Method for producing recombinant proteins

Abstract

The present disclosure relates to methods of producing recombinant proteins. More specifically, the present disclosure relates to methods of making biologies, such as monoclonal antibodies, fusion proteins, or antigen-binding fragments thereof. In particular, the present disclosure relates to the use of methods and feed strategies that improve cell viability and target protein productivity and reduce host cell protein impurities in such manufacturing processes. More specifically, the present disclosure relates to methods of producing tiragolumab.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of Indian application No. 202321079652, filed on November 23, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application contains a sequence list that has been submitted electronically and is hereby incorporated in its entirety by reference. The sequence list submitted with this application is contained in an XML file created on November 21, 2024, with the title “23-1088-WO_Sequence-Listing.xml” and a size of 8,451 bytes. Technical Field

[0005] This disclosure relates to methods for producing recombinant proteins. More specifically, this disclosure relates to methods for preparing biological products such as monoclonal antibodies, fusion proteins, or antigen-binding fragments thereof. In particular, this disclosure relates to methods and feeding protocols for improving cell viability and target protein yield in such manufacturing processes and reducing host cell protein impurities. More specifically, this disclosure relates to a method for producing tildrakizumab. Background Technology

[0006] Recombinant DNA technology is used to produce quantities of proteins that can be used in a range of therapeutic applications. A key task in recombinant protein production is optimizing cell culture feeds and conditions to achieve high protein titers using the most efficient production methods. Any improvement, including incremental improvements, can have significant economic benefits. In the pharmaceutical industry, optimizing protein production for biologics used in therapies to treat diseases is advantageous because any improvement can have a significant impact on productivity when manufacturing biologics at scale. Therefore, there remains a need in the art to maximize protein production from cell cultures expressing biological proteins intended for medical use. Typically, mammalian cell culture feeds are based on commercially available feed formulations. However, these feed formulations are often not optimally enriched to enhance biological protein expression.

[0007] Host cell proteins (HCPs) are complex mixtures of proteins with varying physicochemical properties. Some HCPs are considered high-risk. High-risk HCPs include those with immunogenicity and enzymatic activity, which have the potential to degrade product molecules or excipients used in formulations. Therefore, HCPs are considered critical quality attributes (CQAs) and thus require monitoring during process changes.

[0008] Improvements in antibody titers can be achieved through optimization of culture conditions. Variations in culture conditions can adversely affect the profile of high-risk HCPs. For example, changes in culture medium, feed strategy, process duration, peak cell density, and agitation rate can affect cell viability, ultimately leading to significantly different HCP compositions. Therefore, it is important to develop a process designed to eliminate high-risk HCPs.

[0009] The pharmaceutical industry requires easily produced proteins for use in a range of applications as part of therapeutic research. Commercially viable manufacturing processes are needed to maximize the yield of recombinant proteins produced most efficiently. Therefore, there remains a need for improved cell culture supplements and cell culture methods to maximize protein production. Summary of the Invention

[0010] This disclosure relates to methods for producing recombinant proteins. More specifically, this disclosure relates to methods for preparing biological products such as monoclonal antibodies, fusion proteins, or antigen-binding fragments thereof. In particular, this disclosure relates to feed preparation methods and feed protocols for improving cell viability and protein productivity and reducing host cell protein impurities in such manufacturing processes. More specifically, this disclosure relates to methods for producing tirekizumab.

[0011] Therefore, this disclosure relates to methods for significantly improving the yield of monoclonal antibody titers in commercial production. More specifically, this disclosure relates to methods including improving cell culture feed to improve monoclonal antibody productivity and cell viability at the harvest stage of the process. Increased viability during cell culture reduces the amount of HCP in the cell-free supernatant. This is a result of higher cell viability, which therefore affects cell lysis during culture, leading to lower HCP content at harvest.

[0012] This disclosure provides a manufacturing method for improving cell culture density, viability, and productivity through a combination of feedstocks. This disclosure provides highly advantageous feedstocks because they contain a combination of nutrients in a single formulation. This disclosure provides a method and feedstock that nearly doubles the harvest titer compared to standard processes used for producing recombinant proteins.

[0013] This article provides a method for producing an anti-IL-23p19 antibody, the method comprising: (a) preparing a cell culture by seeding cells capable of expressing a recombinant protein into a container containing a first culture medium; (b) culturing the cells; (c) lowering the temperature of the cell culture; (d) adding a second culture medium to the cell culture; (e) culturing the cells; (f) harvesting the cells from the cell culture; and (g) isolating the recombinant protein from the harvested cells.

[0014] This article also provides a method for producing anti-IL-23p19 antibody, the method comprising: (a) preparing and scaling up seed cell cultures capable of expressing recombinant proteins in a suitable culture medium; (b) inoculating and culturing the seed cell cultures in a suitable production culture medium to prepare cell cultures; (c) lowering the temperature of the cell cultures; (d) periodically adding feed to the cell cultures; (e) culturing the cells; (f) harvesting the cells from the cell cultures; and (g) isolating the recombinant protein from the harvested cells.

[0015] These and other features and advantages of this disclosure will be more fully understood from the following specific embodiments in conjunction with the appended claims. It should be noted that the scope of the claims is defined by the statements therein, and not by the specific discussion of the features and advantages set forth in this specification. Attached Figure Description

[0016] Figure 1 This compares the viable cell density (VCD) during a 22-day incubation period between the standard and improved processes (10). 6 Line graph (cells / mL).

[0017] Figure 2 It is a line graph comparing cell viability (expressed as a percentage) with incubation time (in days) between standard and improved processes.

[0018] Figure 3 It is a line graph comparing the productivity / titer produced in mg / L with incubation time (in days) between the standard process and the improved process.

[0019] Figure 4 It is a bar chart comparing the final titers (in mg / L) produced by the standard process and the improved process.

[0020] Figure 5 The live cell density between the standard process and the improved process (10) 6 A line graph comparing the changes in cells / mL with incubation time (in days).

[0021] Figure 6 This is a line graph comparing cell viability (expressed as a percentage) with incubation time (in days) between the standard and improved processes.

[0022] Figure 7 It is a line graph depicting the change in productivity or titer, measured in mg / L, between standard and improved processes over incubation time (in days).

[0023] Figure 8 This is a bar chart comparing the final titers produced in mg / L between the standard process and the improved process.

[0024] Figure 9 This compares the viable cell density (10⁻⁶) between feed every other day (feeding at 2% v / v post-inoculation volume) and feed every 4 days (feeding at 4% v / v post-inoculation volume). 6 Line graph showing the change in cells / mL over incubation time (in days).

[0025] Figure 10 It is a line graph depicting the change in cell viability (expressed as a percentage) with incubation time (in days) between the improved process using 2% v / v or 4% v / v seeding volume for every other day feeding and feeding every 4 days.

[0026] Figure 11 It is a line graph depicting the change in productivity or titer per mg / L with incubation time (in days) between feeding every other day and feeding every 4 days after inoculation at 2% v / v or 4% v / v.

