Methods of producing ustekinumab

Abstract

The present invention describes methods of making an Ustekinumab product having a targeted level of one or more glycans.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 786,821, filed December 31, 2018, which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on December 20, 2019, named M0168PCT_SL.txt, and is 6,036 bytes in size. Background Art

[0005] Therapeutic antibodies are an important class of therapeutic biological products. Antibody glycosylation and glycan composition can affect antibody activity and effector function. There remains a continuing need for improved methods for controlling the glycan profile of antibody products. Summary of the invention

[0006] The present disclosure provides process means for producing (e.g., manufacturing) Ustekinumab having a target level of one or more glycans. The present disclosure provides the following insight: the relationship between galactose concentration in the culture medium and culture time can be used as a process means to control the target level of one or more glycans (e.g., galactosylation) of Ustekinumab in culture. The present disclosure identifies an inverse correlation between culture time and galactose concentration in the culture medium in controlling the target level of one or more glycans (e.g., galactosylation).

[0007] In certain aspects, the present disclosure provides methods of making Ustekinumab having a target level of one or more glycans. Such methods may include providing (e.g., producing, expressing (e.g., in small-scale or large-scale cell culture) and / or manufacturing) or obtaining (e.g., receiving and / or purchasing from a third party (including a contractually related third party or a non-contractually related (e.g., independent) third party)) a Ustekinumab test protein (e.g., a Ustekinumab drug substance, e.g., a formulation of a Ustekinumab drug substance, e.g., a batch of a test Ustekinumab drug substance).

[0008] In some cases, the present disclosure provides a method of making a pharmaceutical composition comprising Ustekinumab having a target level of one or more glycans, the method comprising: selecting a galactose level and a cell culture time, wherein the galactose level and time are inversely correlated; culturing a cell population genetically engineered to express Ustekinumab under conditions comprising the selected galactose level and time; harvesting Ustekinumab expressed by the cell population, thereby producing a formulation of Ustekinumab; and if the formulation meets the target level of one or more glycans, purifying, concentrating, and / or formulating the Ustekinumab formulation to produce a pharmaceutical composition comprising Ustekinumab. In some embodiments, the target level of one or more glycans is a target level of a glycan selected from GOF, sialylated glycans, and G2F. In some embodiments, the target level of one or more glycans is a target level of GOF.

[0009] In certain cases, the present disclosure provides methods of making a pharmaceutical composition comprising Ustekinumab having a target level of GOF glycans, the method comprising: selecting a target level of GOF glycans; selecting a galactose level and a cell culture time to provide the selected target level of GOF, wherein the galactose level and time are inversely correlated; culturing a cell population genetically engineered to express Ustekinumab under conditions comprising the selected galactose level and time; harvesting Ustekinumab expressed by the cell population, thereby producing a preparation of Ustekinumab; and purifying, concentrating and / or formulating the Ustekinumab preparation if the preparation meets the target level of GOF glycans, to produce a pharmaceutical composition comprising Ustekinumab.

[0010] In some cases, the present disclosure provides a method of producing a Ustekinumab drug product having a target level of one or more glycans, the method comprising: culturing a population of cells genetically engineered to express Ustekinumab under conditions, wherein the conditions are characterized by parameters including a selected galactose level and a cell culture time, and wherein the galactose level and time are inversely correlated; harvesting Ustekinumab expressed by the cells, thereby producing a Ustekinumab preparation; and if the Ustekinumab preparation meets the target level of one or more glycans, purifying, concentrating, and / or formulating the Ustekinumab preparation to produce a Ustekinumab drug product. In some embodiments, the target level of one or more glycans is a target level of glycans selected from GOF, sialylated glycans, and G2F. In some embodiments, the target level of one or more glycans is a target level of GOF.

[0011] In some embodiments, provided methods include culturing a population of mammalian cells genetically engineered to express Ustekinumab. In some embodiments, the mammalian cells are selected from the group consisting of CHO cells, HEK 293 cells, fibrosarcoma HT1080 cells, PER.C6 cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0 cells, and SP 2 / 0 cells.

[0012] In some embodiments, in the provided methods, the culturing step is performed using a continuous culture method. In some embodiments, the provided methods include culturing using a perfusion culture method (e.g., an alternating tangential flow filter (ATF)-based perfusion culture method). In some specific embodiments, the provided methods include culturing mammalian cells genetically engineered to express Ustekinumab (e.g., SP2 / 0 cells expressing Ustekinumab) by a perfusion culture method.

[0013] In some embodiments, methods are provided that include harvesting Ustekinumab expressed by cells two or more times within the time frame of the cell culture period.

[0014] In some embodiments, the target level of G0F glycans is in the range of 20% to 80% G0F relative to total glycans. In some embodiments, the target level of G0F glycans is in the range of 25% to 65% G0F relative to total glycans.

[0015] In some embodiments, provided methods involve manufacturing and / or producing Ustekinumab with a target level of G0F glycans in the range of 20% to up to 40% G0F relative to total glycans. In some embodiments, methods for achieving Ustekinumab with a target level of G0F glycans in the range of 20% to up to 40% G0F relative to total glycans comprise a selected galactose level of 0 mM and a cell culture time in the range of 7 to 15 days.

[0016] In some embodiments, provided methods involve manufacturing and / or producing Ustekinumab having a target level of G0F glycans in the range of 40% to 80% G0F relative to total glycans. In some embodiments, methods for achieving Ustekinumab having a target level of G0F glycans in the range of 40% to 80% G0F relative to total glycans include a selected galactose level in the range of 15 mM to 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 25 days to 42 days.

