Methods for producing ustekinumab

Abstract

To provide methods for producing an ustekinumab product with a target levels of one or more glycans.SOLUTION: Disclosed is a method of manufacturing a pharmaceutical composition comprising ustekinumab having a target level of G0F glycan, the method comprising: (a) selecting a target level of G0F glycan, (b) selecting a level of galactose and a time for cell culture to provide the selected level of G0F glycan, where the level of galactose and the time are inversely related; (c) culturing a population of cells genetically engineered to express ustekinumab under conditions comprising the selected level of galactose and time; (d) harvesting ustekinumab expressed by the population of cells, thereby producing the preparation of ustekinumab; and (e) purifying, concentrating, and / or formulating the ustekinumab preparation to produce pharmaceutical composition comprising ustekinumab if the preparation meets the target level of G0F glycan.SELECTED DRAWING: None

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 786,821, filed on Dec. 31, 2018, which is hereby incorporated by reference in its entirety.

[0002] (Sequence Listing) This application has been electronically filed in ASCII format and includes a sequence listing that is hereby incorporated by reference in its entirety into this specification. The above ASCII copy was created on Dec. 20, 2019, has the name M0168PCT_SL.txt, and is 6,036 bytes in size.

Background Art

[0003] Therapeutic antibodies are an important class of therapeutic biological products. The glycosylation and glycan composition of antibodies can affect antibody activity and effector functions. Improved methods for controlling the glycan profile of antibody products are still needed.

Summary of the Invention

Means for Solving the Problems

[0004] The present disclosure provides a process lever for generating (e.g., manufacturing) ustekinumab having a target level of one or more glycans. The present disclosure provides the insight that the relationship between the galactose concentration in the culture medium and the culture time can be used as a process lever for controlling the target level of one or more glycans (e.g., galactosylation) of ustekinumab in the culture. The present disclosure provides a process for controlling the target level of one or more glycans ​​​​​​​Identify the inverse relationship between the culture time and the galactose concentration in the culture medium in the control of the target level (e.g., galactosylation).

[0005] In certain embodiments, the disclosure provides a method for producing ustekinumab having a target level of one or more glycans. Such a method provides (e.g., generates, expresses (e.g., in small-scale or large-scale cell culture), and / or manufactures) or obtains (receives and / or purchases from a third party (including a contractually related third party or an unrelated (e.g., independent) third party)) an ustekinumab test protein (e.g., an ustekinumab drug substance, e.g., a preparation of an ustekinumab drug substance, e.g., a batch of an ustekinumab drug substance). (e.g., an ustekinumab drug substance, e.g., a preparation of an ustekinumab drug substance, e.g., a batch of an ustekinumab drug substance). (e.g., in cell culture), and / or manufactures), or obtains (receives and / or purchases from a third party (including a contractually related third party or an unrelated (e.g., independent) third party)). It may include receiving and / or purchasing).

[0006] In some examples, the disclosure provides a method for manufacturing a pharmaceutical composition comprising ustekinumab having a target level of one or more glycans, the method comprising selecting the level of galactose and the time of cell culture (wherein the level of galactose and the time are inversely proportional), culturing a population of cells genetically engineered to express ustekinumab under conditions comprising the selected level of galactose and time, collecting the ustekinumab expressed by the population of cells, thereby generating a preparation of ustekinumab, and, if the preparation meets the target level of one or more glycans, purifying, concentrating, and / or formulating the ustekinumab preparation to produce a pharmaceutical composition comprising ustekinumab. (wherein the level of galactose and the time are inversely proportional), culturing a population of cells genetically engineered to express ustekinumab, collecting the ustekinumab expressed by the population of cells, thereby generating a preparation of ustekinumab, and, if the preparation meets the target level of one or more glycans, purifying, concentrating, and / or formulating the ustekinumab preparation to produce a pharmaceutical composition comprising ustekinumab. In some embodiments, the target level of one or more glycans is a glycan target level selected from the group consisting of G0F, sialylated glycans, and G2F. ​​​​​​​​ It is. In some embodiments, the target level of one or more glycans is the target level of G0F is the target level.

[0007] In some examples, the present disclosure provides a method for manufacturing a pharmaceutical composition comprising ustekinumab having a target level of G0F glycans, comprising selecting a target level of G0F glycans, selecting the levels of galactose and the time of cell culture to provide the selected target level of G0F glycans (where the level of galactose and the time are inversely proportional), culturing a population of cells genetically engineered to express ustekinumab under conditions comprising the selected levels of galactose and time, collecting the ustekinumab expressed by the population of cells, thereby generating a preparation of ustekinumab, and if the preparation meets the target level of G0F glycans, purifying, concentrating, and / or formulating the ustekinumab preparation to generate a pharmaceutical composition comprising ustekinumab.

[0008] In some examples, the present disclosure provides a method for generating an ustekinumab drug product having a target level of one or more glycans, comprising culturing a population of cells genetically engineered to express ustekinumab under conditions characterized by parameters comprising the selected level of galactose and the time of cell culture (where the level of galactose and the time are inversely proportional), collecting the ustekinumab expressed by the cells, thereby generating a preparation of ustekinumab, and if the ustekinumab preparation meets the target level of one or more glycans, purifying, concentrating, and / or formulating the ustekinumab preparation. To provide a method, including generating ustekinumab pharmaceutical products. In some embodiments, the target level of one or more glycans is the target level of glycans selected from the group consisting of G0F, sialylated glycans , and G2F. In some embodiments, the target level of one or more glycans is the target level of G0F.

[0009] In some embodiments, the provided method includes culturing a population of mammalian cells genetically engineered to express ustekinumab. In some embodiments, the mammalian cells are selected from CHO cells, HEK 293 cells, fibrosarcoma HT 1080 cells, PER.C6 cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0 cells, and SP 2 / 0 cells.

[0010] In some embodiments, in the provided method, the culturing step is performed using a continuous culture process. In some embodiments, the provided method includes culturing using a perfusion culture process (e.g., an alternating tangential flow filter (ATF)-based perfusion culture process). In some specific embodiments, the provided method includes culturing mammalian cells genetically engineered to express ustekinumab ( e.g., SP 2 / 0 cells expressing ustekinumab) by a perfusion culture process.

[0011] In some embodiments, the provided method includes collecting the ustekinumab expressed by the cells at two or more time points within the time range of the cell culture.

[0012] In some embodiments, the target level of G0F glycan is 20% with respect to total glycans It is within the range of ~80% G0F. In some embodiments, the target level of G0F glycan is within the range of 25% - 65% G0F relative to the total glycan.

[0013] In some embodiments, the provided method is for the production and / or generation of ustekinumab having a target level of G0F glycan within the range of 20% - up to 40% of G0F relative to the total glycan. In some embodiments, to achieve ustekinumab having a target level of G0F glycan within the range of 20% - up to 40% of G0F relative to the total glycan, the method includes a selected galactose level of 0 mM, and the cell culture time is in the range of 7 days to 15 days.

[0014] In some embodiments, the provided method is for the production and / or generation of ustekinumab having a target level of G0F glycan within the range of 40% - 80% G0 F relative to the total glycan. In some embodiments, to achieve ustekinumab having a target level of G0F glycan within the range of 40% - 80% G0F relative to the total glycan, the method includes a selected galactose level in the range of 1 5 mM - 30 mM and a cell culture time in the range of 16 days to 60 days. In some specific embodiments, the cell culture time is in the range of 2 5 days - 42 days.