[0027] Figure 12 This is a bar chart showing the productivity or titer generated in mg / L between improved processes using feed every other day or feed every 4 days (feed volume of 2% v / v or 4% v / v post-inoculation volume).

[0028] Figure 13 This compares the viable cell density (10⁻¹⁰) between the improved process (separate feeding) and the improved process using combined feeding. 6 Line graph showing the change in cells / mL over incubation time (in days).

[0029] Figure 14 It is a line graph depicting the change in cell viability (expressed as a percentage) over incubation time (in days) between the improved process (separate feeding) and the improved process using combined feeding.

[0030] Figure 15 It is a line graph measuring the change in productivity or titer in mg / L with incubation time (in days) between an improved process (separate feeding) and an improved process using combined feeding.

[0031] Figure 16 It is a bar chart depicting a comparison of productivity or titer in mg / L between the improved process (separate feed and individual feeding) and the improved process using combined feeding.

[0032] Figure 17 This compares the viable cell density (10⁻¹⁰) achieved by feeding combinations with intensities of 1.25X, 1.5X, or 1.75X. 6 Line graph showing the change in cells / mL over incubation time (in days).

[0033] Figure 18 It is a line graph depicting the change in cell viability (expressed as a percentage) over incubation time (in days) achieved by feeding combinations at intensities of 1.25X, 1.5X, or 1.75X.

[0034] Figure 19 It is a line graph depicting the change in productivity as a function of incubation time (in days) achieved by feeding with a combination of feed intensities of 1.25X, 1.5X, or 1.75X.

[0035] Figure 20 It is a bar chart comparing the final titer achieved by feeding with feed combinations of 1.25X, 1.5X, or 1.75X intensity as a function of incubation time (in days).

[0036] Figure 21 The live cell density (10⁻¹⁰) of the standard and improved processes was depicted. 6 Line graph showing the change in cells / mL over incubation time (in days).

[0037] Figure 22 Line graphs depicting the change in cell viability (expressed as a percentage) with incubation time (in days) for standard and improved processes.

[0038] Figure 23 It is a line graph showing how the productivity of standard and improved processes changes with incubation time (in days).

[0039] Figure 24 A bar chart depicts the harvest productivity of the standard process and the improved process.

[0040] Figure 25 The live cell density (10⁻¹⁰) of the standard process and the improved process was depicted. 6 The variation of cells / mL with incubation time (in days).

[0041] Figure 26 The cell viability (expressed as a percentage) of both the standard and improved processes was depicted as a function of incubation time (in days).

[0042] Figure 27 The productivity (mg / L) of the standard and improved processes was depicted as a function of incubation time (in days).

[0043] Figure 28 Harvest productivity (mg / L) for both standard and improved processes is described.

[0044] Figure 29 The live cell density (10⁻¹⁰) was depicted using the improved processes of 1X Cell Boost and 2X Cell Boost. 6Changes in cells / mL over incubation time (in days).

[0045] Figure 30 The percentage of cell viability using the improved process with 1X Cell Boost and Feed 4 versus 2X Cell Boost and Feed 4 was depicted as a function of incubation time (in days).

[0046] Figure 31 Line graphs depict the change in productivity (mg / L) as a function of incubation time (in days) for production using the improved processes of 1X Cell Boost and Feed 4 or 2X Cell Boost and Feed 4.

[0047] Figure 32 A bar chart depicts the harvest productivity (mg / L) using 1X Cell Boost 5 versus using an improved process with 2X Cell Boost 5 and 1X Feed 4.

[0048] Figure 33 Viable cell densities (10⁻⁶) were depicted using improved processes of 2X Cell Boost 5 with 0.5%, 0.6%, or 1.0% v / v and 1X Feed 4 with 1%, 1.2%, or 1.5% v / v. 6 The variation of cells / mL with incubation time (in days).

[0049] Figure 34 Line graphs depict the percentage of viability as a function of incubation time (in days) for improved processes using 2X Cell Boost 5 with 0.5%, 0.6%, or 1.0% v / v and 1X Feed 4 with 1%, 1.2%, or 1.5% v / v.

[0050] Figure 35 Line plots depict the productivity (mg / L) of the improved processes using 2X Cell Boost 5 with 0.5%, 0.6%, or 1.0% v / v and 1X Feed 4 with 1%, 1.2%, or 1.5% v / v as a function of incubation time (in days).

[0051] Figure 36 A bar chart depicts the harvest productivity of the improved processes using 2X Cell Boost 5 with 0.5%, 0.6%, or 1.0% v / v and 1X Feed 4 with 1%, 1.2%, or 1.5% v / v.

[0052] Figure 37 It is a flowchart depicting the entire downstream process (DSP) responsible for purification.

[0053] Figure 38 A comparison of common and unique HCP detection methods between standard and modified processes is described.

[0054] Figure 39 This study describes a comparison of common and unique HCP detection between standard and modified processes following the viral inactivation step of tirekizumab.

[0055] Figure 40 This study describes a comparison of common and unique HCP detection between standard and modified processes following cation exchange output of tirekizumab.

[0056] Figure 41 This study describes a comparison of the detection of common and unique HCPs between standard and modified processes after mixed-mode chromatography (MMC) output of tirekizumab.

[0057] Figure 42 This indicates downstream removal of high-risk HCPs during each unit operation of tirecizumab. Detailed Implementation

[0058] This disclosure relates to methods for producing recombinant proteins. More specifically, this disclosure relates to methods for preparing biological products such as antibodies or antigen-binding fragments thereof. In particular, this disclosure relates to feed preparation methods and feed protocols for achieving final protein yield in such manufacturing processes. More specifically, this disclosure relates to methods for producing tgrijujube.

[0059] More specifically, this disclosure relates to methods for significantly improving the yield of monoclonal antibody titers in commercial production. Even more specifically, this disclosure relates to methods including improving cell culture feeds and combinations thereof to improve the productivity of the produced monoclonal antibodies and the viability of the cultures at harvest.

[0060] As used in accordance with this disclosure, unless otherwise indicated or defined, all technical and scientific terms used herein should be understood to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. The following references provide general definitions for many terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale and Marham, The HarperCollins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the meanings assigned to them below.

[0061] Unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. For example, the terms “an” and “the” as used herein shall be understood as either singular or plural unless the context explicitly states otherwise. It should be understood that, unless the context otherwise indicates or specifies, the terms “an” and “a” as used herein mean “one or more” of the listed components. Unless otherwise specified, alternatives ( For example The use of “or” should be understood to mean any one, two, or any combination of the alternatives. Therefore, unless otherwise stated or obvious from the context, the term “or” as used herein should be understood as inclusive.

[0062] As used herein, the terms “comprise” and “comprising” may have the meanings conferred upon them under U.S. patent law and may mean “include,” “including,” “containing,” “having,” etc. Therefore, unless otherwise expressly stated, the terms “comprise” and “comprising” as used herein indicate that additional components or members may optionally be present in addition to the components or members of the list introduced by “comprising.” As used herein, the terms “consisting essentially of” or “consists essentially” also have the meanings conferred upon them under U.S. patent law and allow for the presence of more than those described, provided that the essential or novel features of the described ones are not altered by the presence of the additional ones.