[0017] In some embodiments, the method provided includes selecting a target level of G0F glycans. In some embodiments, the target level of G0F glycans is in the range of 20% to 80% G0F relative to total glycans. In some specific embodiments, the target level of G0F glycans is in the range of 20% to a maximum of 40% G0F relative to total glycans, wherein the method includes a selected galactose level of 0 mM, and a cell culture time in the range of 7 days to 15 days. In some specific embodiments, the target level of G0F glycans is in the range of 40% to 80% G0F relative to total glycans, wherein the method includes a selected galactose level in the range of 15 mM to 30 mM, and a cell culture time in the range of 16 days to 60 days.

[0018] In some embodiments, provided methods control selected galactose levels throughout the culturing step (eg, during the culturing period from t=0 until harvest).

[0019] In some embodiments, provided methods further include measuring the level of G0F glycans. In some embodiments, if the measured level of G0F glycans is in the range of 20% to 80% G0F relative to total glycans (e.g., in the range of 20% to up to 40% G0F relative to total glycans, such as in the range of 40% to 80% G0F relative to total glycans), steps of purifying, concentrating, and / or formulating Ustekinumab formulations are performed.

[0020] These and other aspects of the invention are described in more detail below and in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings included herein consisting of the following figures are for illustration purposes only and are not intended to be limiting.

[0022] Figure 1A comparison of the glycan profiles of various preparations and Ustekinumab sources is shown. The upper left figure provides the abundance of G0F glycans; the upper right figure provides the abundance of G1F-A glycans; the lower left figure provides the abundance of G1F-B glycans; and the lower right figure provides total sialylation. The solid circles represent reference protein products ("RPP") from the United States (black solid circles) and Europe (shaded solid circles). The symbols in the rightmost part of each figure represent Ustekinumab test preparations cultured at different volumes. The solid shaded squares represent samples cultured at 3L, the hollow squares represent samples from satellite culture, the solid triangles represent samples cultured at 100L, and the solid diamonds represent samples cultured at 250L. The relative abundance of each glycan is shown in its corresponding figure, where the samples shown in each figure are divided into four groups, from left to right along the x-axis: Group 1 RPP from the 90 mg / ml reference sample (far left), Group 2 RPP from the 90 mg / ml reference sample (second from the left), 5 mg / mL RPP (third from the left) and Ustekinumab test product (far right).

[0023] Figure 2 The G0F glycan profile is shown as a function of harvest duration. The leftmost column (white) represents the 1st group RPP G0F abundance, the second column from the left (black) represents the 2nd group RPP G0F abundance, and the third column from the left represents the average abundance of G0F for samples cultured for 7 to 42 days. The shaded columns (starting from the fourth column from the left and continuing to the rightmost column) each represent the G0F abundance of samples cultured for a specified number of days ranging from 7 days (D7) to a maximum of 42 days (D42, rightmost column).

[0024] Figure 3 Comparison of major glycan profiles and control variants between various culture conditions is shown. Shown along the x-axis of each graph from left to right: Group 1 RPP (far left), Group 2 RPP, Ustekinumab cultured at 3 LDS, Ustekinumab cultured at 100 L DS, Ustekinumab cultured at 250 L DS, NCM-2 Ustekinumab formulation, and NCM-1 Ustekinumab formulation (far right).

[0025] Some definitions

[0026] Generally speaking, unless otherwise expressly indicated, the terms used herein are according to their meanings understood in the art. Explicit definitions of certain terms are provided below; in specific cases throughout this specification, the meanings of these and other terms will be apparent to those skilled in the art from the context.

[0027] In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout this specification.

[0028] As used herein, the term "about" or "approximately" refers to a value similar to a stated reference value when applied to one or more values ​​of interest. In certain embodiments, the term "about" or "approximately" refers to a value range that falls 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 a stated reference value.

[0029] As used herein, the terms "control", "controlled", "under control" in reference to controlling a target level of one or more glycans (e.g., galactose, e.g., GOF) of Ustekinumab means selecting, maintaining, and / or adjusting one or more culture conditions for producing Ustekinumab. Adjustment may include increasing or decreasing one or more culture conditions for producing Ustekinumab. As used herein, a controlled target level of one or more glycans relates to the production of Ustekinumab having a desired level of one or more glycans with minimal product drift. In some embodiments, the controlled target level of GOF will not vary by more than 20%, 15%, 10%, or 5% 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 production of Ustekinumab (e.g., batch-to-batch consistency, consistency across samples from any particular production process).

[0030] As used herein, "glycan" refers to a compound comprising at least one sugar residue (e.g., monosaccharide). A polysaccharide may be a monomer or polymer of a sugar residue, and may be linear or branched. "Glycan" may include 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'-sulfo N-acetylglucosamine, etc.). The term "glycan" includes homopolymers and heteropolymers of sugar residues. The term "glycan" also encompasses glycan components of glycoconjugates (e.g., glycoproteins, glycolipids, proteoglycans, etc.). The term also encompasses free glycans, including glycans that have been cleaved or otherwise released from glycoconjugates.

[0031] As used herein, "galactosylated glycans" refers to glycans comprising at least one galactose residue. In some embodiments, the galactosylated glycans are G1, G2, G1F, G2F, A1 and / or A2 glycans. Non-galactosylated glycans include G0F or G0. In some embodiments, the target level of galactosylated glycans may refer to the presence of galactosylated glycans (e.g., G2F) and / or the target level of non-galactosylated glycans (e.g., G0F).

