WO2025109545A1 - Methods for producing recombinant proteins - Google Patents

Abstract

The disclosure relates to methods of producing recombinant proteins. More specifically, the disclosure relates to methods of manufacturing biologics, such as monoclonal antibodies, fusion proteins, or antigen-binding fragments thereof. In particular, the disclosure relates to methods and use of feed regimens that improve the cell viability and target protein productivity and lower host cell protein impurities of such manufacturing processes. More specifically, the disclosure relates to methods of producing Tildrakizumab.

Description

METHODS FOR PRODUCING RECOMBINANT PROTEINS

CROSS REFERENCE TO RELATED APPLICATIONS

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

SEQUENCE LISTING

This application contains a sequence listing which is submitted electronically and is hereby incorporated by reference in its entirety. The sequence listing submitted herewith is contained in the XML file created November 21, 2024 entitled "23-1088-WO_Sequence- Listing.xml" and is 8,451 bytes in size.

FIELD OF THE DISCLOSURE

[0001] The disclosure relates to methods of producing recombinant proteins. More specifically, the disclosure relates to methods of manufacturing biologies, such as monoclonal antibodies, fusion proteins, or antigen-binding fragments thereof. In particular, the disclosure relates to methods and use of feed regimens that improve the cell viability and target protein productivity and lower host cell protein impurities of such manufacturing processes. More specifically, the disclosure relates to methods of producing tildrakizumab.

BACKGROUND

[0002] Recombinant DNA technology is used to produce proteins in amounts that allow for use in a spectrum of therapeutic applications. An important task in recombinant protein production is the optimization of cell culture feeds and conditions in order to obtain a high protein titer using the most efficient means of production. Any improvement, including incremental improvements, can have enormous economic benefits. In the pharmaceutical industry, optimization of protein production for biologies used in therapies for the treatment of disease is advantageous, as any improvement can have a significant impact on productivity when the biologic is manufactured on a large scale. As such, there remains a need in the art for maximizing protein production from cell cultures expressing biologic proteins for use in medicine. Typically, mammalian cell culture feed is based on commercially available feed formulations. However, the feed formulations are often not optimally enriched to enhance biologic protein expression. [0003] Host cell proteins (HCPs) are a complex mixture of proteins with diverse physicochemical properties. Some of the HCPs are considered as high-risk HCPs. High-risk HCPs include those that are immunogenic and enzymatically active with the potential to degrade either product molecules or excipients used in formulation. Hence HCPs are considered as a critical quality attribute (CQA) and therefor need to be monitored while making process changes.

[0004] Improvements in antibody titer can be achieved by culture condition optimizations. A change in culture conditions can have an adverse impact on high-risk HCP profile. For example, changes in media, feeding strategy, process duration, peak cell density, and agitation speed can impact cell viability, which would eventually result in considerably different HCP compositions. Hence, it is important to develop a process with the objective to eliminate high-risk HCPs.

[0005] The pharmaceutical industry has a need for readily produced proteins for use in a spectrum of applications as part of therapeutic research. There is a need for commercially viable production processes to maximize the yield of recombinant proteins that are produced in the most efficient manner. Thus, there remains a need for improved cell culture supplements and cell culture methods for maximizing protein production.

SUMMARY

[0006] The disclosure relates to methods of producing recombinant proteins. More specifically, the disclosure relates to methods of manufacturing biologies, such as monoclonal antibodies, fusion proteins, or antigen-binding fragments thereof. In particular, the disclosure relates to feed preparation methods and feeding regimens that improve the cell viability and protein productivity and lower host cell protein impurities of such manufacturing processes. More specifically, the disclosure relates to methods of producing tildrakizumab.

[0007] The disclosure therefore relates to methods for significantly improving the yields of commercially produced monoclonal antibody titers. More specifically, the disclosure relates to methods involving improved cell culture feeds to improve monoclonal antibody productivity and the viability of the cells at the harvesting stage of the process. An increase in viability during cell culture lessens the amount of HCPs in the cell free supernatant. This is a consequence of higher cell viability, which consequently affects cell lysis during culture with resultant lower content of HCPs during harvest. [0008] The disclosure provides a method of manufacture to improve the cell culture density, viability, and productivity through a combination feed. The disclosure provides a combination feed that is highly advantageous, as it contains a combination of nutrients in one formulation. The disclosure provides methods and combination feeds that nearly double the harvest titer in comparison to standard processes for producing recombinant proteins.

[0009] Provided herein is a method of producing an anti-IL-23pl9 antibody comprising: (a) preparing a cell culture by seeding a container containing a first culture medium with cells capable of expressing the recombinant protein; (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.

[0010] Also provided herein is a method of producing an anti-IL-23pl9 antibody comprising: (a) preparing and scaling up a seed cell culture capable of expressing the recombinant protein in a suitable culture medium; (b) inoculating and culturing the seed cell culture in a suitable production medium to prepare a cell culture; (c) lowering the temperature of the cell culture; (d) periodically adding feed 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.

[0011] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.

BRIEF DESCRIPTIONS OF THE DRAWINGS

[0012] FIG. 1 is a line graph comparing the viable cell density (VCD) (10⁶ cells / mb) over an incubation period of 22 days between the standard and improved processes.

[0013] FIG. 2 is a line graph comparing the viability of the cells as a percentage over the incubation time in days between the standard and improved processes.

[0014] FIG. 3 is a line graph comparing the productivity / titer produced in mg/L over the incubation time in days between the standard and improved processes.

[0015] FIG. 4 is a bar graph comparing the final titer produced between the standard and improved process in mg/L. [0016] FIG. 5 is a comparative line graph of the viable cell density (10⁶ cells / mL) over the incubation time in days between the standard and improved processes.

[0017] FIG. 6 is a comparative line graph of the cell viability as a percentage over the incubation time in days between the standard and improved processes.