[0063] Any of the methods or compositions provided herein may be combined with one or more of any other methods or compositions provided herein.

[0064] In this disclosure, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range, and, where appropriate, to include fractions (such as tenths and hundredths of an integer). The ranges provided herein shall be understood as abbreviations of all values ​​within that range. For example, a range of 1 to 50 shall be understood to include any number, combination of numbers, or subrange from the following groups: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0065] Unless specifically stated or obvious from the context, the terms “about” and “approximately” as used herein are understood to mean within normal tolerances in the art, such as within two standard deviations of the average, or within the acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. “About” can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term “about”.

[0066] It should be noted that terms such as “preferredly,” “commonly,” and “typically” are not intended herein to limit the scope of the claimed subject matter or to imply that certain features are critical, necessary, or even important to the structure or function of the claimed subject matter. Rather, these terms are intended only to highlight alternative or additional features that may or may not be used in specific embodiments of this disclosure.

[0067] For purposes of description and limitation of this disclosure, it should be noted that the term "substantially" is used herein to indicate the degree of inherent uncertainty attributable to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also used herein to indicate the degree to which a quantitative representation can be varied from the stated reference without causing a change in the fundamental function of the subject matter under discussion.

[0068] In some implementations, the method for producing anti-IL-23p19 antibody involves: (a) preparing a cell culture by seeding cells capable of expressing the recombinant protein into a container containing a first culture medium, (b) culturing the cells, (c) lowering the temperature of the cell culture, (d) adding a second culture medium to the cell culture, (e) culturing the cells, (f) harvesting the cells from the cell culture, and (g) isolating the recombinant protein from the harvested cells.

[0069] In some implementations, the method for producing anti-IL-23p19 antibody involves: (a) preparing and scaling up seed cell cultures capable of expressing recombinant proteins in a suitable culture medium; (b) inoculating and culturing the seed cell cultures in a suitable production culture medium to prepare cell cultures; (c) lowering the temperature of the cell cultures; (d) periodically adding feed to the cell cultures; (e) culturing the cells; (f) harvesting the cells from the cell cultures; and (g) isolating the recombinant protein from the harvested cells.

[0070] As used herein, the term "antibody" refers to a protein that recognizes and specifically binds to an antigen. Common or conventional mammalian antibodies comprise tetramers, which typically consist of two pairs of identical polypeptide chains, each pair consisting of a "light" chain (typically with a molecular weight of about 25 kDa) and a "heavy" chain (typically with a molecular weight of about 50–70 kDa). As used herein, the terms "heavy chain" and "light chain" refer to any immunoglobulin polypeptide having a variable domain sequence sufficient to confer specificity to the target antigen. The N-terminal portion of each light and heavy chain typically contains a variable domain of about 100 to 110 or more amino acids, which is typically responsible for antigen recognition. The C-terminal portion of each chain typically defines a constant domain responsible for effector function. Therefore, in naturally occurring antibodies, full-length heavy chain immunoglobulin peptides contain a variable domain (VH) and three constant domains (CH1, CH2, and CH3), as well as a hinge region between CH1 and CH2, wherein the VH domain is located at the amino terminus of the peptide and the CH3 domain is located at the carboxyl terminus. Similarly, full-length light chain immunoglobulin peptides contain a variable domain (VL) and a constant domain (CL), wherein the VL domain is located at the amino terminus of the peptide and the CL domain is located at the carboxyl terminus.

[0071] Within both the full-length light and heavy chains, variable and constant domains are typically linked by “J” regions of about 12 or more amino acids, with the heavy chain also containing “D” regions of about 10 or more amino acids. The variable regions of each light / heavy chain pair typically form antigen-binding sites. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FRs) linked by three hypervariable regions (also known as complementarity-determining regions or CDRs). The CDRs from the two chains of each pair are typically aligned by framework regions that enable binding to specific epitopes. From the amino terminus to the carboxyl terminus, both light and heavy chain variable domains typically include domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0072] As used herein, the term "antigen-binding fragment" refers to a portion of a complete antibody and / or to the antigen-determining variable domain of a complete antibody. The antigen-binding function of an antibody is known to be performed by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single-chain antibodies, bifunctional antibodies, and multispecific antibodies formed from antibody fragments.

[0073] In a specific implementation, the anti-IL-23p19 antibody hum13B8-b is tiridgezumab. As used herein, the term "tiridgezumab" refers to a humanized anti-IL-23p19 monoclonal antibody, also known as SCH 900222 or MK-3222. Tiridgezumab is a high-affinity (297 picomol concentration [pM]) humanized immunoglobulin G1 / κ (IgG1 / κ) antibody. The antibody specifically binds to the p19 protein of the IL-23 heterodimer, but does not bind to human IL-12 (IL-12 / 23p40 and IL12p35 heterodimers) or human IL-12 / 23p40.

[0074] In some embodiments, the anti-IL-23p19 antibody tiridgezumab may refer to ILUMYA®. In some embodiments, it is a formulation containing 100 mg of tiridgezumab ( Right now Tigrisuzumab is prepared in a 1 mL single-dose pre-filled syringe (100 mg / mL). In some embodiments, ILUMYA® (tigrisuzumab-asmn) injection for subcutaneous use is a sterile, clear to slightly milky white, colorless to slightly yellow solution. ILUMYA® is provided in a single-dose pre-filled syringe with a glass cylinder and a 29-gauge fixed 1 / 2-inch needle. In some embodiments, tigrisuzumab may be prepared in water for injection containing L-histidine, L-histidine hydrochloride monohydrate, polysorbate 80, and / or sucrose at a pH of 5.7–6.3. In some embodiments, tirekizumab is prepared in a 1 mL single-dose prefilled syringe containing 100 mg of tirekizumab-asmn prepared from the following: L-histidine (0.495 mg), L-histidine hydrochloride monohydrate (1.42 mg), polysorbate 80 (0.5 mg), sucrose (70.0 mg), and water for injection, USP, pH 5.7-6.3.

[0075] In a particular embodiment, tirekizumab comprises a light chain polypeptide containing the amino acid sequence SEQ ID NO: 1 and a heavy chain polypeptide containing the amino acid sequence SEQ ID NO: 2, disclosed in U.S. Patent Nos. 8,404,813 and 8,293,883, the disclosures of which are incorporated herein by reference in their entirety. In other embodiments, tirekizumab or its antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises the CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 3-5, and wherein the light chain variable domain comprises the CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 6-8.