[0032] As used herein, the term "isolated" refers to substances and / or entities that are (1) separated from at least some of the components with which they were originally associated (whether in nature and / or an experimental setting), and / or (2) designed, produced, prepared, and / or manufactured by the hand of a human. An isolated substance and / or entity may be separated from about 10%, about 20%, about 30%, 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 more than about 99% of the other components with which they were originally associated. In some embodiments, the purity of 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%. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as understood by those skilled in the art, a substance may still be considered "isolated" or even "pure" after being combined with, for example, certain other components such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the separation percentage or purity of the substance is calculated without including such carriers or excipients. To give just one example, in some embodiments, a biopolymer such as a polypeptide or polynucleotide occurring in nature is considered "isolated" when: a) by virtue of its origin or source of derivation, it is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species as the species in which it is produced in nature; c) it is expressed by a cell or other expression system that is not the species in which it is produced in nature, or is otherwise associated with components from a cell or other expression system that is not the species in which it is produced in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from that in which it is produced in nature is considered an "isolated" polypeptide. Alternatively or in addition, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered an "isolated" polypeptide to the extent that it has been separated from other components with which it is a) associated in nature; and / or b) with which it was initially produced.

[0033] As used herein, "process means" 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 on an antibody product. The present disclosure provides a novel process means, namely a specific relationship between galactose concentration in a culture medium and culture time. As described herein, the inverse correlation between galactose concentration in a culture medium and culture time in a culture medium can be used as a process means for producing Ustekinumab with a target level of one or more glycans (e.g., galactosylation). In some embodiments, the process means include selected galactose levels and cell culture time 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 a Ustekinumab culture or preparation).

[0034] 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 in the heavy chain.

[0035] In general, as used herein, a "protein" is a polypeptide (i.e., a chain of at least two amino acids linked to each other by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins) and / or may be otherwise processed or modified. One of ordinary skill in the art will appreciate that a "protein" may be a complete polypeptide chain (with or without a signal sequence) as produced by a cell, or may be a functional portion thereof. One of ordinary skill will also appreciate that a protein may sometimes comprise more than one polypeptide chain, for example, linked by one or more disulfide bonds or associated by other means.

[0036] As used herein, "recovering" refers to the process of rendering an agent or entity substantially free of other previously associated components by separation, for example, using purification techniques known in the art. In some embodiments, an agent or entity is recovered from a natural source and / or a source comprising cells.

[0037] As used herein, "sample" refers to a sample produced individually. In some embodiments, evaluation of individual samples includes evaluation of samples from the same culture run (e.g., at different time points during preparation) or from different culture runs (e.g., different rounds of culture).

[0038] As used herein, "target value or target level" 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 one or more specific glycans, such as galactosylated glycans (e.g., G0, G1, G2, G0F, G1F, G2F, or a combination) and / or sialylated glycans (e.g., monosialylated, disialylated, or a combination) described in reference to the Ustekinumab product or in the instructions or masterbatch record of the drug product. In some specific embodiments, the target value is the level of G0F glycans in the Ustekinumab product.

[0039] In some embodiments, the target value refers to the absolute level (e.g., number of moles) of one or more glycans (e.g., galactosylated glycans (e.g., one or more species of galactosylated glycans) and / or sialylated glycans (e.g., one or more species of sialylated glycans)) in the Ustekinumab formulation. In some embodiments, the target value refers to the level of one or more glycans (e.g., galactosylated glycans (e.g., one or more species of galactosylated glycans) and / or sialylated glycans (e.g., one or more species of sialylated glycans)) in the Ustekinumab formulation relative to the total level of glycans in the Ustekinumab formulation. In some embodiments, the target value is expressed as a "percentage", which refers to the number of moles of one or more glycans (e.g., Fc glycans) relative to the total number of moles of glycans (e.g., Fc glycans) in the Ustekinumab formulation. In some embodiments, "percentage" refers to the number of moles of one or more PNGase F-released Fc glycans relative to the total number of moles of PNGase F-released Fc glycans detected.

[0040] As used herein, the term "Ustekinumab preparation" refers to a mixture of Ustekinumab proteins obtained according to a particular production method. The Ustekinumab protein in the Ustekinumab preparation includes multiple copies of Ustekinumab (i.e., having the same or substantially the same amino acid sequence), but has a mixture of glycans associated with the protein. In some cases, the Ustekinumab preparation is prepared using methods and / or systems as provided herein. The production method may include a recombinant preparation step using cultured cells engineered to express Ustekinumab (or to express Ustekinumab at relevant levels or under relevant conditions). In some embodiments, the production method may include a separation step in which Ustekinumab is separated from certain components of the engineered cells (e.g., by lysing the cells and precipitating the protein components by centrifugation). In some embodiments, the production method may also include a purification step in which Ustekinumab is separated from other cellular components (e.g., other proteins or organic components used in the previous steps) (e.g., by chromatography). It should be understood that these steps are non-limiting and may include any number of additional production steps. Different Ustekinumab formulations may be prepared by the same production method but under different circumstances (e.g., different runs or formulations). Alternatively, different Ustekinumab formulations may be prepared by different production methods. The two production methods may differ in any aspect (e.g., expression vector, engineered cell type, culture conditions, separation process, purification conditions, etc.).