[0018] FIG. 7 is a comparative line graph depicting the productivity of titer measured in mg/L over the incubation time in days between the standard and improved processes.

[0019] FIG. 8 is a comparative bar graph comparing the final titer produced in mg/L between the standard and improved processes.

[0020] FIG. 9 is a line graph comparing the viable cell densities (10⁶ cells / mL) over the incubation time in days between the alternative day feeding (individual feeds at 2% v/v of post inoculation volume) and feeding every 4th day (individual feeds at 4% v/v of post inoculation volume).

[0021] FIG. 10 is a line graph depicting the cell viability as a percent over the incubation time in days between the improved process alternate day feeding and every 4th day feeding schedules with either 2% v/v or 4% v/v of post inoculation volume.

[0022] FIG. 11 is a line graph depicting the productivity of titer produced in mg/L over the incubation time in days between alternate day feeding and every 4th day feeding at either 2% v/v or 4% v/v of post inoculation volume.

[0023] FIG. 12 is bar graphs representing the productivity of titer produced in mg/L between the improved process using either alternative day feeding or every 4th day feeding with a feed volume of 2% v/v or 4% v/v of post inoculation volume.

[0024] FIG. 13 is a line graph comparing the viable cell density (10⁶ cells/mL) over the incubation time in days between the improved process (individual feeds fed separately) and the improved process using a combined feed.

[0025] FIG. 14 is a line graph depicting cell viability as a percentage over the incubation time in days between the improved process (individual feeds fed separately) and the improved process using a combined feed.

[0026] FIG. 15 is a line graph measuring productivity of the titer in mg/L over the incubation time in days between the improved process (individual feeds fed separately) and the improved process using a combined feed.

[0027] FIG. 16 is a comparative bar graph depicting the productivity of titer produced in mg/L between the improved process (individual feeds fed separately) and the improved process with a combined feed. [0028] FIG. 17 is a line graph comparing the viable cell density (10⁶ cells / mL) achieved by feeding a combined feed fat a strength of 1.25X, 1.5X, or 1.75X over the incubation time in days.

[0029] FIG. 18 is a line graph depicting the cell viability as a percentage achieved by feeding a combined feed at strength of 1.25X, 1.5X, or 1.75X over the incubation time in days.

[0030] FIG. 19 is a line graph depicting the productivity achieved by feeding a combined feed at a strength of 1.25X, 1 ,5X, or 1.75X over the incubation time in days.

[0031] FIG. 20 is a bar graph comparing the final titer achieved by feeding a combined feed at a strength of 1.25X, 1 ,5X, or 1.75X over the incubation time in days.

[0032] FIG. 21 depicts a line graph of the viable cell density (10⁶ cells / mL) of the standard and improved processes over the incubation time in days.

[0033] FIG. 22 depicts a line graph of the cell viability as a percentage for the standard and improved processes over the incubation time in days.

[0034] FIG. 23 is a line graph representing the productivity of the standard and improved processes over the incubation time in days.

[0035] FIG. 24 depicts a bar graph comparing the harvest productivity of the standard and improved processes.

[0036] FIG. 25 depicts the viable cell density (10⁶ cells / mL) of the standard versus improved processes over the incubation time in days.

[0037] FIG. 26 depicts the cell viability as a percentage for both the standard and improved processes over the incubation time in days.

[0038] FIG. 27 depicts the productivity (mg / L) of the standard and improved processes over the incubation time in days.

[0039] FIG. 28 depicts the harvest productivity (mg / L) of the standard and improved processes.

[0040] FIG. 29 depicts the viable cell density (10⁶ cells / mL) of the improved process with IX Cell Boost versus 2X Cell Boost over the incubation time in days.

[0041] FIG. 30 depicts the percentage of the cell viability for the improved process using IX Cell Boost and Feed 4 versus 2X Cell Boost with Feed 4 over the incubation time in days. [0042] FIG. 31 depicts a line graph of the productivity (mg / L) of the improved process production with IX Cell Boost and Feed 4 or 2X Cell Boost and Feed 4 over the incubation time in days. [0043] FIG. 32 depicts bar graphs of the harvest productivity (mg / L) of the improved process using IX Cell Boost 5 versus 2X Cell Boost 5 with IX Feed 4.

[0044] FIG. 33 depicts the viable cell density (10⁶ cells/ mb) of the improved process using 2X Cell Boost 5 by 0.5%, 0.6% or 1.0% v/v and IX Feed 4 at 1%, 1.2 % or 1.5% v/v over the incubation time in days.

[0045] FIG. 34 depicts a line graph of the viability percentage of the improved process using 2X Cell Boost 5 by 0.5%, 0.6% or 1.0% v/v and IX Feed 4 at 1%, 1.2 % or 1.5% v/v over the incubation time in days.

[0046] FIG. 35 depicts a line graph of the productivity (mg / L) of the improved process using 2X Cell Boost 5 by 0.5%, 0.6% or 1.0% v/v and IX Feed 4 at 1%, 1.2 % or 1.5% v/v over the incubation time in days.

[0047] FIG. 36 depict a bar graph of harvest productivity of the improved process using 2X Cell Boost 5 by 0.5%, 0.6% or 1.0% v/v and IX Feed 4 at 1%, 1.2 % or 1.5% v/v.

[0048] FIG. 37 is a flow chart depicting the overall downstream processes (DSP) responsible for purification.

[0049] FIG. 38 depicts a comparison of the detection of common and unique HCPs between the standard and improved process.

[0050] FIG. 39 depicts a comparison of the detection of common and unique HCPs between the standard and improved process following the virus inactivation step of tildrakizumab.

[0051] FIG. 40 depicts a comparison of the detection of common and unique HCPs between the standard and improved process following the cation exchange output of tildrakizumab.

[0052] FIG. 41 depicts a comparison of the detection of common and unique HCPs between the standard and improved process following the mix mode chromatography (MMC) output of tildrakizumab.

