinant protein, e. and optionally harvesting the recombinant protein, purifying the recombinant protein and formulating the recombinant protein.

4. The method according to any one of the preceding claims, wherein the feed of step (b) is added daily, and wherein the feed is added on day 0 of cultivation or thereafter.

5. A method according to any one of the preceding claims, wherein: i) at least the primary feed of step (b) is added daily in bolus mode, and / or ii) at least one secondary feed of step (b), if any, is added daily in bolus mode.

6. The method according to claim 5, wherein: i) at least the main feed of step (b) is added in bolus mode within about 3 hours or less, within about 2 hours or less, or within about 1 hour or less per day, and / or ii) at least one secondary feed of step (b), if any, is added in bolus mode within about 3 hours or less, within about 2 hours or less, or within about 1 hour or less per day.

7. A method according to any one of the preceding claims, wherein all feeds of step (b) are added daily in bolus mode.

8. The method of claim 7, wherein all feeds of step (b) are added daily in bolus mode within about 3 hours or less, within about 2 hours or less, or within about 1 hour or less.

9. A method according to any one of the preceding claims, wherein the at least one engineering parameter controlled during step (b) is specific power input.

10. The method according to claim 9, wherein the unit power input is controlled daily, and wherein the unit power input in step (b) should reach at least 100 W / m 3 .

11. A method according to any one of the preceding claims, wherein the total amount of feed added during step (b) is higher than about 3.5%, but preferably does not exceed 30%, per the starting volume of the culture.

12. The method according to any one of the preceding claims, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

13. The method according to any one of the preceding claims, wherein the recombinant protein is a cytokine, a growth factor, a hormone, an antibody or a fusion protein.

14. The method according to any one of the preceding claims, wherein the method is performed on a large scale.

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