[0041] All documents and similar materials cited in this patent application, including but not limited to patents, patent applications, articles, books, papers, and web pages, are expressly incorporated by reference in their entirety, regardless of the form of such documents and similar materials. In the event that one or more of the introduced documents and similar materials differs from or conflicts with this patent application, including but not limited to defined terms, term usage, described techniques, etc., this patent application shall prevail. The section headings used herein are for organizational purposes only and should not be interpreted as limiting the subject matter described in any way. DETAILED DESCRIPTION

[0042] The present disclosure describes, at least in part, the discovery of a process approach to produce Ustekinumab with target levels of one or more glycans (e.g., galactosylation, e.g., G0F glycans). Controlling glycan composition and levels during antibody production is an ongoing challenge. 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 glycan composition in Ustekinumab production (e.g., batch-to-batch consistency, consistency across samples from any particular production process). During characterization of the Ustekinumab reference protein product ("RPP"), two different glycan populations of the Ustekinumab RPP were observed.

[0043] The present disclosure describes the development of means for controlling glycan composition during the manufacture of Ustekinumab. Described herein are process means for producing Ustekinumab having target levels of specific glycans (including the glycan profiles of each RPP identified). In the process of developing the means as described, the present disclosure identified the relationship between galactose concentration in the culture medium and the culture time. The present disclosure provides the following insight that the relationship between galactose concentration in the culture medium and the culture (e.g., continuous culture) time can be used as a process means to control the target level of one or more glycans (e.g., galactosylation) of Ustekinumab in the culture.

[0044] The disclosure also provides the insight that for controlling glycan levels (eg, galactosylation), the relationship between galactose levels and time (ie, culture duration) is inversely correlated. The disclosed culture methods for controlling glycan composition include continuous culture methods (eg, perfusion culture).

[0045] Ustekinumab

[0046] The present disclosure provides, in part, methods and processes for making, preparing, controlling, or otherwise generating Ustekinumab with a specific glycan profile.

[0047] Ustekinumab is an antibody that specifically binds to the p-40 subunit 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.

[0048] In some embodiments, Ustekinumab comprises HCDR1, HCDR2, and HCDR sequences that differ by no more than 3 amino acid residues from the HCDR sequence as set forth in SEQ ID NO.:1, and LCDR1, LCDR2, and LCDR3 sequences that differ by no more than 3 amino acid residues from the LCDR sequence as set forth in SEQ ID NO.:2. In some embodiments, Ustekinumab comprises HCDR1, HCDR2, and HCDR sequences that differ by no more than 2 amino acid residues or by no more than 1 amino acid residue from the HCDR sequence as set forth in SEQ ID NO.:1, and LCDR1, LCDR2, and LCDR3 sequences that differ by no more than 2 amino acid residues or by no more than 1 amino acid residue from the LCDR sequence as set forth in SEQ ID NO.:2. In some embodiments, Ustekinumab comprises a heavy chain variable domain that differs by no more than 3 amino acid residues from the sequence as set forth in SEQ ID NO.:1, and a light chain variable domain that differs by no more than 3 amino acid residues from the sequence as set forth in SEQ ID NO.:2. In some embodiments, Ustekinumab comprises a heavy chain comprising a sequence that differs from the sequence as shown in SEQ ID NO.: 1 by no more than 5 amino acids, and a light chain comprising a sequence that differs from the sequence of SEQ ID NO.: 2 by no more than 5 amino acid residues.

[0049] In some embodiments, Ustekinumab comprises the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO.: 1, and the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO.: 2. In some embodiments, Ustekinumab comprises the heavy chain variable domain as set forth in SEQ ID NO.: 1, and the light chain variable domain as set forth in SEQ ID NO.: 2. In some embodiments, Ustekinumab 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.

[0050] SEQ ID NO.: 1—Ustekinumab heavy chain sequence (bold indicates variable sequences with underlined CDR sequences) Domain sequence)

[0051]

[0052] SEQ ID NO.: 2—Ustekinumab light chain sequence (bold indicates variable sequences with underlined CDR sequences) Domain sequence)

[0053]

[0054] Cultivation methods

[0055] In some cases, the present disclosure provides a method of making a pharmaceutical composition comprising Ustekinumab having a target level of one or more glycans, the method comprising: selecting a galactose level and a cell culture time, wherein the galactose level and time are inversely correlated; culturing a cell population genetically engineered to express Ustekinumab under conditions comprising the selected galactose level and time; harvesting Ustekinumab expressed by the cell population, thereby producing a formulation of Ustekinumab; and if the formulation meets the target level of one or more glycans, purifying, concentrating, and / or formulating the Ustekinumab formulation to produce a pharmaceutical composition comprising Ustekinumab. In some embodiments, the target level of one or more glycans is a target level of a glycan selected from GOF, sialylated glycans, and G2F. In some embodiments, the target level of one or more glycans is a target level of GOF.

[0056] In some cases, the present disclosure provides a method of producing a Ustekinumab drug product having a target level of one or more glycans, the method comprising: culturing a population of cells genetically engineered to express Ustekinumab under conditions, wherein the conditions are characterized by parameters including a selected galactose level and a cell culture time, and wherein the galactose level and time are inversely correlated; harvesting Ustekinumab expressed by the cells, thereby producing a Ustekinumab preparation; and if the Ustekinumab preparation meets the target level of one or more glycans, purifying, concentrating, and / or formulating the Ustekinumab preparation to produce a Ustekinumab drug product. In some embodiments, the target level of one or more glycans is a target level of glycans selected from GOF, sialylated glycans, and G2F. In some embodiments, the target level of one or more glycans is a target level of GOF.

[0057] In some embodiments, the target level of G0F glycans is in the range of 20% to 80% G0F relative to total glycans. In some embodiments, the target level of G0F glycans is in the range of 25% to 65% G0F relative to total glycans.