[0053] FIG. 42 represents the downstream removal of high risk HCPs at each unit operation of tildrakizumab.

DETAILED DESCRIPTION

[0054] The disclosure relates to methods of producing recombinant proteins. More specifically, the disclosure relates to methods of manufacturing biologies, such as antibodies or antigen-binding fragments thereof. In particular, the disclosure relates to feed preparation methods and feeding regimens that improve the final protein productivity of such manufacturing processes. More specifically, the disclosure relates to methods of producing tildrakizumab.

[0055] More specifically, the disclosure relates to methods for significantly improving yields of commercially produced monoclonal antibody titers. Even more specifically, this disclosure relates to methods involving improved cell culture feeds and their combinations to improve the productivity of the monoclonal antibodies produced and the viability of the culture at harvest.

[0056] As utilized in accordance with the present disclosure, unless otherwise indicated or defined, all technical and scientific terms used herein shall be understood to have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0057] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. For example, the terms "a," "an," and "the," as used herein, are understood to be singular or plural unless the context clearly dictates otherwise. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components unless otherwise indicated or dictated by its context. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives unless otherwise indicated. Thus, unless specifically stated or apparent from context, the term "or" as used herein is understood to be inclusive.

[0058] The terms "comprises" and "comprising," as used herein, can have the meaning ascribed to them in U.S. patent law and can mean "includes," "including," "containing," "having," and the like. Thus, unless expressly specified otherwise, the terms "comprises" and "comprising," as used herein, indicate that further components or members may optionally be present in addition to the components or members of the list introduced by "comprising." The terms "consisting essentially of' or "consists essentially", as used herein, likewise have the meaning ascribed in U.S. patent law, and allow for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited are not changed by the presence of more than that which is recited. [0059] Any of the methods or compositions provided herein can be combined with one or more of any of the other methods or compositions provided herein.

[0060] In the present disclosure, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 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.

[0061] Unless specifically stated or apparent from context, the terms "about" and "approximately," as used herein, are understood as meaning within a range of normal tolerance in the art, for example within 2 standard deviations of the mean, or mean within an acceptable error range for the 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, z.e., the limitations of the measurement system. "About" can be understood as meaning 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 context, all numerical values provided herein are modified by the term about.

[0062] It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed subject matter or to imply that certain features are critical, essential, or even important to the structure or function of the claimed subject matter. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present disclosure.

[0063] For the purposes of describing and defining the present disclosure it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0064] In some embodiments, a method of producing an anti-IU-23pl9 antibody involves: (a) preparing a cell culture by seeding a container containing a first culture medium with cells capable of expressing the recombinant proteins, (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.

[0065] In some embodiments, a method of producing an anti-IL-23pl9 antibody involves: (a) preparing and scaling up a seed cell culture capable of expressing the recombinant protein in a suitable culture medium; (b) inoculating and culturing the seed cell culture in a suitable production medium to prepare a cell culture; (c) lowering the temperature of the cell culture; (d) periodically adding feed 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.

[0066] The term "antibody" as used herein refers to a protein that is capable of recognizing and specifically binding to an antigen. Ordinary or conventional mammalian antibodies comprise a tetramer, which is typically composed of two identical pairs of polypeptide chains, each pair consisting of one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain," as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino -terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxyl-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CHI, CH2, and CH3) and a hinge region between CHI and CH2, wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.

[0067] Within full-length light and heavy chains, the variable and constant domains typically are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. The variable regions of each light/heavy chain pair typically form an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the amino-terminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0068] The term "antigen binding fragment" as used herein refers to a portion of an intact antibody and/or refers to the antigenic determining variable domains of an intact antibody. It is known that the antigen binding function of an antibody can 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, diabodies, and multispecific antibodies formed from antibody fragments.

[0069] In particular embodiments, the anti-IL-23pl9 antibody huml3B8-b is tildrakizumab. The term "tildrakizumab" as used herein refers to a humanized anti-IL-23pl9 monoclonal antibody, also known as SCH 900222 or MK-3222. Tildrakizumab is a high- affinity (297 picomolar [pM]) humanized immunoglobulin Gl/kappa (IgG I/K) antibody that specifically binds to the pl 9 protein of the IL-23 heterodimer but does not bind human IL- 12 (IL-12/23p40 and IL12p35 heterodimer) or human IL-12/23p40.

[0070] In some embodiments, the anti-IL-23pl9 antibody tildrakizumab can refer to ILUMYA®. In some embodiments, tildrakizumab is formulated in a 1 mL single-dose prefilled syringe containing 100 mg of tildrakizumab (z.e., 100 mg/mL). In some embodiments, ILUMYA® (tildrakizumab-asmn) injection, for subcutaneous use, is a sterile, clear to slightly opalescent, colorless to slightly yellow solution. ILUMYA® is supplied in a single-dose prefilled syringe with a glass barrel and 29-gauge fixed, 1/2-inch needle. In some embodiments, tildrakizumab can be formulated in: L-histidine, L-histidine hydrochloride monohydrate, polysorbate 80, and/or sucrose, in water for injection, with a pH of 5.7-6.3. In some embodiments, tildrakizumab is formulated in a 1 mL single-dose prefilled syringe containing 100 mg of tildrakizumab-asmn formulated in: 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 with a pH of 5.7-6.3.

[0071] In particular embodiments, tildrakizumab comprises a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 2, which are disclosed in U.S. Patent Nos. 8,404,813 and 8,293,883, the disclosures of each of which are hereby incorporated by reference in their entireties. In other embodiments, tildrakizumab or an antigen-binding fragment thereof comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises CDR1, CDR2, and CDR3 sequences of the amino acid sequences of SEQ ID NOs: 3-5, and wherein the light chain variable domain comprises CDR1, CDR2, and CDR3 sequences of the amino acid sequences of SEQ ID NOs: 6-8.