[0076] Hum13B8-b light chain (SEQ ID NO: 1)

[0077] DIQMTQSPSSLSASVGDRVTITCRTSENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGIPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0078] Hum13B8-b heavy chain (SEQ ID NO: 2)

[0079] QVQLVQSGAEVKKPGASVKVSCKASGYIFITYWMTWVRQAPGQGLEWMGQIFPASGSADYNEKFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGGGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0080] Hum13B8-b heavy chain CDR1 (SEQ ID NO: 3)

[0081] GYIFITYWMT

[0082] Hum13B8-b heavy chain CDR2 (SEQ ID NO: 4)

[0083] QIFPASGSADYNEKFE

[0084] Hum13B8-b heavy chain CDR3 (SEQ ID NO: 5)

[0085] GGGGFAY

[0086] Hum13B8-b light chain CDR1 (SEQ ID NO: 6)

[0087] RTSENIYSYLA

[0088] Hum13B8-b light chain CDR2 (SEQ ID NO: 7)

[0089] NAKTLAE

[0090] Hum13B8-b light chain CDR3 (SEQ ID NO: 8)

[0091] QHHYGIPFT

[0092] In some embodiments of the method for producing recombinant proteins disclosed herein, the step of preparing a cell culture by seeding cells capable of expressing the recombinant protein into a container containing a first culture medium may include preparing and scaling up seed cell cultures capable of expressing the recombinant protein in a suitable culture medium. Suitable culture media used in the methods of this disclosure may include any culture medium described herein or known in the art.

[0093] In some embodiments of the method for producing recombinant proteins disclosed herein, the step of preparing a cell culture by seeding cells capable of expressing the recombinant protein into a container containing a first culture medium may further include seeding and culturing the seed cell culture in a suitable production culture medium. Suitable production culture media used in the methods of this disclosure may include any culture medium described herein or known in the art.

[0094] In other embodiments of the method for producing recombinant proteins disclosed herein, the step of adding a second culture medium to the cell culture may include periodically adding feed to the culture. The cell culture feed or tonic used in the methods of this disclosure may include any cell culture feed or tonic described herein or known in the art.

[0095] 1. Methods and compositions

[0096] Standard process

[0097] The standard process for producing recombinant proteins involves using a basal medium, determined by any suitable chemical composition, in a shake flask (or other suitable container), containing 0.3 x 10⁻⁶ ppm. 6 Up to 0.9 x 10 6Initially seed with 100 viable cells / mL, followed by a volume of approximately 100 mL / 500 mL container. Suitable containers include 500 mL shake flasks or 5 L bioreactors. Add L-glutamine to the culture as needed, and begin adding a fixed volume of amino acids to the culture on day 3. When the viable cell count reaches 1.0 x 10⁻⁶ cells / mL... 6 Up to 3.0 x 10 6 When the cell / mL ratio is reached, reduce the process temperature. Add a fixed volume of appropriate feed medium on days 3, 7, 11, and 15 of the fed-batch process. Add a fixed volume of nutrient feed solution on day 9. Harvest the flask when culture viability drops below 75% or on day 22 of the biological process (whichever comes first).

[0098] Regulation of live cell valley density

[0099] When the live cell density reaches 1.0 x 10 6 Up to 3.0 x 10 6 The temperature transition from a standard process to a fed-batch bioprocess begins at 100 viable cells / mL or within 72 hours (whichever occurs first). In the improved process, in addition to 2.5 x 10⁻⁶... 6 A higher viable cell density of [number] cells / mL replaces the 1.0 x 10 [units] in the standard process. 6 Up to 3.0 x 10 6 Apart from the cell / mL ratio, the standard remains the same. In some embodiments of the improved process of this disclosure, the temperature of the cell culture is reduced by 1°C to 4°C. In some embodiments of the improved process of this disclosure, the temperature of the cell culture is reduced by 1°C to 3°C. In a particular embodiment of the improved process of this disclosure, the temperature of the cell culture is reduced by 1°C to 2°C.

[0100] Improved cell feeding

[0101] Standard processes for producing recombinant proteins face challenges such as low productivity (approximately 1300 mg / L) and low cell viability at harvest. A key challenge in developing improved processes for recombinant protein production is increasing productivity and maintaining high cell viability later in the process. The underlying principle behind the new feed addition is to improve the productivity of the standard process.

[0102] To improve the productivity of the standard process, two improved feeds were introduced into the bioprocess: Cell Balan CDCHO Feed 4 (Feed 4) and Cell Boost 5 (CB5). These feeds can be prepared and added independently (alone or separately) or in combination (combined). Details of the feeds used, along with feed intensity, number of days of addition, and feed volume, are summarized in the table below: Table 1: Option A - Separate Feed 4 and CB5 preparations; added separately

[0103] Table 2: Combined feed prepared by mixing two feed ingredients (1.5X)

[0104] Table 3: Combined feed prepared by mixing two feed ingredients (1.0X)

[0105] Table 4: Individual Feed 4 and CB5 preparations; added separately

[0106] Table 5: Individual Feed 4 and CB5 preparations; added separately

[0107] Improve harvest productivity

[0108] The standard process produces a titer of approximately 1300 mg / L at harvest. In some embodiments of the improved process disclosed herein, the process provides relatively high activity and an increased titer of 2000 mg / L to over 10000 mg / L at harvest. In some embodiments of the improved process disclosed herein, the process provides relatively high activity and an increased titer of 2000 mg / L to 5000 mg / L at harvest. In a particular embodiment of the improved process disclosed herein, the process provides relatively high activity and an increased titer of at least 2000 mg / L at harvest.

[0109] 2. Example

[0110] Example 1: Feeding Selection

[0111] The standard process produces titers of approximately 1.0 to 1.3 g / L. To improve the production yield of the standard process, commercially available feedstocks were evaluated. The following feedstocks were considered for feedstock evaluation: BalanCD CHO Feed 4 (Fujifilm-Irvine Scientific) and Cell Boost 5 (Cytiva). Starting from day 2, feedstock at 2% v / v post-inoculation volume was added every other day. Except for the introduction of feedstocks and the standard of higher VCD at temperature transition, the process parameters in this study remained similar to the standard process. Shake flasks were incubated at 36.5°C with shaking at 140 rpm in a CO2-controlled shaker incubator with 5% CO2 in the shaker. When the titer reached 2.5 x 10⁻⁶ g / L... 6Apply a temperature change to the culture at 1 cell / mL VCD or at 72 hours (whichever comes first).

[0112] Results: Shake-flask studies showed that productivity doubled when using BalanCD CHO Feed 4 and Cell Boost 5. The addition of feed supported higher viable cell densities over a 3-week production period. Figure 1 and Figure 21 ), and also maintain a higher percentage of cell viability until harvest ( Figure 2 and Figure 22 Furthermore, the increased productivity achieved through standard feeding is gradual compared to improved feeding. Figure 3 and Figure 23 The final protein yield was significantly higher than the standard process after the introduction of the new feedstock. Figure 4 and Figure 24 ).

[0113] Example 2: Standard Process and Improved Process

[0114] Based on the results obtained, the addition of the improved feedstock increased the productivity of the standard process. To further confirm the effectiveness of the improved feedstock, both the standard and improved processes were run in a 5L bioreactor. Except for the feedstock addition at temperature transitions and the VCD standard, the bioreactor process parameters remained identical in both programs.