[0058] In some embodiments, provided methods involve manufacturing and / or producing Ustekinumab with a target level of G0F glycans in the range of 20% to up to 40% G0F relative to total glycans. In some embodiments, methods for achieving Ustekinumab with a target level of G0F glycans in the range of 20% to up to 40% G0F relative to total glycans comprise a selected galactose level of 0 mM, and a cell culture time in the range of 7 days to 15 days.

[0059] In some embodiments, provided methods involve manufacturing and / or producing Ustekinumab having a target level of G0F glycans in the range of 40% to 80% G0F relative to total glycans. In some embodiments, methods for achieving Ustekinumab having a target level of G0F glycans in the range of 40% to 80% G0F relative to total glycans include a selected galactose level in the range of 15 mM to 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 25 days to 42 days.

[0060] In some embodiments, the method provided also includes selecting a target level of G0F glycans. In some embodiments, the target level of G0F glycans is in the range of 20% to 80% G0F relative to total glycans. In some specific embodiments, the target level of G0F glycans is in the range of 20% to a maximum of 40% G0F relative to total glycans, wherein the method includes a selected galactose level of 0 mM, and a cell culture time in the range of 7 days to 15 days. In some specific embodiments, the target level of G0F glycans is in the range of 40% to 80% G0F relative to total glycans, wherein the method includes a selected galactose level in the range of 15 mM to 30 mM, and a cell culture time in the range of 16 days to 60 days.

[0061] In some embodiments, provided methods further include measuring the level of G0F glycans. In some embodiments, if the measured level of G0F glycans is in the range of 20% to 80% G0F relative to total glycans (e.g., in the range of 20% to up to 40% G0F relative to total glycans, such as in the range of 40% to 80% G0F relative to total glycans), steps of purifying, concentrating, and / or formulating Ustekinumab formulations are performed.

[0062] In some embodiments, provided methods for making and / or producing Ustekinumab having target glycan levels include target levels of sialylated glycans and / or G2F glycans. Table 1 below provides conditions for Ustekinumab having target levels of sialylated glycans and / or G2F glycans.

[0063] Table 1 - Exemplary methods for producing Ustekinumab formulations with targeted G2F and sialylated glycan levels condition

[0064] Glycan range G2F Total sialylation NCM-1 (0 mM galactose, culture time 7-15 days) >5％-10％ >15％-30％ NCM-2 (15-30 mM galactose, culture time 25-60 days) 1％-5％ 5％-15％

[0065] In some embodiments, provided methods are directed to manufacturing and / or producing Ustekinumab having a target level of sialylated glycans in the range of 15% to 30% sialylated glycans relative to total glycans. In some embodiments, methods for achieving Ustekinumab having a target level of sialylated glycans in the range of 15% to 30% sialylated glycans relative to total glycans comprise a selected galactose level of 0 mM and a cell culture time in the range of 7 days to 15 days.

[0066] In some embodiments, provided methods involve manufacturing and / or producing Ustekinumab having a target level of sialylated glycans in the range of 5% to up to 15% sialylated glycans relative to total glycans. In some embodiments, methods for achieving Ustekinumab having a target level of G0F glycans in the range of 5% to up to 15% sialylated glycans relative to total glycans include a selected galactose level in the range of 15 mM to 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 25 days to 42 days.

[0067] In some embodiments, provided methods involve manufacturing and / or producing Ustekinumab with a target level of G2F glycans in the range of 5% to 10% G2F relative to total glycans. In some embodiments, methods for achieving Ustekinumab with a target level of G2F glycans in the range of 5% to 10% G2F relative to total glycans comprise a selected galactose level of 0 mM, and a cell culture time in the range of 7 days to 15 days.

[0068] In some embodiments, provided methods involve manufacturing and / or producing Ustekinumab with a target level of G2F glycans in the range of 1% to up to 5% G0F relative to total glycans. In some embodiments, methods for achieving Ustekinumab with a target level of G2F glycans in the range of 1% to up to 5% G2F relative to total glycans include a selected galactose level in the range of 15 mM to 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 25 days to 42 days.

[0069] In some embodiments, provided methods control selected galactose levels throughout the culturing step (eg, during the culturing period from t=0 until harvest).

[0070] In some embodiments, provided methods include culturing a population of mammalian cells genetically engineered to express Ustekinumab. In some embodiments, the mammalian cells are selected from the group consisting of CHO cells, HEK 293 cells, fibrosarcoma HT1080 cells, PER.C6 cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0 cells, and SP 2 / 0 cells.

[0071] In some embodiments, in the provided methods, the culturing step is performed using a continuous culture method. In some embodiments, the provided methods include culturing using a perfusion culture method (e.g., an alternating tangential flow filter (ATF)-based perfusion culture method). In some specific embodiments, the provided methods include culturing mammalian cells genetically engineered to express Ustekinumab (e.g., SP2 / 0 cells expressing Ustekinumab) by a perfusion culture method.

[0072] In some embodiments, methods are provided that include harvesting Ustekinumab expressed by cells two or more times within the time frame of the cell culture period.

[0073] In some embodiments, the target value is a predetermined pharmaceutical product specification or a quality control standard for a pharmaceutical preparation, such as a certificate of analysis (CofA), a certificate of testing (CofT), or a master batch record. In some embodiments, the product specification is a product description in an FDA label, a physician insert, a USP monograph, or an EP monograph.