[0072] Huml3B8-b Light Chain (SEQ ID NO: 1)

DIQMTQSPSSLSASVGDRVTITCRTSENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQHHYGIPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC

[0073] Huml3B8-b Heavy Chain (SEQ ID NO: 2)

QVQLVQSGAEVKKPGASVKVSCKASGYI FITYWMTWVRQAPGQGLEWMGQI FPASGSADYNE KFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGGGFAYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK

[0074] Huml3B8-b Heavy Chain CDR1 (SEQ ID NO: 3)

GYIFITYWMT

[0075] Huml3B8-b Heavy Chain CDR2 (SEQ ID NO: 4)

QI FPASGSADYNEKFE

[0076] Huml3B8-b Heavy Chain CDR3 (SEQ ID NO: 5)

GGGGFAY

[0077] Huml3B8-b Light Chain CDR1 (SEQ ID NO: 6)

RTSENIYSYLA

[0078] Huml3B8-b Light Chain CDR2 (SEQ ID NO: 7)

NAKTLAE

[0079] Huml3B8-b Light Chain CDR3 (SEQ ID NO: 8)

QHHYGIPFT

[0080] In certain embodiments of the methods of producing a recombinant protein of the disclosure, the step of preparing a cell culture by seeding a container containing a first culture medium with cells capable of expressing the recombinant proteins can include the preparation and scale-up of a seed cell culture capable of expressing the recombinant protein in a suitable culture medium. The suitable culture medium used in the methods of the disclosure can include any medium described herein or known in the art.

[0081] In some embodiments of the methods of producing a recombinant protein of the disclosure, the step of preparing a cell culture by seeding a container containing a first culture medium with cells capable of expressing the recombinant proteins can also include inoculating and culturing the seed cell culture in a suitable production medium. The suitable production medium used in the methods of the disclosure can include any medium described herein or known in the art.

[0082] In other embodiments of the methods of producing a recombinant protein of the disclosure, the step of adding a second culture medium to the cell culture can include the periodic addition of feed to the culture. The cell culture feed or feed used in the methods of the disclosure can include any cell culture feed or feed described herein or known in the art.

1. Methods and Compositions

Standard Process

[0083] A standard process for producing recombinant proteins uses any suitable chemically defined basal medium in a shake flask (or other appropriate container) containing an initial seeding of between 0.3 x 10⁶ and 0.9 x 10⁶ viable cells/mL in a post inoculation volume of about 100 mb per 500 mb of container volume. Suitable containers include a 500 mb shake flask or 5L bioreactor. L-glutamine was added to the culture as needed and a fixed volume of amino acids were added to the culture beginning at Day 3. When the viable cell count reached between 1.0 x 10⁶ and 3.0 x 10⁶ cells/mL, the process temperature was lowered. A fixed volume of a suitable feed medium was added on Days 3, 7, 11, and 15 of the fed batch process. A fixed volume of nutrient feed solution was added on Day 9. Flasks were harvested when culture viability dropped below 75% or on Day 22 of the bioprocess, whichever occurred first.

Viable Cell Dell Density Adjustment

[0084] The temperature shift to production fed-batch bioprocess was initiated in the standard process when viable cell density reached 1.0 x 10⁶ to 3.0 x 10⁶ viable cells/mL or within 72 hours, whichever occurred first. In the improved process, the criteria remained the same other than a higher viable cell density of 2.5 x 10⁶ cells/mL instead of 1.0 x 10⁶ to 3.0 x 10⁶ cells/mL in the standard process. In some embodiments of the improved process of the disclosure, the temperature of the cell culture is lowered by between 1°C and 4°C. In certain embodiments of the improved process of the disclosure, the temperature of the cell culture is lowered by between 1°C and 3°C. In particular embodiments of the improved process of the disclosure, the temperature of the cell culture is lowered by between 1°C and 2°C.

Improved Cell Feeds

[0085] Standard processes for producing recombinant proteins face the issue of low productivity (-1300 mg/L) and viability at harvest. The main challenge in developing improved processes for producing recombinant proteins was to enhance the productivity and maintain cell viability at higher levels in the latter days of the process. The rationale behind new feed addition was to improve the productivity of the standard process.

[0086] In order to enhance the productivity of the standard process, two improved feeds were introduced into the bioprocess: Cell Balan CD CHO Feed 4 (Feed 4) and Cell Boost 5 (CB5). These feeds can be prepared and added independently (individually or separately) or in a combined manner (in combination). The details of the feeds that were used , as well as the feed strength, days of addition, and feed volumes are summarized in the tables below:

Table 1: Option A - individual Feed 4 and CB5 preparation; added separately

Table 2: Combined feed prepared by mixing both feeds (1.5X)

Table 3: Combined feed prepared by mixing both feeds (1.0X)

Table 4: Individual Feed 4 and CB5 preparation; added separately

Table 5: Individual Feed 4 and CB5 preparation; added separately

Enhanced Productivity at Harvest

[0087] The standard process yields a titer of approximately 1300 mg/L at harvest. In some embodiments of the improved process of the disclosure, the process provides comparatively higher viability and increased titers of between 2000 mg/L and more than 10000 mg/L at harvest. In certain embodiments of the improved process of the disclosure, the process provides comparatively higher viability and increased titers of between 2000 mg/L and 5000 mg/L at harvest. In particular embodiments of the improved process of the disclosure, the process provides comparatively higher viability and increased titers of at least 2000 mg/L at harvest.