[0115] Results: These findings indicate that the introduction of the novel feedstock improved protein yield. A significant difference in viable cell density was observed between the standard and improved processes after the first two days of cell inoculation. The viable cell density was significantly higher than the standard process, sometimes reaching up to twice the cell count in certain cases. Figure 5 and Figure 25 During the process improvement, the percentage of cell viability was generally higher throughout the production period. Figure 6 and Figure 26 Compared to the standard process, the improved process has higher productivity, especially in the initial stages when titer levels are similar. Figure 7 and Figure 27 However, with the introduction of improved feed, the final titer nearly doubled, with a yield of 2157 mg / L compared to 1261 mg / L. Figure 8 and Figure 28 ).

[0116] Example 3: Optimization of Feed Addition Days

[0117] Preliminary studies on the new feedstock confirmed that adding feedstock significantly improved productivity. Feedstock was added to the bioreactor every other day, starting from day 2 of the bioprocess. To further investigate the effect of the feedstock addition protocol on protein productivity, shake flasks and the bioreactor were run every 4 days from day 2 with feedstock added at a rate of 4% v / v post-inoculation volume, whereas the standard process was run with a lower feedstock addition rate from day 2 of the protocol.

[0118] Result: Changing the feeding schedule from every other day to every 4 days resulted in similar cell densities in the production process. Figure 9 However, the percentage of cell viability was lower than the standard feeding schedule. Figure 10 Compared to the improved process using every-other-day feeding, less protein (mg / L) accumulated during the production period. Figure 11 Finally, compared to the alternating-day schedule, the total harvest yield increased only minimally with the 4-day feeding schedule. Figure 12 ).

[0119] Example 4: Concentrated Feeding Composition

[0120] When the feed additives BalanCD CHO Feed 4 (112 g / L) and Cell boost 5 (35 g / L) are added independently, the resulting combination results in a high volume of feed additive in this process. Therefore, blending feed additives at higher concentrations is the next step in developing effective feed additive formulations. The concentrated combination feed additive containing 112 g / L Feed 4 and 35 g / L CB5 is referred to as the 2X feed additive.

[0121] Results: Shake flask studies were conducted using either the individual feed or a combination of 2X concentrated feeds. Interestingly, despite higher nutrient density compared to high-volume mixed feeds, the combined feeds did not improve cell density, cell viability, or productivity. Viable cell density was significantly lower throughout the production process compared to adding the individual feed. Figure 13 The cell viability percentage was slightly lower but similar to the single-feed regimen. Figure 14 Compared to individual feeding methods, the daily protein titer was significantly lower in the combined feeding scheme within this process. Figure 15 The final titer after using combined feeding was about one-third lower than that after individual feeding. Figure 16 ).

[0122] Example 5: Optimizing Feed Concentration

[0123] Additional feed variants were prepared at feed intensities of 1.25X, 1.5X, or 1.75X. Flask and bioreactor studies were conducted to evaluate the effect of feed intensity adjustments on protein productivity. These feed combinations were added alternately at 2% v / v starting from day 2.

[0124] Results: Viable cell densities were found to be roughly similar across all three intensities throughout the production process, indicating that these intensities had no individual residual effect on cell density. Figure 17 Cell viability percentages were also similar, but it was found that viability was maintained slightly higher at 1.75X intensity for almost the entire process than at other intensity levels. Figure 18 Although similar during the production period, the 1.75X strength again exhibited a higher overall production period than the other two feed strengths. Figure 19 Finally, the 1.75X intensity yielded the best final yield of 2269 mg / L, representing an approximately 25% increase in yield compared to similar yields from feed intensities of 1.25X (1716 mg / L) and 1.5X (1681 mg / L). Figure 20 The resulting feed solution can increase volumetric production. A single, high-intensity feed is advantageous for batch protein production in cell culture because it reduces the amount of feed and the total volume of feed to be added.

[0125] Example 6: Purification of products from high-risk host cell proteins (HCP)

[0126] Chinese hamster ovary (CHO) cells were used to produce tirezumab. The process involved recovering the product from the production bioreactor to harvest cell culture medium (HCCF), followed by a series of downstream processing (DSP) steps. Figure 21 Tcrifenzumab DSP optimizes product recovery while focusing on removing process-related impurities (such as host cell proteins, DNA, RNA, and lipids) as well as product-related impurities (such as aggregates). HCPs in tcrifenzumab solutions can increase the risk of immunogenicity by inducing humoral and cell-dependent immune responses. Co-purification of protein-hydrolyzed HCPs with drug solutions, even in trace amounts, can induce time-dependent product cleavage, thereby affecting product stability and bioactivity. Jones et al. , “‘High‐risk’ host cell proteins (HCPs): A multi‐companycollaborative view,” Biotechnol. Bioeng. 118(8): 2870-85 (2021). High-risk HCPs are presented in Table 6. Table 6: High-risk host cell proteins (HCPs)

[0127] Process improvements to tiregizumab were designed to improve product titers and cell viability at harvest. The latter was found to affect HCP levels in the clarified harvest.

[0128] Results: LC-MS / MS combined with the CHO cell HCP database enabled the identification of individual HCP species. These HCPs were then classified as risky or risk-free. Improved cell culture feed increased harvest viability from approximately 65% ​​to >93%, leading to a reduction in risky HCPs and an increase in tirezhizumab yield in the harvest. The reduction in HCPs in the harvest also reduced HCP contamination during purification, ensuring a cleaner, purer drug substance (Table 6).

[0129] Risky hepatocellular carcinomas (HCPs) identified in standard and improved antibody manufacturing processes are shown in Table 7. Improved processes eliminate HCPs such as 78 kDa glucose-regulated protein (GRP78, BiP), lysosomal phospholipase A2 (LPLA2), and hepatic carboxylesterase 1. These carboxylesterases and lipases can potentially degrade the tiregizumab product and further erode product stability by digesting polysorbate, an emulsifier often added to improve drug product solubility. Jones et al. (2021).

[0130] Table 7: Host cell proteins identified using LC-MS / MS

[0131] In addition to step-specific HCP analysis, a comprehensive HRMS-based HCP workflow has been used to analyze drug substance batches from both standard and modified processes, enabling direct process comparison. Figures 22-25 This comparative analysis is crucial for assessing HCP-related risks associated with process improvements and is conducted as required by regulatory authorities. This analysis ensures that any modifications to the process do not introduce new risks related to residual HCP, supporting the safety and quality of the final product.

[0132] Table 8: Number of HCPs detected in each batch and the cumulative total for standard and modified processes.

[0133] Example 7: Optimizing Feed Concentration

[0134] Additional fed variants were prepared for Cell boost 5 (35 g / L) at feed intensities of 1X (35 g / L) and 2X (70 g / L), while the concentration of BalanCD CHO Feed 4 was maintained at 1X (112 g / L). Flask and bioreactor studies were conducted to evaluate the effect of feed concentration adjustments on protein productivity.