[0074] Generally, the cell culture methods of the present disclosure include culturing at a temperature in the range of 25°C to 40°C and under gravity encountered on Earth. In some embodiments, the methods of culturing cell populations provided are sufficient to express the Ustekinumab product. The cell culture medium typically contains an appropriate energy source and compounds that regulate the cell cycle. Generally, the culture medium contains amino acids, vitamins, inorganic salts, and glucose, such as those known to those skilled in the art. In some embodiments, the cell culture medium has a pH of 6 to 8. Culture media for animal cell culture are well known in the art and are routinely optimized by the skilled artisan for specific purposes and / or cell types.

[0075] In some embodiments, the methods of the present disclosure include culturing a cell population 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 culture medium supply, and a discharge waste collection. In some embodiments, the continuous cell culture system includes a cell population (e.g., a cell population engineered to express Ustekinumab, e.g., a cell population composed of cells engineered to express Ustekinumab) and a cell culture medium.

[0076] In some embodiments, the cell retention device is or includes a continuous centrifuge, an alternating tangential flow filter (ATF), a tangential flow membrane filter (TFF), a dynamic filter, a spin filter, an ultrasound and dielectrophoresis separator, or a gravity sedimentation tank. In some specific embodiments, the cell retention device is or includes an ATF.

[0077] In some embodiments, the bioreactor system includes a stirred tank bioreactor, a cell retention device, a media supply, and effluent waste collection.

[0078] In some embodiments, the bioreactor system (e.g., a perfusion bioreactor system) includes a sprinkler. In some embodiments, the bioreactor system includes a drilled sprinkler. In some embodiments, the bioreactor system includes an open tube sprinkler. In some embodiments, the bioreactor system includes a sintered sprinkler.

[0079] In some embodiments, the methods of the present disclosure include culturing a cell population (e.g., a cell population engineered to express Ustekinumab, e.g., a cell population consisting of cells engineered to express Ustekinumab) at a volume ranging from 1 L to 3000 L. In some embodiments, provided methods include 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, provided methods include culturing mammalian cells genetically engineered to express Ustekinumab at a volume of about 25 L to about 250 L.

[0080] Glycan Assessment

[0081] In some embodiments, the glycans of Ustekinumab are analyzed (e.g., measured) by any available suitable method. In some cases, the glycan structures and compositions as described herein are analyzed, for example, by one or more of enzymatic, chromatography, mass spectrometry (MS), chromatography followed by MS, electrophoretic methods, electrophoretic methods followed by MS, nuclear magnetic resonance (NMR) methods, and combinations thereof. Exemplary enzymatic methods include contacting the Ustekinumab formulation with one or more enzymes under conditions and times sufficient to release one or more glycans (e.g., one or more exposed glycans). In some cases, the one or more enzymes include PNGase F. Exemplary chromatographic methods include, but are not limited to, high performance anion exchange chromatography (SAX-PAD), liquid chromatography (LC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UPLC), thin layer chromatography (TLC), amide column chromatography, and combinations thereof using pulsed amperometric detection. Exemplary mass spectrometry (MS) includes, but is not limited to, tandem MS, LC-MS, LC-MS / MS, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation-mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), and combinations thereof. Exemplary electrophoresis methods include, but are not limited to, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using antibodies that recognize specific glycan structures, and combinations thereof. Exemplary nuclear magnetic resonance (NMR) include, but are not limited to, one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopy magnetic angle rotation NMR (COSY-NMR), total correlation spectroscopy NMR (TOCSY-NMR), heteronuclear single quantum coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence (HMQC-NMR), rotating nuclear Overhauser effect spectroscopy NMR (ROESY-NMR), nuclear Overhauser effect spectroscopy (NOESY-NMR), and combinations thereof.

[0082] cell

[0083] Any host cell useful for expressing Ustekinumab can be used in the methods described herein. In some embodiments, cells engineered to express Ustekinumab include one or more nucleic acids encoding a Ustekinumab product comprising a heavy chain variable domain as set forth in SEQ ID NO.:1 and / or a light chain variable domain as set forth in SEQ ID NO.:2. In some embodiments, cells engineered to express Ustekinumab include one or more nucleic acids encoding a Ustekinumab product comprising a heavy chain comprising the sequence of SEQ ID NO.:1 and / or a light chain comprising the sequence of SEQ ID NO.:2. In some embodiments, cells engineered to express Ustekinumab include one or more nucleic acids encoding a Ustekinumab product comprising a HCDR1, HCDR2, and HCDR3 sequence as set forth in SEQ ID NO.:1 and a LCDR1, LCDR2, and LCDR3 sequence as set forth in SEQ ID NO.:2.

[0084] In some embodiments, cells engineered to express Ustekinumab include one or more nucleic acids encoding a Ustekinumab product having the same primary amino acid sequence as a protein approved for therapeutic or diagnostic use in humans or animals, e.g., under a secondary approval process. In some embodiments, cells engineered to express Ustekinumab include one or more nucleic acids encoding a Ustekinumab product that differs from an approved therapeutic or diagnostic protein by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues. In some embodiments, cells engineered to express Ustekinumab include one or more nucleic acids encoding a protein having at least 90%, 95%, 98%, 99%, or 100% sequence identity to an approved therapeutic or diagnostic protein. The terms "identical primary amino acid sequence," "primary amino acid sequences that differ by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues," "sequences having at least 98% or greater sequence identity," or similar terms refer to the level of identity between primary amino acid sequences. In some embodiments, protein preparations or products include amino acid variants, such as species that differ at terminal residues (e.g., at one or both terminal residues). In some embodiments of such cases, the sequence identities compared are the identities between the primary amino acid sequences of the most abundant (e.g., most abundant active) species in each product being compared. In some embodiments, the sequence identity refers to the amino acid sequence encoded by a nucleic acid that can be used to prepare the ustekinumab product.