2. Examples

Example 1: Feeds selection

[0088] The standard process yields titers around 1.0 to 1.3 g/L. In order to enhance the productivity of the standard process, commercially available feeds were evaluated. The following feeds were considered for feed evaluation: BalanCD CHO Feed 4 (Fujifilm -Irvine Scientific) and Cell Boost 5 (Cytiva). Feeds were added 2% v/v of post inoculation volume on alternate days from Day 2. The process parameters of this study were kept similar to the standard process except for the introduction of the feeds and the criteria of higher VCD at the time of temperature shift. Shake flasks were incubated in a CO2 orbital shaker incubator set at a temperature of 36.5°C, shaking at 140 rpm, and at 5% CO2 within the shaker. Temperature shift was administered to the culture when a VCD of 2.5 xlO⁶ cells/mL was attained or at 72 hours, whichever occurred first.

[0089] Results: Shake flask study results indicate a two-fold increment of productivity by using BalanCD CHO Feed 4 and Cell Boost 5. The addition of the feeds supported a higher viable cell density over the 3 -week production period (Figures 1 and 21), and also maintained a higher percentage of cell viability until harvest (Figures 2 and 22). In addition, the increased productivity achieved by the standard feed was gradual as compared to the improved feed (Figures 3 and 23). The final productivity of the protein after introduction of the new feeds was significantly higher than the standard process (Figures 4 and 24).

Example 2: Standard process versus improved process

[0090] Based on the results obtained, the addition of improved feed improved the productivity of the standard process. To further confirm the effect of the improved feed, 5L bioreactor runs were performed for both the standard and improved processes. The bioreactor process parameters were kept the same in both procedures except for the feed addition and VCD criteria at temperature shift.

[0091] Results: The findings demonstrate that the introduction of the new feeds improved the yields of protein. After the initial two days of seeding the cells, there was a distinct divergence of the viable cell densities between the standard and improved processes. The viable cell density was considerably higher, sometimes as much as double the cell count in certain cases as compared to the standard process (Figures 5 and 25). The percentage of cell viability was higher overall throughout the entire production period during the improved process (Figures 6 and 26). The productivity of the improved process was higher as compared to the standard process especially in the initial stages when the titer levels were similar (Figures 7 and 27). However, the final titer was nearly double with the introduction of the improved feeds with yields as high as 2157 mg/L in comparison to 1261 mg/L (Figures 8 and 28).

Example 3: Feed addition days optimization

[0092] Initial studies with the new feeds confirmed that the addition of the feeds significantly improves productivity. Feeds were added to bioreactors on alternate days beginning from Day 2 of the bioprocess. To further study the impact of the feed addition regimens on protein productivity, the shake flask and bioreactor were run with the feeds added at 4% v/v of post inoculation volume on every 4th day from Day 2 versus a lower addition of feeds in the standard process from Day 2 of the protocol.

[0093] Results: Changing the feed schedule from every other day to every 4th day resulted in a similar cell density over the production process (Figure 9), but the percentage of cell viability was lower than the standard feed schedule (Figure 10). Productivity of the protein over the production period also amassed less protein (mg/L) than the improved process did with alternative day feeding (Figure 11). Finally, the overall harvest yields were only minimally increased by the 4th day feeding schedule as compared to alternative day scheduling (Figure 12).

Example 4: Concentrated feed composition

[0094] When feeds BalanCD CHO Feed 4 (112 g/L) and Cell boost 5 (35 g/L) were added independently, the resulting combination resulted in high-volume feeds in the process. Therefore, blending the feeds at higher concentration was the next step in developing an effective feed formulation. The concentrated, combined feed containing 112 g/L Feed 4 and 35 g/L CB5 was termed the 2X feed.

[0095] Results: Shake flask studies were performed with individual feeds or the combined 2X concentrated feed. Interestingly, the combined feed did not improve cell density, cell viability, and productivity despite having higher nutrient density as compared to the high volume mix feed. The viable cell density was distinctly lower throughout the production process as compared to the addition of individual feeds (Figure 13), while the percentage of cell viability was slightly lower but similar to the individual feed protocol (Figure 14). The protein titer each day over the process was notably less in the combined feed protocol as compared to the individual feeds (Figure 15). The final titer after using the combined feed was about one third less in comparison to feeds fed individually (Figure 16).

Example 5: Optimizing feed concentration

[0096] Further feed variants were prepared at feed strength of 1.25X, 1.5X, or 1.75X. Flasks and bioreactor studies were performed to assess the impact of adjustments to the strength of feeds on protein productivity. These combined feeds were added at 2% v/v alternatively from Day 2.

[0097] Results: The viable cell densities of all three strengths were found to be largely similar throughout the production process, indicative that these strengths are not having any individual residual effects on the cell density (Figure 17). The percentage of cell viability also was similar, but the viability maintained in 1.75X strength was found to be slightly above the others for nearly the entire process (Figure 18). The productivity, while similar over the production period, of the 1.75X strength again had an overall higher production period than the other two feed strengths (Figure 19). Finally, the 1.75X strength had the best final yield of 2269 mg/L, an approximate 25% increase in yield in comparison to the similar yielding 1.25X (1716 mg/L) and 1.5X (1681 mg/L) feed strengths (Figure 20). The resulting feed solutions were capable of increasing the volumetric production. A single, high strength feed is favorable for cell culture fed-batch protein manufacturing, because it reduces the number of feeds and total volume of the feed to be added.

Example 6: Purifying product of high-risk host cell proteins (HCPs)

[0098] Chinese hamster ovary (CHO) cells were used for production of tildrakizumab. Recovery of product from the production bioreactor was initiated with harvest of the cell culture fluid (HCCF), followed by a subsequent series of downstream processing (DSP) steps (Figure 21). Tildrakizumab DSP optimizes product recovery while focusing on the removal of process-related impurities, for example host cell proteins, DNA, RNA, and lipids, as well as product related impurities, such as aggregates. HCPs in the tildrakizumab solution can increase the risk of immunogenicity by inducing humoral and cell-dependent immune responses. Co-purification of proteolytic HCPs with the drug solution, even in trace amounts can induce cleavage of the product over time, thereby affecting product stability and biological activity. Jones et al., "'High-risk' host cell proteins (HCPs): A multi-company collaborative view," Biotechnol. Bioeng. 118(8): 2870-85 (2021). High risk HCPs are presented in Table 6.