[0015] In some embodiments, the provided method includes selecting a target level of G0F glycan. In some embodiments, the target level of G0F glycan is within the range of 20% - 80% G0F relative to the total glycan. In some specific embodiments, the G0F gly ​​​​​​The target level of the can is within the range of 20% to a maximum of 40% G0F for all glycans, The method includes a level of selected galactose of 0 mM and a cell culture time within the range of 7 days to 15 days. In some specific embodiments, the target level of G0F glycan is within the range of 40% to 80% G0F for all glycans, and the method includes a selected galactose level within the range of 15 mM to 30 m M and a cell culture time within the range of 16 days to 60 days.

[0016] In some embodiments, in the provided method, the selected galactose level is controlled throughout the culture process (e.g., controlled during culture from t = 0 to collection) .

[0017] In some embodiments, the provided method further includes measuring the target level of G0F glycan. In some embodiments, when the measured level of G0F glycan is within the range of 20% to 80% G0F for all glycans (e.g., within the range of 20% to a maximum of 40% G0F for all glycans, e.g., within the range of 40% to 80% G0F for all glycans) , a step of purifying, concentrating, and / or formulating the ustekinumab preparation is performed. These and other aspects of the invention are described in more detail below and in the claims.

[0018] These and other aspects of the invention are described in more detail below and in the claims.

Brief Description of the Drawings

[0019] The drawings included herein, which are composed of the following figures, are for illustrative purposes only and not for limiting purposes.

Figure 1

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Figure 3-2

[0020] Specific definitions In general, the terms used in this specification shall follow the meanings understood in the relevant technical field, unless otherwise clearly indicated. Explicit definitions of specific terms are provided below. Throughout this specification, the meanings of these and other terms in specific examples will be apparent to those skilled in the art from the context. Throughout this specification, the meanings of these and other terms in specific examples will be apparent to those skilled in the art from the context.

[0021] To make the present invention more easily understood, specific terms are defined initially below. Further definitions of the following terms and other terms are described throughout this specification.

[0022] As used herein, the term “about” or “approximately” when applied to one or more values of interest refers to a value similar to the specified reference value. In certain embodiments, the term “about” or “approximately” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the specified reference value.

[0023] The terms “control, controlled, controlling” as used herein, when referring to one or more glycans (such as galactose, such as G0F) of ustekinumab, refer to the control of the target level of the glycans. ​​​​​​As used herein with respect to, selecting, maintaining, and / or adjusting one or more culture conditions for generating ustekinumab. Adjusting may include increasing or decreasing one or more culture conditions for generating ustekinumab. As used herein, a controlled target level of one or more glycans relates to generating ustekinumab having a desired level of one or more glycans with minimal product drift. In some embodiments, the controlled target level of G0F varies by 20%, 15%, 10%, or 5% or less between samples in the same production run and / or batch. In some embodiments, controlling the target level of one or more glycans ensures consistency in the generation of ustekinumab (e.g., consistency between batches, consistency across samples from any particular production process).

[0024] As used herein, "glycan" refers to a compound containing at least one sugar residue (e.g., monosaccharide). A glycan may be a monomer or polymer of sugar residues and may be linear or branched. "Glycan" includes natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6'- sulfonato N-acetylglucosamine, etc.). The term glycan includes homopolymers and heteropolymers of sugar residues. The term "glycan" also includes , including glycan components such as glycolipids and proteoglycans. This term also encompasses free glycans that have been cleaved from or otherwise released from glycoconjugates.

[0025] As used herein, "galactosylated glycan" refers to a glycan that contains at least one galactose -sugar residue. In some embodiments, the galactosylated glycan is , G1, G2, G1F, G2F, A1, and / or A2 glycan. Non-galactosylated glycans include G0F or G0. In some embodiments, the target level of galactosylated glycan can refer to the presence of galactosylated glycan (e.g., G2F) and / or the target level of non-gal actosylated glycan (e.g., G0F).

[0026] The term "isolated", as used herein, (1) is separated from at least a portion of the components it was associated with when first produced, and / or (2) refers to a substance and / or entity that has been designed, generated, prepared, and / or manufactured by human hands. An isolated substance and / or entity can be separated from about 10%, about 20%, about 30% of the other components it was originally associated with, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92% , about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 9 9%. In some embodiments, the isolated agent is about 80%, about 85 %, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97 %, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" when it substantially does not contain other components. In some embodiments, those skilled in the art ... ... As will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., , buffer, solvent, water, etc.). In such embodiments, the isolation rate or purity of the substance is calculated without including such carriers or excipients. By way of example, in some embodiments, a biological polymer such as a polypeptide or polynucleotide that occurs in nature is "isolated" if a) it is not associated with some or all of the components that are naturally associated with it in its natural state due to its origin or source, b) it does not substantially contain other polypeptides or nucleic acids of the same species that produce it in nature, and c) it is expressed by components from a cell or other expression system that is not of the species that produces it in nature or is otherwise associated with it. Thus, for example, in some embodiments , a polypeptide that is chemically synthesized or synthesized in a cell line different from the one that produces it in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques is considered an "isolated" polypeptide as long as it is separated from a) components that are naturally associated with it and / or b) other components that were associated with it when it was first produced. As used herein, a "process lever" refers to an element of a culture process (e.g., one or more culture conditions) that can be controlled to increase or decrease the abundance of one or more glycans in an antibody product. The present disclosure relates to galactose in a culture medium . .

[0027] As used herein, a "process lever" refers to an element of a culture process (e.g., one or more culture conditions) that can be controlled to increase or decrease the abundance of one or more glycans in an antibody product. Provide a novel process lever that is a specific relationship between glucose concentration and culture time. This specification As described herein, the inverse relationship between the glucose concentration in the culture medium and the time of culture in the culture medium is used as a process lever to produce ustekinumab having a target level of one or more glycans (e.g., gal actosylation). In some embodiments, the process lever includes a selected galactose level and the time of cell culture to control the level of one or more glycans (e.g., galactosylation, e.g., G0F) of ustekinumab (e.g., the level of one or more glycans in the culture or preparation of ustekinumab).

[0028] As used herein, "N-glycosylation site of the Fc region" refers to an amino acid residue within the Fc region to which a glycan is N-linked. In some specific embodiments, the N-glycosylation site of ustekinumab is located at position Asn299 of the heavy chain.

[0029] Generally, "protein" as used herein is a polypeptide (i.e., a chain of at least two amino acids linked to each other by peptide bonds). A protein may contain moieties other than amino acids (e.g., it may be a glycoprotein), and / or may be processed or modified in other ways. One of ordinary skill in the art will understand that "protein" may be a complete polypeptide chain produced by a cell (with or without a signal sequence), or a functional portion thereof. One of ordinary skill in the art will understand that a protein may sometimes be linked, for example, by one or more disulfide bonds ​​​​and may comprise more than one polypeptide chain that is associated by covalent bonding or otherwise will be further understood

[0030] As used herein, "recovering" refers to, for example, using purification techniques known in the art to make an agent or entity substantially free of other previously associated components, e.g., by isolation. In some embodiments, the agent or entity is recovered from a natural source and / or a source comprising cells

[0031] As used herein, "sample" refers to separately generated samples. In some embodiments, the evaluation of separate samples includes the evaluation of samples from the same culture run or different culture runs (e.g., different culture rounds) at different times during preparation (e.g.,)

[0032] "Target value or target level" as used herein refers to a predetermined level of one or more specific glycans, such as galactosylated glycans and / or sialylated glycans. In some embodiments, the target value is the level of galactosylated glycans (e.g., G0, G1, G2, G0F, G1F, G2F, or combinations) and / or sialylated glycans (e.g., monosialylated, disialylated, or combinations) described in the specifications or master batch records for a reference ustekinumab product or for a pharmaceutical product. In some specific embodiments, the target value is the level of G0F glycan in an ustekinumab product

[0033] ​​​​​​​​​​​​​In some embodiments, the target value is one or more in the ustekinumab preparation of glycans (e.g., galactosylated glycans (e.g., one of the galactosylated glycans or two or more species) and / or sialylated glycans (e.g., one of the sialylated glycans or two or more species)) absolute level (e.g., number of moles). In some embodiments , the target value is relative to the total level of glycans in the ustekinumab preparation, one or more glycans in the ustekinumab preparation (e.g., galactosylated glycans (e.g. for example, one or more species of galactosylated glycans) and / or sialylated glycans (e.g., one or more species of sialylated glycans)). In some embodiments, the target value is expressed as a "percentage", which is one or more glycans relative to the total moles of glycans (e.g., Fc glycans) in the ustekinumab preparation (e.g., Fc glycans). In some embodiments, the "percentage" is , one or more PNGase F released relative to the total moles of Fc glycans released by the detected PNGase F number of moles of Fc glycans.