[0085] In some embodiments, the cell engineered to express Ustekinumab is a mammalian cell.

[0086] In some embodiments, the cells engineered to express Ustekinumab are mouse cells. In some embodiments, the cells engineered to express Ustekinumab are from a murine cell line. Mouse (e.g., mouse) cell lines include, for example, murine myeloma cell lines, such as NS0 cells and SP 2 / 0 cells.

[0087] In some embodiments, the cells engineered to express Ustekinumab are human cells. In some embodiments, the cells engineered to express Ustekinumab are from human cell lines. Human cell lines include, for example, HEK 293: human embryonic kidney 293; HT-1080: from a fibrosarcoma with an epithelioid phenotype; PER.C6: from human embryonic retinal cells immortalized via transfection with an adenovirus E1 gene; CAP: from human amniocytes immortalized by an adenovirus type 5 E1 gene; HKB-11: created by polyethylene glycol fusion of HEK293-S with a human B cell line; and HuH-7: from a 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.

[0088] In some embodiments, a cell population expressing a Ustekinumab product as described herein is produced using recombinant methods. Recombinant expression of a gene (such as a gene encoding a polypeptide such as an antibody reagent described herein) can include constructing an expression vector containing a polynucleotide encoding the polypeptide. Once the polynucleotide is obtained, a vector for producing the polypeptide can be produced by recombinant DNA technology using techniques known in the art. Known methods can be used to construct an expression vector containing a polypeptide coding sequence and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0089] Once the Ustekinumab product described herein is produced by recombinant expression, it can be purified by any method known in the art for purification, such as by chromatography (e.g., ion exchange, affinity, and fractionated column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. For example, Ustekinumab can be isolated and purified by appropriately selecting and combining affinity columns such as Protein A columns with chromatographic columns, filtration, ultrafiltration, salting out, and dialysis processes (see Antibodies:A Laboratory Manual , Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). In addition, as described herein, the Ustekinumab product can be fused to a heterologous polypeptide sequence to facilitate purification.

[0090] Pharmaceutical composition

[0091] The Ustekinumab product produced or manufactured using any method, system and / or process described herein can be introduced into a pharmaceutical composition. Such pharmaceutical compositions can be used to prevent and / or treat diseases. Pharmaceutical compositions comprising Ustekinumab can be prepared by methods known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 20th edition, Lippincott Williams & Wilkins, 2000). Pharmaceutical compositions can be administered parenterally in the form of injectable preparations (including sterile solutions or suspensions in water or another pharmaceutically acceptable liquid). For example, cell products (e.g., recombinant proteins, such as glycoproteins, such as antibody reagents) can be appropriately combined with pharmaceutically acceptable carriers or media (such as sterile water and saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring excipients, diluents, vehicles, preservatives, binders), and then mixed with the unit dosage form required for recognized pharmaceutical practice to prepare pharmaceutical compositions. The amount of active ingredient contained in the pharmaceutical preparation is such that a suitable dose within a specified range is provided.

[0092] In some embodiments, the formulation of Ustekinumab comprises sucrose as a stabilizer / tonicity modifier. In some embodiments, the formulation of Ustekinumab comprises histidine (e.g., L-histidine) as a buffer. In some embodiments, the formulation of Ustekinumab comprises polysorbate 80 as a surfactant. In some specific embodiments, the formulation of Ustekinumab comprises histidine (e.g., L-histidine), sucrose, and polysorbate 80.

[0093] In some embodiments, the formulation of Ustekinumab is formulated for parenteral administration, e.g., intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the formulation of Ustekinumab is formulated for subcutaneous administration.

[0094] The present disclosure is further illustrated by the following examples. These examples are provided only for illustrative purposes. They should not be construed as limiting the scope or content of the present disclosure in any way.

[0095] Example

[0096] Example 1: Identification of changes in glycan composition of Ustekinumab products

[0097] This example identifies significant variation in glycan composition among commercially available Ustekinumab Reference Protein Product ("RPP") batches. Specifically, this example demonstrates the variability in glycan composition among commercially available Ustekinumab batches separated into two distinct glycan profiles. These two groups of glycan variants are referred to herein as "Group 1," which is characterized by relatively high levels of G0F glycans, and "Group 2," which is characterized by relatively low levels of G0F glycans.

[0098] Thirty (30) batches of Ustekinumab RPPs were analyzed. Of these 30 batches, 28 were from 90 mg / mL formulations and 2 were 5 mg / mL formulations. Of the 28 batches of 90 mg / mL Ustekinumab RPPs, eight (8) of these batches were identified as having Group 1 glycan profiles and twenty (20) were identified as having Group 2 glycan profiles. See Figure 1 .

[0099] A summary of the mean abundance of each of the major glycan species for Group 1 and Group 2 samples is summarized in Table 2 below.