Table 6: High-Risk Host Cell Proteins (HCPs)

[0099] Process modification of tildrakizumab was intended to improve the product titer and the cell viability at the time of harvest. The latter was found to influence the levels of HCP in the clarified harvest.

[0100] Results: LC-MS/MS in conjunction with the CHO cell HCP database enabled identification of individual HCP species. These HCPs were then classified as risky and non- risky HCPs. Viability improved at the time of harvest from -65% to >93% by improving cell culture feedings, resulting in a reduction of risky HCPs with gain in yield of tildrakizumab in the harvest. Reduction of HCPs in the harvest also reduced HCP contaminants in the purification process, thus ensuring a cleaner, pure drug substance (Table 6). [0101] Risky HCPs found in the standard and improved antibody production processes are shown in Table 7. The improved process eliminated HCPs such as 78 kDa glucose regulated protein (GRP78, BiP), lysosomal phospholipase A2 (LPLA2), and liver carboxylesterase 1. These carboxyesterases and lipases can potentially degrade the tildrakizumab product and further erode product stability by digestion of polysorbate, an emulsifier often added to improve solubility of the drug product. Jones et al. (2021).

Table 7: Host Cell Proteins Identified Using LC-MS/MS

[0102] Beyond step-specific HCP profiling, drug-substance batches from both the standard and improved processes have been comprehensively analyzed using the HRMS- based HCP workflow to enable direct process comparison (Figures 22-25). This comparative profiling is essential for assessing HCP-related risks associated with the improved process and is conducted in alignment with regulatory agency requirements. Such analysis ensures that any modifications in the process do not introduce new risks related to residual HCPs, supporting the safety and quality of the final product.

Table 8: Number of HCPs Detected Per Batch and Cumulative Totals for Standard and

Improved Process

Example 7: Optimizing feed concentration

[0103] Further feed variants were prepared at feed strengths of IX (35 g/L) and 2X (70 g/L) for Cell boost 5 (35 g/L) and the concentration of BalanCD CHO Feed 4 was maintained at IX (112 g/L). Flasks and bioreactor studies were performed to assess the impact of adjustments to the concentration of feed on protein productivity.

[0104] Results: The viable cell densities of both the strengths of Cell boost 5 showed a difference in growth profile. 2X strength Cell Boost 5 showed better maintenance of cell count during the stationary phase of the production process (Figure 29). The percentage of cell viability also was similar, but the viability maintained in Cell Boost 5 of 2X strength was found to be slightly above the other for nearly the entire process (Figure 30). No significant difference in the productivity was observed even when the concentration of feed was varied (Figure 31). Finally, the 2X strength had a final yield of 2170 mg/L in comparison to the similar yield obtained for IX (z.e., 2084 mg/L) (Figure 32).

Example 8: Optimization of Feed Volume

[0105] To further study the impact of the feed volume addition on protein productivity, the shake flask and bioreactor were run with a different feed volume of 2X Cell Boost 5 (0.5, 0.6, 1% v/v ) and IX BalanCD CHO Feed 4 (1, 1.2, 1.5% v/v).

[0106] Results: Changing the feed volume showed a difference in cell density. During the stationary phase, a higher count was maintained where 2X Cell Boost 5 and IX BalanCD CHO were added at 0.6% v/v and 1.2% v/v, respectively (Figure 33). The percentage of cell viability also was similar, but the viability was maintained when 2X Cell Boost 5 and IX BalanCD CHO were added at 0.6% v/v and 1.2% v/v, respectively, viability was found to be slightly above the other for nearly the entire process (Figure 34). Feeding 2X Cell boost 5 and IX BalanCD CHO Feed 4 at 0.6% v/v and 1.2% v/v, respectively, showed maximum productivity (2520 mg/L) (Figures 35 and 36). 3. Embodiments

[0107] Embodiment 1: A method of producing an anti -IL-23 antibody comprising:

(a) preparing a cell culture by seeding a container containing a first culture medium with cells capable of expressing the recombinant protein;

(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.

[0108] Embodiment 2: A method of producing an anti -IL-23 antibody comprising:

(a) preparing and scaling up a seed cell culture capable of expressing the recombinant protein in a suitable culture medium;

(b) inoculating and culturing the seed cell culture in a suitable production medium to prepare a cell culture;

(c) lowering the temperature of the cell culture;

(d) periodically adding feed 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.

[0109] Embodiment 3: A method of producing an anti-IL-23pl9 antibody comprising:

(a) preparing a cell culture by seeding a container containing a first culture medium with cells capable of expressing the recombinant protein;

(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.

[0110] Embodiment 4: A method of producing an anti-IL-23pl9 antibody comprising:

(a) preparing and scaling up a seed cell culture capable of expressing the recombinant protein in a suitable culture medium;

(b) inoculating and culturing the seed cell culture in a suitable production medium to prepare a cell culture; (c) lowering the temperature of the cell culture;

(d) periodically adding feed 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.

[oni] Embodiment 5 : The method of either embodiment 3 or embodiment 4, wherein the anti-IL-23pl9 antibody comprises:

(i) a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 1, and

(ii) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

[0112] Embodiment 6: The method of either embodiment 3 or embodiment 4, wherein the anti-IL-23pl9 antibody comprises:

(i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 3, 4, and 5; and

(ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 6, 7, and 8.

[0113] Embodiment 7: The method of either embodiment 1 or embodiment 3, wherein the container is a 500 mb shake flask or 5L bioreactor.

[0114] Embodiment 8: The method of either embodiment 1 or embodiment 3, wherein the container is seeded with 0.3 xlO⁶ to 0.9 xlO⁶ viable cells/mL.