[0034] The term "ustekinumab preparation" as used herein refers to a mixture of ustekinumab proteins obtained according to a specific production method. The ustekinumab protein in the ustekinumab preparation contains multiple copies of ustekinumab (i.e., having the same or substantially the same amino acid sequence), but includes a mixture of glycans associated with the protein. In some examples, using the methods and / or systems provided herein, usteki numab Prepare a numab preparation. The production method involves genetically engineered cultured cells that express (or express numab under related levels or related conditions). It may include a recombinant preparation step using For example, by lysing the cells and pelleting the protein components by centrifugation), it may include an isolation step of isolating numab from specific components of the genetically engineered cells. In some embodiments, the production method may also involve other cell components used in earlier steps, such as other proteins or organic components, to separate numab (e.g., by chromatography). These steps are non-limiting, and it will be understood that any number of additional production steps may be included. Different opportunities (e.g., different runs or preparations), but the same production method can be used to prepare different numab preparations. Alternatively, different production methods can be used to prepare different numab preparations. Two production methods may differ in some aspects (e.g., expression vectors, genetically engineered cell types, culture conditions, isolation procedures, purification conditions, etc.).

[0035] All documents and similar materials cited in this application, including but not limited to patents, patent applications, papers, books, monographs, and web pages, are hereby expressly incorporated by reference in their entirety, regardless of the format of such documents and similar materials. For those including but not limited to defined terms, usage of terms, described techniques, etc., if one or more of the incorporated documents and similar materials are different from or conflict with this application, ... If so, this application is given priority. The headings of the chapters used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described in any way. should not be construed as limiting the subject matter described in any way. should not be construed as limiting the subject matter described in any way.

Best Mode for Carrying Out the Invention

[0036] The present disclosure describes, at least in part, the discovery of a lever for generating ustekinumab having a target level of one or more glycans (e.g., galactosylation, e.g., G0F glycan). Controlling the glycan composition and levels during antibody production is an ongoing challenge. The glycosylation of therapeutic antibodies can affect their safety and / or efficacy. (Zhang et al. (2016) Drug Discovery Today 21(5):740 - 765). Therefore, it is important to be able to ensure consistency in the glycan composition in the production of ustekinumab (e.g., batch - to - batch consistency, consistency across the entire sample from any particular production process). During the characterization of the ustekinumab reference protein product ("RPP"), two distinct glycan populations of the ustekinumab RPP were observed. lactosylation, e.g., G0F glycan). Controlling the glycan composition and levels during antibody production is an ongoing challenge. The glycosylation of therapeutic antibodies can affect their safety and / or efficacy. (Zhang et al. (2016) Drug Discovery Today 21(5):740 - 765). Therefore, it is important to be able to ensure consistency in the glycan composition in the production of ustekinumab (e.g., batch - to - batch consistency, consistency across the entire sample from any particular production process). During the characterization of the ustekinumab reference protein product ("RPP"), two distinct glycan populations of the ustekinumab RPP were observed. lactosylation, e.g., G0F glycan). Controlling the glycan composition and levels during antibody production is an ongoing challenge. The glycosylation of therapeutic antibodies can affect their safety and / or efficacy. (Zhang et al. (2016) Drug Discovery Today 21(5):740 - 765). Therefore, it is important to be able to ensure consistency in the glycan composition in the production of ustekinumab (e.g., batch - to - batch consistency, consistency across the entire sample from any particular production process). During the characterization of the ustekinumab reference protein product ("RPP"), two distinct glycan populations of the ustekinumab RPP were observed. lactosylation, e.g., G0F glycan). Controlling the glycan composition and levels during antibody production is an ongoing challenge. The glycosylation of therapeutic antibodies can affect their safety and / or efficacy. (Zhang et al. (2016) Drug Discovery Today 21(5):740 - 765). Therefore, it is important to be able to ensure consistency in the glycan composition in the production of ustekinumab (e.g., batch - to - batch consistency, consistency across the entire sample from any particular production process). During the characterization of the ustekinumab reference protein product ("RPP"), two distinct glycan populations of the ustekinumab RPP were observed. lactosylation, e.g., G0F glycan). Controlling the glycan composition and levels during antibody production is an ongoing challenge. The glycosylation of therapeutic antibodies can affect their safety and / or efficacy. (Zhang et al. (2016) Drug Discovery Today 21(5):740 - 765). Therefore, it is important to be able to ensure consistency in the glycan composition in the production of ustekinumab (e.g., batch - to - batch consistency, consistency across the entire sample from any particular production process). During the characterization of the ustekinumab reference protein product ("RPP"), two distinct glycan populations of the ustekinumab RPP were observed. lactosylation, e.g., G0F glycan). Controlling the glycan composition and levels during antibody production is an ongoing challenge. The glycosylation of therapeutic antibodies can affect their safety and / or efficacy. (Zhang et al. (2016) Drug Discovery Today 21(5):740 - 765). Therefore, it is important to be able to ensure consistency in the glycan composition in the production of ustekinumab (e.g., batch - to - batch consistency, consistency across the entire sample from any particular production process). During the characterization of the ustekinumab reference protein product ("RPP"), two distinct glycan populations of the ustekinumab RPP were observed. lactosylation, e.g., G0F glycan). Controlling the glycan composition and levels during antibody production is an ongoing challenge. The glycosylation of therapeutic antibodies can affect their safety and / or efficacy. (Zhang et al. (2016) Drug Discovery Today 21(5):740 - 765). Therefore, it is important to be able to ensure consistency in the glycan composition in the production of ustekinumab (e.g., batch - to - batch consistency, consistency across the entire sample from any particular production process). During the characterization of the ustekinumab reference protein product ("RPP"), two distinct glycan populations of the ustekinumab RPP were observed. lactosylation, e.g., G0F glycan). Controlling the glycan composition and levels during antibody production is an ongoing challenge. The glycosylation of therapeutic antibodies can affect their safety and / or efficacy. (Zhang et al. (2016) Drug Discovery Today 21(5):740 - 765). Therefore, it is important to be able to ensure consistency in the glycan composition in the production of ustekinumab (e.g., batch - to - batch consistency, consistency across the entire sample from any particular production process). During the characterization of the ustekinumab reference protein product ("RPP"), two distinct glycan populations of the ustekinumab RPP were observed. lactosylation, e.g., G0F glycan). Controlling the glycan composition and levels during antibody production is an ongoing challenge. The glycosylation of therapeutic antibodies can affect their safety and / or efficacy. (Zhang et al. (2016) Drug Discovery Today 21(5):740 - 765). Therefore, it is important to be able to ensure consistency in the glycan composition in the production of ustekinumab (e.g., batch - to - batch consistency, consistency across the entire sample from any particular production process). During the characterization of the ustekinumab reference protein product ("RPP"), two distinct glycan populations of the ustekinumab RPP were observed. lactosylation, e.g., G0F glycan). Controlling the glycan composition and levels during antibody production is an ongoing challenge. The glycosylation of therapeutic antibodies can affect their safety and / or efficacy. (Zhang et al. (2016) Drug Discovery Today 21(5):740 - 765). Therefore, it is important to be able to ensure consistency in the glycan composition in the production of ustekinumab (e.g., batch - to - batch consistency, consistency across the entire sample from any particular production process). During the characterization of the ustekinumab reference protein product ("RPP"), two distinct glycan populations of the ustekinumab RPP were observed.