[0135] Results: The viable cell density of the two intensities of Cell Boost 5 showed differences in growth curves. The 2X intensity of Cell Boost 5 demonstrated better cell count maintenance during the stationary phase of the production process. Figure 29 Cell viability percentage was also similar, but it was found that cell viability was maintained slightly better at 2X intensity Cell Boost 5 than at other intensities for almost the entire process. Figure 30 Even when the feed concentration changed, no significant difference in productivity was observed. Figure 31 Finally, compared with the similar yield obtained for 1X ( Right now Compared to 2084 mg / L, the 2X strength has a final yield of 2170 mg / L. Figure 32 ).

[0136] Example 8: Optimization of Feed Volume

[0137] To further investigate the effect of feed volume addition on protein productivity, shake flasks and bioreactors were run with different feed volumes of 2X Cell Boost 5 (0.5%, 0.6%, 1% v / v) and 1X BalanCD CHO Feed 4 (1%, 1.2%, 1.5% v / v).

[0138] Results: Variations in feed volume showed differences in cell density. Higher cell counts were maintained during the stationary phase with additions of 2X Cell Boost 5 at 0.6% v / v and 1X BalanCD CHO at 1.2% v / v, respectively. Figure 33 Cell viability percentages were also similar, but viability was maintained when 2X Cell Boost 5 and 1X BalanCD CHO were added at 0.6% v / v and 1.2% v / v, respectively, with viability found to be slightly higher than the other two throughout almost the entire process. Figure 34 Maximum productivity (2520 mg / L) was observed with 2X Cell Boost 5 and 1X BalanCD CHO Feed 4 at 0.6% v / v and 1.2% v / v, respectively. Figure 35 and Figure 36 ).

[0139] 3. Implementation Plan

[0140] Implementation Scheme 1: A method for producing anti-IL-23 antibody, the method comprising: (a) A cell culture is prepared by seeding cells capable of expressing recombinant proteins into a container containing a first culture medium; (b) Culture the cells; (c) Lower the temperature of the cell culture; (d) Add a second culture medium to the cell culture; (e) Culture the cells; (f) Harvesting the cells from the cell culture; and (g) Isolate the recombinant protein from the harvested cells.

[0141] Implementation Scheme 2: A method for producing anti-IL-23 antibody, the method comprising: (a) Prepare and scale up seed cell cultures capable of expressing recombinant proteins in a suitable culture medium; (b) Inoculating and culturing the seed cell culture in a suitable production medium to prepare a cell culture; (c) Lower the temperature of the cell culture; (d) Periodically add feed to the cell culture; (e) Culture the cells; (f) Harvesting the cells from the cell culture; and (g) Isolate the recombinant protein from the harvested cells.

[0142] Implementation Scheme 3: A method for producing anti-IL-23p19 antibody, the method comprising: (a) A cell culture is prepared by seeding cells capable of expressing recombinant proteins into a container containing a first culture medium; (b) Culture the cells; (c) Lower the temperature of the cell culture; (d) Add a second culture medium to the cell culture; (e) Culture the cells; (f) Harvesting the cells from the cell culture; and (g) Isolate the recombinant protein from the harvested cells.

[0143] Implementation Scheme 4: A method for producing anti-IL-23p19 antibody, the method comprising: (a) Prepare and scale up seed cell cultures capable of expressing recombinant proteins in a suitable culture medium; (b) Inoculating and culturing the seed cell culture in a suitable production medium to prepare a cell culture; (c) Lower the temperature of the cell culture; (d) Periodically add feed to the cell culture; (e) Culture the cells; (f) Harvesting the cells from the cell culture; and (g) Isolate the recombinant protein from the harvested cells.

[0144] Implementation Scheme 5: The method as described in Implementation Scheme 3 or Implementation Scheme 4, wherein the anti-IL-23p19 antibody comprises: (i) a light chain polypeptide comprising the amino acid sequence SEQ ID NO: 1, and (ii) A heavy chain polypeptide comprising the amino acid sequence SEQ ID NO: 2.

[0145] Implementation Scheme 6: The method as described in Implementation Scheme 3 or Implementation Scheme 4, wherein the anti-IL-23p19 antibody comprises: (i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences having amino acid sequences SEQ ID NO: 3, 4, and 5; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences having amino acid sequences SEQ ID NO: 6, 7 and 8.

[0146] Implementation Scheme 7: The method as described in Implementation Scheme 1 or Implementation Scheme 3, wherein the container is a 500 mL shake flask or a 5 L bioreactor.

[0147] Implementation Scheme 8: The method as described in Implementation Scheme 1 or Implementation Scheme 3, wherein 0.3 x 10⁻⁶ ppm is inoculated into the container. 6 Up to 0.9 x 10 6 live cells / mL.

[0148] Implementation Scheme 9: The method as described in any one of Implementation Schemes 1 to 6, wherein when the viable cell count in the cell culture reaches 2.3 x 10⁻⁶ 6 Up to 3.0 x 10 6 When the cell / mL ratio is reached, the temperature is lowered.

[0149] Implementation Scheme 10: The method as described in Implementation Scheme 9, wherein when the viable cell count in the cell culture reaches 2.5 x 10⁻⁶ 6 When the cell / mL ratio is reached, the temperature is lowered.

[0150] Implementation Scheme 11: The method of any one of Implementation Schemes 1 to 6, wherein the temperature is lowered before culturing the cells or about 72 hours after culturing the cells.

[0151] Implementation Scheme 12: The method as described in Implementation Scheme 1 or Implementation Scheme 3, wherein the second culture medium is added every other day starting from the second day of culturing the cells.

[0152] Implementation Scheme 13: The method as described in Implementation Scheme 12, wherein approximately 2% v / v of the second culture medium is added to the cell culture.

[0153] Implementation Scheme 14: The method as described in Implementation Scheme 1 or Implementation Scheme 3, wherein the second culture medium is added every four days starting from the second day of culturing the cells.

[0154] Implementation Scheme 15: The method as described in Implementation Scheme 14, wherein approximately 4% v / v of the second culture medium is added to the cell culture.

[0155] Implementation Scheme 16: The method as described in Implementation Scheme 1 or Implementation Scheme 3, wherein the second culture medium is DME / F12, nutrient supplementation solution, BalanCD CHO Feed 4, or Cell Boost 5.

[0156] Implementation Scheme 17: The method as described in Implementation Scheme 1 or Implementation Scheme 3, wherein the second culture medium is a combination feed containing BalanCD CHO Feed 4 and Cell Boost 5.

[0157] Implementation Scheme 18: The method as described in Implementation Scheme 17, wherein the combined feed has a feed intensity of 1.25X, 1.5X, 1.75X, or 2X.

[0158] Implementation Scheme 19: The method as described in Implementation Scheme 18, wherein the combined feeding has a feeding intensity of 2X.

[0159] Implementation Scheme 20: The method as described in Implementation Scheme 19, wherein the combined feed contains approximately 112 g / L AllanCD CHO Feed 4 and approximately 35 g / L Cell Boost 5.

[0160] Implementation Scheme 21: The method as described in Implementation Scheme 20, wherein the combined feed is added every other day starting from the second day of culturing the cells.

[0161] Implementation Scheme 22: The method as described in Implementation Scheme 21, wherein the combined feed is added every four days starting from day 2 of culturing the cells.