[0115] Embodiment 9: The method of any one of embodiments 1 to 6, wherein the temperature is lowered when the viable cell count in the cell culture reaches between 2.3 x 10⁶ and 3.0 x 10⁶ cells/mL.

[0116] Embodiment 10: The method of embodiment 9, wherein the temperature is lowered when the viable cell count in the cell culture reaches 2.5 x 10⁶ cells/mL.

[0117] Embodiment 11 : The method of any one of embodiments 1 to 6, wherein the temperature is lowered prior to or at about 72 hours of culturing the cells.

[0118] Embodiment 12: The method of either embodiment 1 or embodiment 3, wherein the second culture medium is added every other day from day 2 of culturing the cells.

[0119] Embodiment 13: The method of embodiment 12, wherein about 2% v/v of the second culture medium is added to the cell culture.

[0120] Embodiment 14: The method of either embodiment 1 or embodiment 3, wherein the second culture medium is added every fourth day from day 2 of culturing the cells. [0121] Embodiment 15: The method of embodiment 14, wherein about 4% v/v of the second culture medium is added to the cell culture.

[0122] Embodiment 16: The method of either embodiment 1 or embodiment 3, wherein the second culture medium is DME/F12, Nutrient feed solution, BalanCD CHO Feed 4, or Cell Boost 5.

[0123] Embodiment 17: The method of either embodiment 1 or embodiment 3, wherein the second culture medium is a combined feed containing BalanCD CHO Feed 4 and Cell Boost 5.

[0124] Embodiment 18: The method of embodiment 17, wherein the combined feed has a feed strength of 1.25X, 1 ,5X, 1.75X, or 2X.

[0125] Embodiment 19: The method of embodiment 18, wherein the combined feed has a feed strength of 2X.

[0126] Embodiment 20: The method of embodiment 19, wherein the combined feed contains about 112 g/L BalanCD CHO Feed 4 and about 35 g/L Cell Boost 5.

[0127] Embodiment 21 : The method of embodiment 20, wherein the combined feed is added every other day from day 2 of culturing the cells.

[0128] Embodiment 22: The method of embodiment 21, wherein the combined feed is added every fourth day from day 2 of culturing the cells.

[0129] Embodiment 23 : The method of embodiment 22, wherein the combined feed is added after 2, 6, 10, 14, and 18 days of culturing the cells.

[0130] Embodiment 24: The method of embodiment 23, wherein about 2% to about 4% v/v of the combined feed is added to the cell culture.

[0131] Embodiment 25 : The method of embodiment 24, wherein about 4% v/v of the combined feed is added to the cell culture.

[0132] Embodiment 26: The method of embodiment 18, wherein the combined feed has a feed strength of 1.75X.

[0133] Embodiment 27 : The method of embodiment 26, wherein the combined feed contains about 84.0 g/L BalanCD CHO Feed 4 and about 26.25 g/L Cell Boost 5.

[0134] Embodiment 28: The method of embodiment 26, wherein the combined feed is added every other day from day 2 of culturing the cells.

[0135] Embodiment 29: The method of embodiment 26, wherein the combined feed is added every fourth day from day 2 of culturing the cells.

[0136] Embodiment 30: The method of embodiment 29, wherein the combined feed is added after 2, 6, 10, 14, and 18 days of culturing the cells. [0137] Embodiment 31 : The method of embodiment 30, wherein about 2% to about 4% v/v of the combined feed is added to the cell culture.

[0138] Embodiment 32: The method of embodiment 31, wherein about 4% v/v of the combined feed is added to the cell culture.

[0139] Embodiment 33: The method of embodiment 21, wherein the combined feed has a feed strength of 1 ,5X.

[0140] Embodiment 34: The method of embodiment 33, wherein the combined feed contains about 56.1 g/L BalanCD CHO Feed 4 and about 17.5 g/L Cell Boost 5.

[0141] Embodiment 35: The method of embodiment 33, wherein the combined feed is added every other day from day 2 of culturing the cells.

[0142] Embodiment 36: The method of embodiment 33, wherein the combined feed is added every fourth day from day 2 of culturing the cells.

[0143] Embodiment 37: The method of embodiment 36, wherein the combined feed is added after 2, 6, 10, 14, and 18 days of culturing the cells.

[0144] Embodiment 38: The method of embodiment 37, wherein about 2% to about 4% v/v of the combined feed is added to the cell culture.

[0145] Embodiment 39: The method of embodiment 38, wherein about 4% v/v of the combined feed is added to the cell culture.

[0146] Embodiment 40: The method of embodiment 16, wherein the second culture medium is Nutrient feed solution.

[0147] Embodiment 41 : The method of embodiment 40, wherein a fixed volume of Nutrient feed solution is added to the cell culture.

[0148] Embodiment 42: The method of any one of embodiments 1 to 4, wherein the cells are harvested from the cell culture when culture viability drops below 75% or on Day 22 of the bioprocess, whichever occurs earlier.

[0149] Embodiment 43 : The method of any one of embodiments 1 to 4, wherein at least 1.0 to 3.0 g/L of recombinant protein is isolated from the harvested cells in the cell culture.

[0150] Embodiment 44: The method of embodiment 43, wherein at least 2.2 g/L of recombinant protein is isolated from the harvested cells in the cell culture.

[0151] Embodiment 45: The method of embodiment 43, wherein at least 2.2 g/L of recombinant protein is isolated from the harvested cells in the cell culture.

[0152] Embodiment 46: The method of embodiment 43, wherein at least 2.5 g/L of recombinant protein is isolated from the harvested cells in the cell culture. [0153] Embodiment 47: The method of embodiment 43, wherein at least 3.0 g/L of recombinant protein is isolated from the harvested cells in the cell culture.

[0154] Embodiment 48: The method of any one of embodiments 1 to 4, wherein host cell protein (HCP) contaminants in the harvested cells are reduced as compared with HCP contaminants in cells harvested in a standard process.