[0037] The present disclosure describes the development of a lever for controlling the glycan composition during the manufacture of ustekinumab. A process lever for generating ustekinumab having a target level of a particular glycan, including each RPP for which a glycan profile has been identified, is described herein. In the process of developing the lever as described, the present disclosure identified a relationship between the galactose concentration in the culture medium and the time of culture. The present disclosure describes the relationship between the galactose concentration in the culture medium and the time of culture. The present disclosure describes the development of a lever for controlling the glycan composition during the manufacture of ustekinumab. A process lever for generating ustekinumab having a target level of a particular glycan, including each RPP for which a glycan profile has been identified, is described herein. In the process of developing the lever as described, the present disclosure identified a relationship between the galactose concentration in the culture medium and the time of culture. The present disclosure describes the relationship between the galactose concentration in the culture medium and the time of culture. The present disclosure describes the development of a lever for controlling the glycan composition during the manufacture of ustekinumab. A process lever for generating ustekinumab having a target level of a particular glycan, including each RPP for which a glycan profile has been identified, is described herein. In the process of developing the lever as described, the present disclosure identified a relationship between the galactose concentration in the culture medium and the time of culture. The present disclosure describes the relationship between the galactose concentration in the culture medium and the time of culture. The present disclosure describes the development of a lever for controlling the glycan composition during the manufacture of ustekinumab. A process lever for generating ustekinumab having a target level of a particular glycan, including each RPP for which a glycan profile has been identified, is described herein. In the process of developing the lever as described, the present disclosure identified a relationship between the galactose concentration in the culture medium and the time of culture. The present disclosure describes the relationship between the galactose concentration in the culture medium and the time of culture. The present disclosure describes the development of a lever for controlling the glycan composition during the manufacture of ustekinumab. A process lever for generating ustekinumab having a target level of a particular glycan, including each RPP for which a glycan profile has been identified, is described herein. In the process of developing the lever as described, the present disclosure identified a relationship between the galactose concentration in the culture medium and the time of culture. The present disclosure describes the relationship between the galactose concentration in the culture medium and the time of culture. The relationship between the sialic acid concentration and the time of culture (e.g., continuous culture) is used as a process lever for controlling the target level of one or more glycans (e.g., galactosylation) in the culture. This provides the insight that it can be used.

[0038] The present disclosure further provides the insight that the relationship between the galactose level and the time (i.e., the culture period) is inversely proportional in order to control the glycan level (e.g., galactosylation). The culture method of the present disclosure for controlling the glycan composition includes a continuous culture method (e.g., perfusion culture).

[0039] ustekinumab The present disclosure provides, in part, methods and processes for manufacturing, formulating, controlling, or otherwise making ustekinumab having a particular glycan profile.

[0040] Ustekinumab is an antibody that specifically binds to the p-40 subunits of both IL-12 and IL-23. Ustekinumab has been studied in a number of human diseases including psoriasis, psoriatic arthritis, Crohn's disease, and multiple sclerosis.

[0041] In some embodiments, ustekinumab comprises HCDR1, HCDR2, and HCDR3 sequences that differ from the HCDR sequences set forth in SEQ ID NO: 1 by three or fewer amino acid residues, and LCDR1, LCDR2, and LCDR3 sequences that differ from the LCDR sequences set forth in SEQ ID NO: 2 by three or fewer amino acid residues. In some embodiments, ustekinumab comprises HCDR1, HCDR2, and HCDR3 sequences that differ from the HCDR sequences set forth in SEQ ID NO: 1 by two or fewer amino acid residues or one or fewer amino acid residues, and LCDR1, LCDR2, and LCDR3 sequences that differ from the LCDR sequences set forth in SEQ ID NO: 2 by two or fewer amino acid residues. ​​​​​​​​​ LCDR1, LCDR2, and LCDR3 sequences that differ by one or fewer amino acid residues or by one or fewer amino acid residues. In some embodiments, ustekinumab comprises a heavy chain variable domain that differs from the sequence set forth in SEQ ID NO: 1 by three or fewer amino acid residues and a light chain variable domain that differs from the sequence set forth in SEQ ID NO: 2 by three or fewer amino acid residues. In some

[0042] embodiments, ustekinumab comprises an HCDR1, HCD R2, and HCDR3 sequences set forth in SEQ ID NO: 1 and an LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NO: 2. In some embodiments, ustekinumab comprises a heavy chain variable domain set forth in SEQ ID NO: 1 and a light chain variable domain set forth in SEQ ID NO: 2. In some embodiments, ustekinumab

[0043] comprises a heavy chain comprising the sequence of SEQ ID NO: 1 and / or a light chain comprising the sequence of SEQ ID NO: 2.

[0044]

Chemical Structure

[0045] SEQ ID NO: 2 - Ustekinumab light chain sequence (bold indicates the variable domain sequence, and the CDR sequences are underlined)

[0046]

Chemical Structure

[0047] Culture method In some examples, the present disclosure provides a method for the preparation of a mouse model of a mouse having a target level of one or more glycans. A method for producing a pharmaceutical composition comprising tekinumab, comprising: Selecting the time of feeding (where galactose level and time are inversely proportional), The antibody was designed to express ustekinumab under selected conditions, including selected levels of galactose and time. By culturing a population of genetically engineered cells, the population of cells expresses the Ustekinuma and collecting the samples, thereby producing a preparation of ustekinumab, and Purify, concentrate, and / or enrich ustekinumab preparations if they meet target levels of two or more glycans. and / or formulating to produce a pharmaceutical composition comprising ustekinumab. In some embodiments, the target level of one or more glycans is G Target levels of glycans selected from the group consisting of 0F, sialylated glycans, and G2F. In some embodiments, the target level of one or more glycans is G0F This is the target level.

[0048] In some examples, the present disclosure provides a method for the preparation of a mouse model of a mouse having a target level of one or more glycans. A method for producing a tekinumab drug product comprising the steps of: The cells express ustekinumab under conditions characterized by parameters including the time of culture. Cultivating a population of cells genetically engineered to produce a galactose-dependent The cells were then harvested to collect the expressed ustekinumab, which was then used to Generating a ustekinumab preparation, and purifying, concentrating, and / or formulating the ustekinumab preparation to produce a ustekinumab drug product when the target level of one or more glycans is met. Provided is a method. In some embodiments, the target level of one or more glycans is the target level of a glycan selected from the group consisting of G0F, sialylated glycan, and G2F. In some embodiments, the target level of one or more glycans is the target level of G0F. In some embodiments, the target level of G0F glycan is within the range of 20% to 80% G0F with respect to total glycans. In some embodiments, the target level of G0F glycan is within the range of 25% to 65% G0F with respect to total glycans. In some embodiments, the provided method aims to manufacture and / or generate ustekinumab having a target level of G0F glycan within the range of 20% to a maximum of 40% G0F with respect to total glycans. In some embodiments, the method for achieving ustekinumab having a target level of G0F glycan within the range of 20% to a maximum of 40% G0F with respect to total glycans includes a level of selected galactose of 0 mM and a cell culture time within the range of 7 days to 15 days. In some embodiments, the provided method aims to manufacture and / or generate ustekinumab having a target level of G0F glycan within the range of 40% to 80% G0F with respect to total glycans. In some embodiments, the range of 40% to 80% G0F with respect to total glycans

[0049]

[0050]

[0051] ​​​​​​​​​​​​​A method for realizing ustekinumab having a target level of G0F glycan within a circle is as follows: 1 It includes a selected galactose level within the range of 5 mM to 30 mM and a cell culture time within the range of 16 days to 60 days. In some specific embodiments, the cell culture time is within the range of 2 25 days to 42 days.