[0162] Implementation Scheme 23: The method as described in Implementation Scheme 22, wherein the combined feed is added after 2, 6, 10, 14 and 18 days of culturing the cells.

[0163] Implementation Scheme 24: The method as described in Implementation Scheme 23, wherein the combined feed of about 2% v / v to about 4% v / v is added to the cell culture.

[0164] Implementation Scheme 25: The method as described in Implementation Scheme 24, wherein approximately 4% v / v of the combined feed is added to the cell culture.

[0165] Implementation Scheme 26: The method as described in Implementation Scheme 18, wherein the combined feed has a feed intensity of 1.75X.

[0166] Implementation Scheme 27: The method as described in Implementation Scheme 26, wherein the combined feed contains approximately 84.0 g / L AllanCD CHO Feed 4 and approximately 26.25 g / L Cell Boost 5.

[0167] Implementation Scheme 28: The method as described in Implementation Scheme 26, wherein the combined feed is added every other day starting from the second day of culturing the cells.

[0168] Implementation Scheme 29: The method as described in Implementation Scheme 26, wherein the combined feed is added every four days starting from day 2 of culturing the cells.

[0169] Implementation Scheme 30: The method as described in Implementation Scheme 29, wherein the combined feed is added after 2, 6, 10, 14 and 18 days of culturing the cells.

[0170] Implementation Scheme 31: The method as described in Implementation Scheme 30, wherein the combined feed of about 2% v / v to about 4% v / v is added to the cell culture.

[0171] Implementation Scheme 32: The method as described in Implementation Scheme 31, wherein approximately 4% v / v of the combined feed is added to the cell culture.

[0172] Implementation Scheme 33: The method as described in Implementation Scheme 21, wherein the combined feeding has a feeding intensity of 1.5X.

[0173] Implementation Scheme 34: The method as described in Implementation Scheme 33, wherein the combined feed contains approximately 56.1 g / L AlanCD CHO Feed 4 and approximately 17.5 g / L Cell Boost 5.

[0174] Implementation Scheme 35: The method as described in Implementation Scheme 33, wherein the combined feed is added every other day starting from the second day of culturing the cells.

[0175] Implementation Scheme 36: The method as described in Implementation Scheme 33, wherein the combined feed is added every four days starting from day 2 of culturing the cells.

[0176] Implementation Scheme 37: The method as described in Implementation Scheme 36, wherein the combined feed is added after 2, 6, 10, 14 and 18 days of culturing the cells.

[0177] Implementation Scheme 38: The method as described in Implementation Scheme 37, wherein the combined feed of about 2% v / v to about 4% v / v is added to the cell culture.

[0178] Implementation Scheme 39: The method as described in Implementation Scheme 38, wherein approximately 4% v / v of the combined feed is added to the cell culture.

[0179] Implementation Scheme 40: The method as described in Implementation Scheme 16, wherein the second culture medium is a nutrient supplement solution.

[0180] Implementation Scheme 41: The method as described in Implementation Scheme 40, wherein a fixed volume of nutrient feed solution is added to the cell culture.

[0181] Implementation Scheme 42: The method of any one of Implementation Schemes 1 to 4, wherein the cells are harvested from the cell culture when the culture viability drops below 75% or on day 22 of the biological process, whichever occurs first.

[0182] Implementation Scheme 43: The method of any one of Implementation Schemes 1 to 4, wherein at least 1.0 to 3.0 g / L of recombinant protein is isolated from cells harvested in the cell culture.

[0183] Implementation Scheme 44: The method as described in Implementation Scheme 43, wherein at least 2.2 g / L of recombinant protein is isolated from cells harvested from the cell culture.

[0184] Implementation Scheme 45: The method as described in Implementation Scheme 43, wherein at least 2.2 g / L of recombinant protein is isolated from cells harvested from the cell culture.

[0185] Implementation Scheme 46: The method as described in Implementation Scheme 43, wherein at least 2.5 g / L of recombinant protein is isolated from cells harvested from the cell culture.

[0186] Implementation Scheme 47: The method as described in Implementation Scheme 43, wherein at least 3.0 g / L of recombinant protein is isolated from cells harvested from the cell culture.

[0187] Implementation Scheme 48: The method of any one of Implementation Schemes 1 to 4, wherein the host cell protein (HCP) contaminants in the harvested cells are reduced compared to the HCP contaminants in cells harvested in a standard process.

[0188] Implementation Scheme 49: The method as described in Implementation Scheme 48, wherein the HCP contaminant is a high-risk HCP contaminant.

[0189] Implementation Scheme 50: The method as described in Implementation Scheme 49, wherein the high-risk HCP contaminant includes one or more of 78 kDa glucose regulatory protein (GRP78, BiP), lysosomal phospholipase A2 (LPLA2), and / or hepatic carboxylesterase 1.

[0190] Implementation Scheme 51: A method for producing an anti-IL-23p19 antibody, the method comprising: (a) A cell culture is prepared by seeding cells capable of expressing recombinant proteins into a container containing a first culture medium; (b) Culture the cells; (c) Lower the temperature of the cell culture; (d) Add a second culture medium to the cell culture; (e) Culture the cells; (f) Harvesting the cells from the cell culture; and (g) Isolate the recombinant protein from the harvested cells; The anti-IL-23p19 antibody comprises: (i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences having amino acid sequences SEQ ID NO: 3, 4, and 5; and (ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences having amino acid sequences SEQ ID NO: 6, 7, and 8; and The number of host cell protein (HCP) contaminants in the harvested cells was reduced compared to the number of HCP contaminants in cells harvested using standard processes.

Claims

1. A method for producing an anti-IL-23 antibody, the method comprising: (a) A cell culture is prepared by seeding cells capable of expressing recombinant proteins into a container containing a first culture medium; (b) Culture the cells; (c) Lower the temperature of the cell culture; (d) Add a second culture medium to the cell culture; (e) Culture the cells; (f) Harvesting the cells from the cell culture; and (g) Isolate the recombinant protein from the harvested cells.

2. A method for producing an anti-IL-23 antibody, the method comprising: (a) Prepare and scale up seed cell cultures capable of expressing recombinant proteins in a suitable culture medium; (b) Inoculating and culturing the seed cell culture in a suitable production medium to prepare a cell culture; (c) Lower the temperature of the cell culture; (d) Periodically add feed to the cell culture; (e) Culture the cells; (f) Harvesting the cells from the cell culture; and (g) Isolate the recombinant protein from the harvested cells.

3. A method for producing an anti-IL-23p19 antibody, the method comprising: (a) A cell culture is prepared by seeding cells capable of expressing recombinant proteins into a container containing a first culture medium; (b) Culture the cells; (c) Lower the temperature of the cell culture; (d) Add a second culture medium to the cell culture; (e) Culture the cells; (f) Harvesting the cells from the cell culture; and (g) Isolate the recombinant protein from the harvested cells.