[0100] Table 2: Abundance of major glycans in Group 1 and Group 2 Ustekinumab samples

[0101] Polysaccharide (%) Group 1 Group 2 4,3,1,0,0(G0F) 27.7 62.1 4,4,1,0,0A(G1F-A) 28.1 14.1 4,4,1,0,0 B(G1F-B) 6.6 3.5 4，4，1，0，1 A 5.4 4.1 4，4，1，0，1 B 3.2 2.9 4,5,1,0,0(G2F) 8.4 2.3 4,6,1,0,0 1.4 0.2 4,5,1,0,1 6.7 2.1 4,5,1,0,2 2.0 0.9 4,6,1,0,1 B 3.4 0.7 The sum of the above 92.9 92.9

[0102] Ustekinumab test products generated by culturing with 5 mM galactose (e.g., by an ATF-based perfusion culture method) at different volumes ranging from 3 L to 250 L for a duration of 42 days were also analyzed. The glycan abundance of these Ustekinumab test products is depicted in Figure 1 The glycan profiles of the Ustekinumab test products are within the range of those of Group 1 and Group 2 RPPs. Thus, this example identifies two distinct groups of glycan profiles of commercially available Ustekinumab RPPs and demonstrates that concurrent culture conditions produce Ustekinumab test products with glycan abundances intermediate between the ranges of these two groups.

[0103] Example 2: Influence of culture time on the glycan composition of Ustekinumab

[0104] This example demonstrates that the timing of harvest is a factor in controlling the glycan composition of Ustekinumab. Figure 2 As depicted, the abundance of G0F glycans relative to total glycans generally decreased with culture duration. For example, Ustekinumab preparations harvested at 13 days of culture had almost 50% abundance of G0F glycans, while samples harvested after 34 days or more of culture had less than 30% G0F glycans relative to the total glycan composition.

[0105] Interestingly, the present disclosure provides the following insights: a decrease in culture duration (ie, time until harvest) is accompanied by an increase in GOF levels; whereas an increase in culture duration (ie, time until harvest) is coupled with a decrease in GOF levels.

[0106] Thus, this example demonstrates that increasing incubation time can reduce the abundance of G0F glycans relative to the total glycan composition, and also describes that incubation time can be varied to control Ustekinumab glycan composition.

[0107] Example 3: Generation of Ustekinumab Products with Group 1 and Group 2 Glycan Profiles

[0108] This example identifies an inverse correlation between culture time and galactose concentration in the culture medium, which can serve as a process means to control the level of Ustekinumab glycans (e.g., galactosylation). This example demonstrates the generation of non-consistent materials (i.e., non-consistent Ustekinumab formulations) with engineered glycan profiles. Specifically, this example demonstrates the generation of two non-consistent Ustekinumab formulations, which are referred to herein as NCM-1 and NCM-2, and which have been engineered to have glycan profiles matching Group 1 RPPs and Group 2 RPPs, respectively (as described in Example 1 above).

[0109] Specifically, NCM-1 preparations were generated by culturing in a medium lacking galactose (0 mM galactose) (e.g., by an ATF-based perfusion culture method) and harvesting Ustekinumab before day 16 of culture (e.g., harvesting at a time point in the range of 7 to 15 days). In contrast, NCM-2 preparations were generated by culturing with 15 mM galactose (e.g., by an ATF-based perfusion culture method) and harvesting Ustekinumab after day 25 of culture (e.g., harvesting at a time point in the range of 34 to 45 days). Figure 3 As described, for each glycan class analyzed (including G0F, G1FA, G1FB, G2F, total sialylated, major, acidic, and basic glycans), the NCM-1 glycan profile correlated well with those of Group 1 RPPs, and the NCM-2 glycan profile correlated well with those of Group 2 RPPs. Table 3 provides exemplary target ranges for each glycan engineered by process means of controlling galactose concentration and culture time.

[0110] Table 3 - Exemplary target glycan ranges for NCM-1 and NCM-2 Ustekinumab formulations

[0111]

[0112] Thus, this example demonstrates that the inverse correlation of galactose concentration and incubation time can be used as a process tool to control glycan composition and can be used to generate heterogeneous Ustekinumab formulations with target glycan levels.

[0113] Equivalent solution

[0114] It should be understood that while the disclosure has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. A method for manufacturing a pharmaceutical composition comprising Ustekinumab having a target level of G0F glycans, the method comprising: (a) selecting a target level of G0F glycans, wherein the target level of G0F glycans is in the range of 40% to 80% relative to total glycans, (b) selecting a galactose level and a cell culture time to provide a selected G0F glycan level, wherein the galactose level is in the range of 15 mM to 30 mM, and the cell culture time is in the range of 16 days to 60 days; (c) culturing a population of cells genetically engineered to express Ustekinumab under conditions including a selected level of galactose and for a selected period of time; (d) harvesting Ustekinumab expressed by the cell population, thereby producing a preparation of Ustekinumab; as well as (e) if the preparation meets the target level of GOF glycans, purifying, concentrating and / or formulating the Ustekinumab preparation to produce a pharmaceutical composition comprising Ustekinumab.

2. The method according to claim 1, wherein the culturing is performed using a perfusion culturing method.

3. The method according to claim 1, wherein the cell culture time is in the range of 25 to 60 days. The method according to claim 1 , wherein the cell culture time is in the range of 25 to 42 days.

5. The method of claim 1 or 2, further comprising measuring the level of G0F glycans.

6. The method of claim 1 or 2, wherein the population of cells genetically engineered to express Ustekinumab are mammalian cells.

7. The method according to claim 6, wherein the mammalian cell is selected from CHO cells, HEK 293 cells, fibrosarcoma HT 1080 cells, PER.C6 cells, CAP cells, HKB-11 cells, HuH-7 cells, NS0 cells or SP 2 / 0 cells.

8. The method of claim 1 or 2, wherein the selected galactose level is controlled during the cultivation period from t=0 until harvest.

9. The method of claim 1 or 2, wherein Ustekinumab expressed by the cells is harvested two or more times within the time frame of the cell culture time.

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