[0052] In some embodiments, the provided method further includes selecting a target level of G0F glycan. In some embodiments, the target level of G0F glycan is within the range of 20% to 80% G0F relative to the total glycan. In some specific embodiments, the target level of G0 F glycan is within the range of 20% to a maximum of 40% G0F relative to the total glycan, and the method includes a selected galactose level of 0 mM and a cell culture time within the range of 7 days to 15 days. In some specific embodiments, the target level of G0F glycan is within the range of 40% to 80% G0F relative to the total glycan, and the method includes a selected galactose level within the range of 15 mM to 30 mM and a cell culture time within the range of 16 days to 60 days. In some embodiments, the provided method further includes measuring the target level of G0F glycan. In some embodiments, when the measured level of G0F glycan is within the range of 20% to 80% G0F relative to the total glycan (for example, within the range of 20% to a maximum of 40% G0F relative to the total glycan, for example, within the range of 40% to 80% G0F relative to the total glycan), steps of purifying, concentrating, and / or formulating the ustekinumab preparation are carried out.

[0053] In some embodiments, the provided method further includes measuring the target level of G0F glycan. In some embodiments, when the measured level of G0F glycan is within the range of 20% to 80% G0F relative to the total glycan (e.g., within the range of 20% to a maximum of 40% G0F relative to the total glycan, e.g., within the range of 40% to 80% G0F relative to the total glycan), the step of purifying, concentrating, and / or formulating the ustekinumab preparation is performed. 40% G0F relative to the total glycan, for example, within the range of 40% to 80% G0F relative to the total glycan), steps of purifying, concentrating, and / or formulating the ustekinumab preparation are carried out. When the measured level of G0F glycan is within the range of 20% to 80% G0F relative to the total glycan (for example, within the range of 20% to a maximum of

[0054] In some embodiments, provided are methods for manufacturing and / or generating ustekinumab having a target glycan level, which include target levels of sialylated glycan and / or G2F glycan. Table 1 below provides conditions for ustekinumab having target levels of sialylated glycan and / or G2F glycan. In some embodiments, provided are methods for manufacturing and / or generating ustekinumab having a target glycan level, which include target levels of sialylated glycan and / or G2F glycan. Table 1 below provides conditions for ustekinumab having target levels of sialylated glycan and / or G2F glycan. In some embodiments, provided are methods for manufacturing and / or generating ustekinumab having a target glycan level, which include target levels of sialylated glycan and / or G2F glycan. Table 1 below provides conditions for ustekinumab having target levels of sialylated glycan and / or G2F glycan.

[0055] [Table 1]

[0056] In some embodiments, the provided methods are for manufacturing and / or generating ustekinumab having a target level of sialylated glycan within the range of 15% - 30% sialylated glycan relative to total glycan. In some embodiments, the method for achieving ustekinumab having a target level of sialylated glycan within the range of 15% - 30% sialylated glycan relative to total glycan includes a level of selected galactose of 0 mM and a cell culture time within the range of 7 days to 15 days. In some embodiments, the provided methods are for manufacturing and / or generating ustekinumab having a target level of sialylated glycan within the range of 15% - 30% sialylated glycan relative to total glycan. In some embodiments, the method for achieving ustekinumab having a target level of sialylated glycan within the range of 15% - 30% sialylated glycan relative to total glycan includes a level of selected galactose of 0 mM and a cell culture time within the range of 7 days to 15 days. In some embodiments, the provided methods are for manufacturing and / or generating ustekinumab having a target level of sialylated glycan within the range of 15% - 30% sialylated glycan relative to total glycan. In some embodiments, the method for achieving ustekinumab having a target level of sialylated glycan within the range of 15% - 30% sialylated glycan relative to total glycan includes a level of selected galactose of 0 mM and a cell culture time within the range of 7 days to 15 days. In some embodiments, the provided methods are for manufacturing and / or generating ustekinumab having a target level of sialylated glycan within the range of 15% - 30% sialylated glycan relative to total glycan. In some embodiments, the method for achieving ustekinumab having a target level of sialylated glycan within the range of 15% - 30% sialylated glycan relative to total glycan includes a level of selected galactose of 0 mM and a cell culture time within the range of 7 days to 15 days.

[0057] In some embodiments, the provided methods are for manufacturing and / or generating ustekinumab having a target level of sialylated glycan within the range of 5% - up to 15% sialylated glycan relative to total glycan. In some embodiments, the method for achieving ustekinumab having a target level of G0F glycan within the range of 5% - up to 15% sialylated glycan relative to total glycan includes a level of selected galactose within the range of 15 mM - 30 mM and a cell culture time within the range of 16 days to 60 days. In some specific embodiments, the cell culture time is within the range of 25 days to 42 days. In some embodiments, the provided methods are for manufacturing and / or generating ustekinumab having a target level of sialylated glycan within the range of 5% - up to 15% sialylated glycan relative to total glycan. In some embodiments, the method for achieving ustekinumab having a target level of G0F glycan within the range of 5% - up to 15% sialylated glycan relative to total glycan includes a level of selected galactose within the range of 15 mM - 30 mM and a cell culture time within the range of 16 days to 60 days. In some embodiments, the provided methods are for manufacturing and / or generating ustekinumab having a target level of sialylated glycan within the range of 5% - up to 15% sialylated glycan relative to total glycan. In some embodiments, the method for achieving ustekinumab having a target level of G0F glycan within the range of 5% - up to 15% sialylated glycan relative to total glycan includes a level of selected galactose within the range of 15 mM - 30 mM and a cell culture time within the range of 16 days to 60 days. In some embodiments, the provided methods are for manufacturing and / or generating ustekinumab having a target level of sialylated glycan within the range of 5% - up to 15% sialylated glycan relative to total glycan. In some embodiments, the method for achieving ustekinumab having a target level of G0F glycan within the range of 5% - up to 15% sialylated glycan relative to total glycan includes a level of selected galactose within the range of 15 mM - 30 mM and a cell culture time within the range of 16 days to 60 days. In some specific embodiments, the cell culture time is within the range of 25 days to 42 days. In some embodiments, the provided methods are for manufacturing and / or generating ustekinumab having a target level of sialylated glycan within the range of 5% - up to 15% sialylated glycan relative to total glycan. In some embodiments, the method for achieving ustekinumab having a target level of G0F glycan within the range of 5% - up to 15% sialylated glycan relative to total glycan includes a level of selected galactose within the range of 15 mM - 30 mM and a cell culture time within the range of 16 days to 60 days. In some specific embodiments, the cell culture time is within the range of 25 days to 42 days.

[0058] In some embodiments, the provided method is for the production and / or generation of ustekinumab having a target level of G2F glycan within the range of 5% to 10% G2F relative to total glycan and / or generation. In some embodiments, the method for achieving ustekinumab having a target level of G2F glycan within the range of 5% to 10% G2F relative to total glycan includes a selected galactose level of 0 mM and a cell culture time within the range of 7 days to 15 days. and a cell culture time within the range of 7 days to 15 days. and a cell culture time within the range of 7 days to 15 days.