[0155] Embodiment 49: The method of embodiment 48, wherein the HCP contaminants are high-risk HCP contaminants.

[0156] Embodiment 50: The method of embodiment 49, wherein the high-risk HCP contaminants include one or more of 78 kDa glucose regulated protein (GRP78, BiP), lysosomal phospholipase A2 (LPLA2), and/or liver carboxylesterase 1.

[0157] Embodiment 51: A method of producing an anti-IL-23p 19 antibody comprising :

(a) preparing a cell culture by seeding a container containing a first culture medium with cells capable of expressing the recombinant protein;

(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;

[0158] wherein the anti-IL-23pl9 antibody comprises:

(i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 3, 4, and 5; and

(ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 6, 7, and 8; and

[0159] wherein host cell protein (HCP) contaminants in the harvested cells are reduced as compared with HCP contaminants in cells harvested in a standard process.

Claims

WHAT IS CLAIMED IS:

Claim 1: A method of producing an anti-IL-23 antibody comprising:

(a) preparing a cell culture by seeding a container containing a first culture medium with cells capable of expressing the recombinant protein;

(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.

Claim 2: A method of producing an anti-IL-23 antibody comprising:

(a) preparing and scaling up a seed cell culture capable of expressing the recombinant protein in a suitable culture medium;

(b) inoculating and culturing the seed cell culture in a suitable production medium to prepare a cell culture;

(c) lowering the temperature of the cell culture;

(d) periodically adding feed 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.

Claim 3: A method of producing an anti-IL-23pl9 antibody comprising:

(a) preparing a cell culture by seeding a container containing a first culture medium with cells capable of expressing the recombinant protein;

(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. Claim 4: A method of producing an anti-IL-23pl9 antibody comprising:

(a) preparing and scaling up a seed cell culture capable of expressing the recombinant protein in a suitable culture medium;

(b) inoculating and culturing the seed cell culture in a suitable production medium to prepare a cell culture;

(c) lowering the temperature of the cell culture;

(d) periodically adding feed 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.

Claim 5: The method of either claim 3 or claim 4, wherein the anti-IL-23pl9 antibody comprises:

(i) a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 1, and

(ii) a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

Claim 6: The method of either claim 3 or claim 4, wherein the anti-IL-23pl9 antibody comprises:

(i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 3, 4, and 5; and

(ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 6, 7, and 8.

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

Claim 8: The method of either claim 1 or claim 3, wherein the container is seeded with

0.3 xlO⁶ to 0.9 xlO⁶ viable cells/mL.

Claim 9: The method of any one of claims 1 to 6, wherein the temperature is lowered when the viable cell count in the cell culture reaches between 2.3 x 10⁶ and 3.0 x 10⁶ cells/mL. Claim 10: The method of claim 9, wherein the temperature is lowered when the viable cell count in the cell culture reaches 2.5 x 10⁶ cells/mL.

Claim 11 : The method of any one of claims 1 to 6, wherein the temperature is lowered prior to or at about 72 hours of culturing the cells.

Claim 12: The method of either claim 1 or claim 3, wherein the second culture medium is added every other day from day 2 of culturing the cells.

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

Claim 14: The method of either claim 1 or claim 3, wherein the second culture medium is added every fourth day from day 2 of culturing the cells.

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

Claim 16: The method of either claim 1 or claim 3, wherein the second culture medium is DME/F12, Nutrient feed solution, BalanCD CHO Feed 4, or Cell Boost 5.

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

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

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

Claim 20: The method of claim 19, wherein the combined feed contains about 112 g/L

BalanCD CHO Feed 4 and about 35 g/L Cell Boost 5. Claim 21 : The method of claim 20, wherein the combined feed is added every other day from day 2 of culturing the cells.

Claim 22: The method of claim 21, wherein the combined feed is added every fourth day from day 2 of culturing the cells.

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

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

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

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

Claim 27: The method of claim 26, wherein the combined feed contains about 84.0 g/L BalanCD CHO Feed 4 and about 26.25 g/L Cell Boost 5.

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

Claim 29: The method of claim 26, wherein the combined feed is added every fourth day from day 2 of culturing the cells.

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

Claim 31 : The method of claim 30, wherein about 2% to about 4% v/v of the combined feed is added to the cell culture. Claim 32: The method of claim 31, wherein about 4% v/v of the combined feed is added to the cell culture.

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

Claim 34: The method of claim 33, wherein the combined feed contains about 56.1 g/L BalanCD CHO Feed 4 and about 17.5 g/L Cell Boost 5.

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

Claim 36: The method of claim 33, wherein the combined feed is added every fourth day from day 2 of culturing the cells.

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

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

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

Claim 40: The method of claim 16, wherein the second culture medium is Nutrient feed solution.

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

Claim 42: The method of any one of claims 1 to 4, wherein the cells are harvested from the cell culture when culture viability drops below 75% or on Day 22 of the bioprocess, whichever occurs earlier. Claim 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 the harvested cells in the cell culture.

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

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

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

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

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

Claim 49: The method of claim 48, wherein the HCP contaminants are high-risk HCP contaminants.

Claim 50: The method of claim 49, wherein the high-risk HCP contaminants include one or more of 78 kDa glucose regulated protein (GRP78, BiP), lysosomal phospholipase A2 (LPLA2), and/or liver carboxylesterase 1.

Claim 51: A method of producing an anti-IL-23p 19 antibody comprising :

(a) preparing a cell culture by seeding a container containing a first culture medium with cells capable of expressing the recombinant protein;

(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; wherein the anti-IL-23pl9 antibody comprises:

(i) a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 3, 4, and 5; and

(ii) a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 6, 7, and 8; and wherein host cell protein (HCP) contaminants in the harvested cells are reduced as compared with HCP contaminants in cells harvested in a standard process.