4. A method for producing an anti-IL-23p19 antibody, the method comprising: (a) Prepare and scale up seed cell cultures capable of expressing recombinant proteins in a suitable culture medium; (b) Inoculating and culturing the seed cell culture in a suitable production medium to prepare a cell culture; (c) Lower the temperature of the cell culture; (d) Periodically add feed to the cell culture; (e) Culture the cells; (f) Harvesting the cells from the cell culture; and (g) Isolate the recombinant protein from the harvested cells.

5. The method of claim 3 or claim 4, wherein the anti-IL-23p19 antibody comprises: (i) a light chain polypeptide comprising the amino acid sequence SEQ ID NO: 1, and (ii) A heavy chain polypeptide comprising the amino acid sequence SEQ ID NO:

2.

6. The method of claim 3 or claim 4, wherein the anti-IL-23p19 antibody comprises: (i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences having amino acid sequences SEQ ID NO: 3, 4, and 5; and (ii) a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences having amino acid sequences SEQ ID NO: 6, 7 and 8.

7. The method of claim 1 or claim 3, wherein the container is a 500 mL shake flask or a 5 L bioreactor.

8. The method of claim 1 or claim 3, wherein 0.3 x 10⁻⁶ ppm is inoculated into the container. 6 Up to 0.9 x 10 6 live cells / mL.

9. The method of any one of claims 1 to 6, wherein the viable cell count in the cell culture reaches 2.3 x 10⁻⁶. 6 Up to 3.0 x 10 6 When the cell / mL ratio is reached, the temperature is lowered.

10. The method of claim 9, wherein the viable cell count in the cell culture reaches 2.5 x 10⁻⁶. 6 When the cell / mL ratio is reached, the temperature is lowered.

11. The method of any one of claims 1 to 6, wherein the temperature is lowered before culturing the cells or about 72 hours after culturing the cells.

12. The method of claim 1 or claim 3, wherein the second culture medium is added every other day starting from the second day of culturing the cells.

13. The method of claim 12, wherein about 2% v / v of the second culture medium is added to the cell culture.

14. The method of claim 1 or claim 3, wherein the second culture medium is added every four days starting from day 2 of culturing the cells.

15. The method of claim 14, wherein about 4% v / v of the second culture medium is added to the cell culture.

16. The method of claim 1 or claim 3, wherein the second culture medium is DME / F12, nutrient supplementation solution, BalanCD CHO Feed 4, or Cell Boost 5.

17. The method of claim 1 or claim 3, wherein the second culture medium is a combined feed containing BalanCD CHOFeed 4 and Cell Boost 5.

18. The method of claim 17, wherein the combined feed has a feed intensity of 1.25X, 1.5X, 1.75X, or 2X.

19. The method of claim 18, wherein the combined feed has a feed intensity of 2X.

20. The method of claim 19, wherein the combined feed contains about 112 g / L BalanCD CHO Feed4 and about 35 g / L Cell Boost 5.

21. The method of claim 20, wherein the combined feed is added every other day starting from day 2 of culturing the cells.

22. The method of claim 21, wherein the combined feed is added every four days starting from day 2 of culturing the cells.

23. The method of claim 22, wherein the combined feed is added after 2, 6, 10, 14 and 18 days of culturing the cells.

24. The method of claim 23, wherein the combined feed of about 2% v / v to about 4% v / v is added to the cell culture.

25. The method of claim 24, wherein about 4% v / v of the combined feed is added to the cell culture.

26. The method of claim 18, wherein the combined feed has a feed strength of 1.75X.

27. The method of claim 26, wherein the combined feed contains about 84.0 g / L BalanCD CHO Feed4 and about 26.25 g / L Cell Boost 5.

28. The method of claim 26, wherein the combined feed is added every other day starting from day 2 of culturing the cells.

29. The method of claim 26, wherein the combined feed is added every four days starting from day 2 of culturing the cells.

30. The method of claim 29, wherein the combined feed is added after 2, 6, 10, 14 and 18 days of culturing the cells.

31. The method of claim 30, wherein the combined feed of about 2% v / v to about 4% v / v is added to the cell culture.

32. The method of claim 31, wherein about 4% v / v of the combined feed is added to the cell culture.

33. The method of claim 21, wherein the combined feed has a feed intensity of 1.5X.

34. The method of claim 33, wherein the combined feed contains about 56.1 g / L BalanCD CHO Feed4 and about 17.5 g / L Cell Boost 5.

35. The method of claim 33, wherein the combined feed is added every other day starting from day 2 of culturing the cells.

36. The method of claim 33, wherein the combined feed is added every four days starting from day 2 of culturing the cells.

37. The method of claim 36, wherein the combined feed is added after 2, 6, 10, 14 and 18 days of culturing the cells.

38. The method of claim 37, wherein the combined feed of about 2% v / v to about 4% v / v is added to the cell culture.

39. The method of claim 38, wherein about 4% v / v of the combined feed is added to the cell culture.

40. The method of claim 16, wherein the second culture medium is a nutrient supplement solution.

41. The method of claim 40, wherein a fixed volume of nutrient feed solution is added to the cell culture.

42. The method of any one of claims 1 to 4, wherein the cells are harvested from the cell culture when the culture viability drops below 75% or on day 22 of the biological process, whichever occurs first.

43. The method of any one of claims 1 to 4, wherein at least 1.0 to 3.0 g / L of recombinant protein is isolated from cells harvested from the cell culture.

44. The method of claim 43, wherein at least 2.2 g / L of recombinant protein is isolated from cells harvested from the cell culture.

45. The method of claim 43, wherein at least 2.2 g / L of recombinant protein is isolated from cells harvested from the cell culture.

46. ​​The method of claim 43, wherein at least 2.5 g / L of recombinant protein is isolated from cells harvested from the cell culture.

47. The method of claim 43, wherein at least 3.0 g / L of recombinant protein is isolated from cells harvested from the cell culture.

48. The method of any one of claims 1 to 4, wherein the host cell protein (HCP) contaminants in the harvested cells are reduced compared to the HCP contaminants in cells harvested in a standard process.

49. The method of claim 48, wherein the HCP contaminant is a high-risk HCP contaminant.

50. The method of claim 49, wherein the high-risk HCP contaminant comprises one or more of 78 kDa glucose regulatory protein (GRP78, BiP), lysosomal phospholipase A2 (LPLA2), and / or hepatic carboxylesterase 1.

51. A method for producing an anti-IL-23p19 antibody, the method comprising: (a) A cell culture is prepared by seeding cells capable of expressing recombinant proteins into a container containing a first culture medium; (b) Culture the cells; (c) Lower the temperature of the cell culture; (d) Add a second culture medium to the cell culture; (e) Culture the cells; (f) Harvesting the cells from the cell culture; and (g) Isolate the recombinant protein from the harvested cells; The anti-IL-23p19 antibody comprises: (i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences having amino acid sequences SEQ ID NO: 3, 4, and 5; and (ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences having amino acid sequences SEQ ID NO: 6, 7, and 8; and The number of host cell protein (HCP) contaminants in the harvested cells was reduced compared to the number of HCP contaminants in cells harvested using standard processes.

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