[0059] In some embodiments, the provided method is for the production and / or generation of ustekinumab having a target level of G2F glycan within the range of 1% to up to 5% G2F relative to total glycan and / or generation. In some embodiments, the method for achieving ustekinumab having a target level of G2F glycan within the range of 1% to up to 5% G2F relative to total glycan includes a selected galactose level within the range of 15 mM to 30 mM and a cell culture time within the range of 16 days to 60 days. In some particular embodiments, the cell culture time is within the range of 25 days to 42 days. and a cell culture time within the range of 16 days to 60 days. In some particular embodiments, the cell culture time is within the range of 25 days to 42 days. and a cell culture time within the range of 16 days to 60 days. In some particular embodiments, the cell culture time is within the range of 25 days to 42 days. and a cell culture time within the range of 16 days to 60 days. In some particular embodiments, the cell culture time is within the range of 25 days to 42 days.

[0060] In some embodiments, in the provided method, the selected galactose level is controlled over the entire culture process (e.g., controlled during culture from t = 0 until harvest).

[0061] In some embodiments, the provided method includes culturing a population of mammalian cells genetically engineered to express ustekinumab. In some embodiments, the mammalian cells are CHO cells, HEK 293 cells, fibrosarcoma HT 1080 cells, PER.C6 cells, fibrosarcoma HT 1080 cells, PER.C6 cells, fibrosarcoma HT 1080 cells, PER.C6 ​​​​Cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0 cells, and SP 2 / 0 selected from the group consisting of cells.

[0062] In some embodiments, in the provided method, the culturing step is performed using a continuous culture process is carried out. In some embodiments, the provided method is a perfusion culture process (e.g., an alternating tangential flow filter (ATF)-based perfusion culture process) is used including culturing. In some specific embodiments, the provided method is a perfusion culture process by which mammalian cells genetically engineered to express ustekinumab ( e.g., SP 2 / 0 cells expressing ustekinumab) are cultured.

[0063] In some embodiments, the provided method includes collecting the ustekinumab expressed by the cells at two or more time points within the time range of the cell culture time.

[0064] In some embodiments, the target value is a predetermined pharmaceutical product specification or quality control standard for a pharmaceutical preparation, e.g., a certificate of analysis (CofA), a certificate of test (CofT), or a master batch record is. In some embodiments, the product specification is the product description of an FDA label, a package insert for physicians, a US P monograph, or an EP monograph.

[0065] Generally, the cell culture method of the present disclosure includes culturing at a temperature within the range of 25°C to 40°C and under the gravity encountered on the ground. In some embodiments, the provided method of culturing a population of cells is sufficient for the expression of the ustekinumab product. The cell culture medium generally contains an appropriate energy source and a compound that regulates the cell cycle. Generally, the culture medium contains, for example, those commonly used in the art such as an energy source and a compound that regulates the cell cycle. Generally, the culture medium contains, for example, those commonly used in the art It contains amino acids, vitamins, inorganic salts, and glucose known to those skilled in the art. In some embodiments the cell culture medium has a pH of 6 to 8. Media for animal cell culture are well established in the art and are routinely optimized by those skilled in the art for specific purposes and / or cell types.

[0066] In some embodiments, the method of the present disclosure includes culturing a population of cells via a continuous cell culture system (e.g., a perfusion cell culture system). In some embodiments, the continuous cell culture system includes a bioreactor tank and a cell retention device. In some embodiments, the continuous cell culture system includes a bioreactor tank, a cell retention device, a medium supply, and a bleed waste collection. In some embodiments, the continuous cell culture system includes a population of cells (e.g., a population of cells genetically engineered to express ustekinumab, e.g., a population of cells consisting of cells genetically engineered to express ustekinumab) and a cell culture medium.

[0067] In some embodiments, the cell retention device is a continuous centrifuge, an alternating tangential flow filtration (ATF) device, a tangential flow membrane filter (TFF), a dynamic filter, a spin filter, an ultrasonic and dielectrophoretic separator, or a gravity sedimentation device, or includes these. In some specific embodiments, the cell retention device is an ATF or includes an ATF.

[0068] In some embodiments, the bioreactor system includes a stirred tank bioreactor, a cell retention device, a medium supply, and a bleed waste collection.

[0069] In some embodiments, a bioreactor system (e.g., a perfusion bioreactor system) includes a sparger. In some embodiments, the bioreactor system includes a drilled hole sparger. In some embodiments, the bioreactor system includes an open pipe sparger. In some embodiments, the bioreactor system includes a sintered sparger.

[0070] In some embodiments, the methods of the present disclosure involve culturing a population of cells having a volume in the range of 1 L to 3000 L (e.g., a population of cells genetically engineered to express ustekinumab, e.g., a population of cells consisting of cells genetically engineered to express ustekinumab). In some embodiments, the provided methods involve culturing mammalian cells genetically engineered to express ustekinumab at a volume of at least 25 L, 50 L, 100 L, 200 L, 250 L, 400 L, 500 L, 600 L, 800 L, 1000 L, or 2000 L. In some embodiments, the provided methods involve culturing mammalian cells genetically engineered to express ustekinumab at

[0071] a volume of about 25 L to about 250 L. Glycan assessment In some embodiments, the glycans of ustekinumab are analyzed (e.g., measured) by any available suitable method. In some examples, the glycan structures and compositions described herein are analyzed, for example, Kinetic methods, nuclear magnetic resonance (NMR) methods, and combinations thereof are analyzed. Exemplary enzymatic methods include contacting a ustekinumab preparation with one or more enzymes under conditions and for a time sufficient to release one or more glycans (e.g., one or two or more exposed glycans). In some examples, one or two or more enzymes include PNGase F. Exemplary chromatographic methods include strong anion exchange chromatography using pulsed amperometric detection (S AX-PAD), liquid chromatography (LC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), thin layer chromatography (TLC), amide column chromatography, and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation by mass spectrometry (IMS), and combinations thereof, but are not limited thereto. Exemplary mass spectrometry (MS) methods include tandem MS, LC-MS, LC-MS / MS, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry ( separation with mass spectrometry, IMS-MS), electron transfer dissociation (ETD-MS), and combinations thereof, but not limited thereto. Exemplary electrophoresis methods include capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) following Western blotting using antibodies that recognize specific glycan structures, and combinations thereof, but not limited thereto. Exemplary nuclear magnetic resonance (NMR) includes one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopy magnetic-angle spinning NMR (COSY-NMR), total correlated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR (ROESY-NMR), nuclear overhauser effect spectroscopy NMR (NOESY-NMR), etc. fer dissociation, ETD-MS), and combinations thereof, but not limited thereto. Limited. Exemplary electrophoresis methods include capillary electrophoresis (capillary electr ophoresis, CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide a mid gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) following Western blotting using antibodies that recognize specific glycan structures, and combinations thereof, but not limited thereto. ng continues SDS-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electro phoresis, SDS-PAGE), and combinations thereof, but not limited to these. Exemplary nuclear magnetic resonance (NMR) includes one-dimensional NMR (one-dimensional NM R, 1D-NMR), two-dimensional NMR (two-dimensional NMR, 2D-NMR), correlation spectroscopy magnetic angle spinning NMR (correlation spectroscopy magnetic-angle spinning NMR, C OSY-NMR), total correlated spectroscopy NMR (total correlated spectroscopy NMR, TOCS Y-NMR), heteronuclear single-quantum coherence NMR (heteronuclear single-quantum coh erence NMR, HSQC-NMR), heteronuclear multiple quantum coherence (heteronuclear multiple quantum coherence, HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR (ro It should be noted that there seems to be some incomplete or incorrect expressions in the original text, which may affect the accuracy of the overall translation. You may want to check and correct the original text for a more precise translation.Nuclear Overhauser effect spectroscopy (NOESY-N MR), and combinations thereof, including but not limited to these.

[0072] Cell Any host cell that can be used to express ustekinumab can be used in the methods described herein. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that includes the heavy chain variable domain set forth in SEQ ID NO: 1 and / or the light chain variable domain set forth in SEQ ID NO: 2. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that includes a heavy chain containing the sequence of SEQ ID NO: 1 and / or a light chain containing the sequence of SEQ ID NO: 2. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that includes the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NO: 1 and the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NO: 2. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that has the same primary amino acid sequence as an approved protein, for example, under a secondary approval process, for therapeutic or diagnostic use in humans or animals. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that is identical to an approved therapeutic or diagnostic protein in its primary amino acid sequence. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that includes the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NO: 1 and the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NO: 2. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that includes the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NO: 1 and the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NO: 2. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that includes the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NO: 1 and the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NO: 2. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that includes the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NO: 1 and the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NO: 2. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that includes the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NO: 1 and the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NO: 2.

[0073] In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that has the same primary amino acid sequence as an approved protein, for example, under a secondary approval process, for therapeutic or diagnostic use in humans or animals. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that has the same primary amino acid sequence as an approved protein, for example, under a secondary approval process, for therapeutic or diagnostic use in humans or animals. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that has the same primary amino acid sequence as an approved protein, for example, under a secondary approval process, for therapeutic or diagnostic use in humans or animals. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that has the same primary amino acid sequence as an approved protein, for example, under a secondary approval process, for therapeutic or diagnostic use in humans or animals. In some embodiments, the cells genetically engineered to express ustekinumab contain one or more nucleic acids encoding a ustekinumab product that has the same primary amino acid sequence as an approved protein, for example, under a secondary approval process, for therapeutic or diagnostic use in humans or animals. 、2、3、4、5、6、7、8、9、10、15, or 20 residues different from, ustekinumab comprises one or more nucleic acids encoding the ustekinumab product. In some embodiments, cells genetically engineered to express ustekinumab have at least 90, 95, 98, 99% or 100% sequence identity with an approved therapeutic or diagnostic protein and comprise one or more nucleic acids encoding the protein. The terms "same primary amino acid sequence ", "primary amino acid sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues different ", "sequence having at least 98% or more sequence identity", or similar terms relate to the level of identity between primary amino acid sequences. In some embodiments, preparations or products of the protein include amino acid variants, e.g., species in which the terminal residues, e.g., one or two terminal residues, are different. In some embodiments of such cases, the sequence identity being compared is the identity between the primary amino acid sequences of the most abundant (e.g., most abundant active) species in each of the products being compared. In some embodiments, sequence identity refers to the amino acid sequence encoded by a nucleic acid that can be used to produce the ustekinumab product. In some embodiments, cells genetically engineered to express ustekinumab are mammalian cells. In some embodiments, cells genetically engineered to express ustekinumab are rodent cells. In some embodiments, the gene

[0074] In some embodiments, cells genetically engineered to express ustekinumab are mammalian cells.

[0075] In some embodiments, cells genetically engineered to express ustekinumab are rodent cells. In some embodiments, the gene engineered to express ustekinumab The engineered cells are derived from a murine cell line. Examples of murine (e.g., mouse) cell lines include, for example, mouse myeloma cell lines such as NS0 cells and SP 2 / 0 cells .

[0076] In some embodiments, the cells engineered to express ustekinumab are human cells. In some embodiments, the cells engineered to express ustekinumab are derived from a human cell line. Examples of human cell lines include, for example, HEK 293: human embryonic kidney 293; HT-1080: derived from fibrosarcoma with an epitheloid phenotype; PER.C6 : derived from human embryonic retinal cells immortalized via transfection of the adenovirus E1 gene ; CAP: derived from human amniotic cells immortalized via the adenovirus type 5 E1 gene ; HKB-11: produced by polyethylene glycol fusion of HEK293-S and a human B cell line ; and HuH-7: derived from human hepatocellular carcinoma. In some specific embodiments , the shear-sensitive cells are selected from HEK 293 cells, fibrosarcoma HT 1080 cells, PER.C6 cells, CAP cells, HKB-11 cells, and HuH-7 cells .

[0077] In some embodiments, a population of cells expressing an ustekinumab product as described herein is generated using recombinant methods. Recombinant expression of a gene encoding a polypeptide such as an antibody agent described herein can include construction of an expression vector containing a polynucleotide encoding the polypeptide. Once the polynucleotide is obtained, recombinant DNA techniques known in the art can be used to generate a population of cells expressing an ustekinumab product as described herein. Recombinant expression of a gene encoding a polypeptide such as an antibody agent described herein can include construction of an expression vector containing a polynucleotide encoding the polypeptide. Once the polynucleotide is obtained, recombinant DNA techniques known in the art can be used to generate a population of cells expressing an ustekinumab product as described herein. Recombinant expression of a gene encoding a polypeptide such as an antibody agent described herein can include construction of an expression vector containing a polynucleotide encoding the polypeptide. Once the polynucleotide is obtained, recombinant DNA techniques known in the art can be used to generate a population of cells expressing an ustekinumab product as described herein. Recombinant expression of a gene encoding a polypeptide such as an antibody agent described herein can include construction of an expression vector containing a polynucleotide encoding the polypeptide. Once the polynucleotide is obtained, recombinant DNA techniques known in the art can be used to generate a population of cells expressing an ustekinumab product as described herein. Recombinant expression of a gene encoding a polypeptide such as an antibody agent described herein can include construction of an expression vector containing a polynucleotide encoding the polypeptide. Once the polynucleotide is obtained, recombinant DNA techniques known in the art can be used By using known techniques, vectors for producing polypeptides can be produced. Using the method, a polypeptide coding sequence and appropriate transcriptional and translational control signals are It is possible to construct an expression vector containing the gene. These include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0078] Once the ustekinumab products described herein are produced by recombinant expression, they can be purified. Any method known in the art for isolating the hydroxyl group can be used, such as chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility or any other standard technique for purifying proteins. For example, an affinity column such as a Protein A column can be appropriately selected and used for chromatography. By combining column, filtration, ultrafiltration, salting out, and dialysis procedures, Mabs can be isolated and purified (Antibodies: A Laboratories y Manual,Ed Harlow,David Lane,Cold Sprin (See Harvard Harbor Laboratory, 1988). The ustekinumab product may be fused to a heterologous polypeptide sequence and purified as described in can be promoted.

[0079] Pharmaceutical Compositions Using any of the methods, systems, and / or processes described herein The ustekinumab product produced or manufactured can be incorporated into a pharmaceutical composition. Such pharmaceutical compositions may be useful for the prevention and / or treatment of diseases. The pharmaceutical composition containing can be formulated by methods known to those skilled in the art (for example, Re mington’s Pharmaceutical Sciences, 20th E d., Lippincott Williams & Wilkins, 2000 may be referred to ). The pharmaceutical composition can be administered parenterally in the form of an injectable preparation containing a sterile solution or suspension in water or another pharmaceutically acceptable liquid . For example, the pharmaceutical composition can be formulated by suitably combining a cell product (for example, a recombinant protein, for example, a glycoprotein, for example, an antibody agent) with a pharmaceutically acceptable vehicle or medium such as sterile water and physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, etc., and then mixed in a generally recognized unit dosage form required for pharmaceutical practice. The amount of the active ingredient contained in the pharmaceutical preparation is an amount such that a suitable dosage within the specified range is provided . In some embodiments, the preparation of ustekinumab contains sucrose as a stabilizer / isotonic agent .

[0080] In some embodiments, the preparation of ustekinumab contains histidine (for example, L-histidine) as a buffer . In some embodiments, the preparation of ustekinumab contains polysorbate 80 as a surfactant . In some specific embodiments, the preparation of ustekinumab contains histidine (for example, L-histidine), sucrose, and polysorbate 80 . In some embodiments, the preparation of ustekinumab is formulated for parenteral administration, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection

[0081] . In some embodiments, the preparation of ustekinumab... Preparations of ustekinumab are formulated for subcutaneous administration.

[0082] The present disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. The examples are not to be construed as limiting the scope or content of the present disclosure in any way.

Example

[0083] Example 1: Identification of shifts in the glycan composition of ustekinumab products This example identifies significant shifts in the glycan composition between lots of a commercially available ustekinumab reference protein product (“RPP”). Specifically, this example demonstrates the variation in glycan composition between lots of commercially available ustekinumab that falls into two distinct glycan profiles. These two groups of glycan variants are referred to herein as “Group 1,” which is characterized by a relatively high level of G0F glycan, and “Group 2,” which is characterized by a relatively low level of G0F glycan. Thirty lots of ustekinumab RPP were analyzed. Of these 30 lots, 28 lots were 90 mg / mL preparations and 2 lots were 5 mg / mL preparations. Of the 28 lots of 90 mg / mL ustekinumab RPP, 8 of these lots were identified as having the glycan profile of Group 1 and 20 lots were identified as having the glycan profile of Group 2. See Figure 1. The summary of the average abundance of each of the major glycan species for samples of Group 1 and Group 2 is summarized in Table 2 below.

[0084]

[0085]

[0086] ​​​​​​​​​​​

Table 2

[0087] Prepared by culturing for 42 days with 5 mM galactose at various volumes within the range of 3 L to 250 L (e.g., , by an ATF-based perfusion culture process), the ustekinumab test products were also analyzed. The glycan abundance of these ustekinumab test products is shown in the group at the right end of each panel in Figure 1. The glycan profiles of the ustekinumab test products were within the range of the RPP of Group 1 and Group 2. Therefore, this example demonstrated the identification of two distinct groups of glycan profiles for commercially available ustekinumab RPP, and that, depending on the co-culture conditions, ustekinumab test products with intermediate glycan abundances within the ranges of these two groups can be obtained.

[0088] Example 2: Culture time affects the glycan composition of ustekinumab This example demonstrates that the timing of collection is an element for controlling the glycan composition of ustekinumab. As shown in Figure 2, the abundance of G0F glycan relative to total glycan generally decreases with the culture period. For example, an ustekinumab preparation collected on day 13 of culture had approximately 50% abundance of G0F glycan, while a sample collected after culturing for 34 days or more had less than 30% G0F glycan relative to the total glycan composition.

[0089] Interestingly, the present disclosure shows that a decrease in the culture period (i.e., the time until collection) is associated with an increase in the G0F level, while an increase in the culture period (i.e., the time until collection) is associated with a decrease in the G0F gives the perception of being associated with a decrease in level.

[0090] Thus, this example demonstrates that an increase in culture time can decrease the abundance of G0F glycan in the total glycan composition, and further demonstrates that by varying the culture time, the glycan composition of ustekinumab can be controlled.

[0091] Example 3: Preparation of ustekinumab products having glycan profiles of Group 1 and Group 2 This example identifies an inverse relationship between culture time and the concentration of galactose in the culture medium, which can function as a process lever for controlling the glycan level (e.g., galactosylation) of ustekinumab. This example demonstrates the generation of non-conforming materials (i.e., non-conforming ustekinumab preparations) having a genetically engineered glycan profile. Specifically, this example demonstrates the generation of two non-conforming ustekinumab preparations, herein referred to as NCM-1 and NCM-2, which are genetically engineered to have a glycan profile that matches Group 1 RPP and Group 2 RPP as described above in Example 1, respectively.

[0092] Specifically, the NCM-1 preparation was made by culturing with 15 mM galactose (e.g., by an ATF-based perfusion culture process) and collecting ustekinumab before day 16 of the culture (e.g., at a time point within the range of days 7 to 15). In contrast, by culturing with a medium without galactose (0 mM galactose) (e.g., by an ATF-based perfusion culture process) and collecting ustekinumab after day 25 of the culture, ​ For example, NCM-2 was prepared by collecting at a point within the range of day 34 to day 45. As shown in Figure 3, for each of the analyzed glycan species containing G0F, G1FA, G1FB, G2F, fully sialylated, main, acidic, and basic glycans, the NCM-1 glycan profile showed good correlation with Group 1 RPP, and the NCM-2 glycan profile showed good correlation with Group 2 RPP. Table 3 provides exemplary target ranges for each glycan genetically engineered by controlling the process levers of galactose concentration and culture time. -1 glycan profile was in good correlation with Group 1 RPP, and the NCM-2 gly can profile was in good correlation with Group 2 RPP. Table 3 provides the galactose concentration and the exemplary target ranges of each glycan genetically engineered by controlling the process levers of culture time. provides.

[0093]

Table 3

[0094] Therefore, this example demonstrates that factors inversely proportional to galactose concentration and culture time can be used as process levers for controlling glycan composition and can be used to generate an inapt ustekinumab preparation with a target glycan level. It has been demonstrated that it can be used to generate an inapt ustekinumab preparation having a target glycan level. It has been demonstrated that it can be used to generate an inapt ustekinumab preparation having a target glycan level. demonstrated.

[0095] Equivalents Although the present disclosure has been described in connection with its detailed description, the foregoing description is intended to illustrate the scope of the invention and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Although the present disclosure has been described in connection with its detailed description, the foregoing description is intended to illustrate the scope of the invention and is not intended to limit the scope of the invention as defined by the appended claims. It should be understood that it is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the following claims.

Claims

1. A method for manufacturing a pharmaceutical composition comprising ustekinumab having a target level of G0F glycan comprising: (a) selecting a target level of G0F glycan; (b) selecting the level of galactose and the time of cell culture to provide the selected level of G0F glycan (wherein the level of said galactose and said time are inversely proportional); (c) culturing a population of cells genetically engineered to express ustekinumab under conditions comprising the selected level of galactose and time; (d) collecting the ustekinumab expressed by the population of cells, thereby generating a preparation of ustekinumab; and (e) purifying, concentrating, and / or formulating the ustekinumab preparation to produce a pharmaceutical composition comprising ustekinumab when the preparation meets the target level of G0F glycan. (f) The method as described above.

2. The method according to claim 1, wherein the culturing is carried out using a perfusion culture process.

3. The method according to claim 1 or 2, wherein the target level of G0F glycan is in the range of 20% to 80% G0F relative to total glycan.

4. The method according to claim 1 or 2, wherein the target level of G0F glycan is in the range of 25% to 65% G0F relative to total glycan.

5. The method according to any one of claims 1 to 4, wherein the target level of G0F glycan is in the range of 20% to a maximum of 40% G0F relative to total glycan, the selected level of galactose is 0 mM, and the time of cell culture is in the range of 7 days to 15 days.

6. The method according to any one of claims 1 to 4, wherein the target level of G0F glycan is in the range of 40% to 80% G0F relative to total glycan, the selected level of galactose is in the range of 15 mM to 30 mM, and the time of cell culture is in the range of 16 days to 60 days.

7. The method according to claim 6, wherein the time of cell culture is in the range of 25 days to 42 days.

8. The method according to any one of claims 1 to 7, further comprising measuring the level of G0F glycan.

9. The method according to any one of claims 1 to 8, wherein the population of cells genetically engineered to express ustekinumab is mammalian cells.

10. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ the mammalian cell is a CHO cell, HEK 293 cell, fibrosarcoma HT 1080 cell, PER.C6 cell, CAP cell, HKB-11 cell, HuH-7 cell, NS0 cell, and SP 2 / 0 cell, the method according to claim 9.

11. the selected galactose level is controlled during the culture from t = 0 until collection, the method according to any one of claims 1 to 10.

12. the ustekinumab expressed by the cell is collected two or more times within the time range of the time for cell culture the method according to any one of claims 1 to 11.

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