Reducing α-GAL

Abstract

The present disclosure relates to methods for producing glycoproteins with reduced a-Gal content by lowering the pH from 7.20 to 6.90, by increasing the GlcNAc or zinc concentration and / or by decreasing the uridine or manganese concentration. Also provided are glycoproteins obtained or obtainable by the methods.

Description

[0001] This disclosure relates to a method for producing glycoproteins with reduced α-Gal content. Glycoproteins that can be obtained or obtained by said method are also provided. Background Technology

[0002] Galactose-α-1,3-galactose (alpha-Gal or α-Gal) is a common carbohydrate structure expressed in mammalian tissues and synthesized by the glycosylation enzyme α-1,3-galactosyltransferase (Figure 1). α-gal modifies extracellular proteins and lipids and is expressed to varying degrees in all organs, tissues, and cell types in mammals (except for humans and Old World primates, which lack the functional gene encoding α-1,3-galactosyltransferase).

[0003] The deletion of this gene in humans leads to the production of anti-Gal antibodies specific to the α-Gal antigen, reaching a level where these anti-Gal antibodies account for approximately 1% of all circulating immunoglobulins in humans. Therefore, exposure to α-Gal-containing glycans elicits an immunogenic response. Furthermore, individuals possessing IgE isotype anti-Gal antibodies develop allergic reactions as a response to exposure to α-Gal.

[0004] Because some drugs and vaccines contain α-Gal glycans, such biotherapeutic agents need to be carefully screened to avoid unwanted immunogenicity.

[0005] The purpose of this invention is to provide a new method for reducing the α-Gal content in glycoprotein production. Summary of the Invention

[0006] This invention provides a method for producing glycoproteins with reduced α-Gal content.

[0007] According to a first aspect of the invention, a method for producing a glycoprotein having a reduced α-Gal content is provided, the method comprising: culturing a eukaryotic cell line containing a polynucleotide encoding a polypeptide portion of the glycoprotein under conditions suitable for producing the glycoprotein, wherein the conditions suitable for production include one or more of the following: (a) a pH below about 7.1; (b) a GlcNAc concentration in the culture of at least about 10 mM; (c) a zinc concentration in the culture of at least about 400 µM; (d) a uridine concentration in the culture of less than about 15 mM; and (e) a manganese concentration in the culture of less than about 400 nM.

[0008] It is not desired to be bound by theory, but it is believed that controlling one or more of the conditions (a) to (e) in the method of this disclosure results in the production of glycoproteins with a reduced α-Gal content compared to glycoproteins produced without controlling said conditions.

[0009] According to a second aspect of the invention, a glycoprotein is provided that can be obtained or acquired by the method of the first aspect. Attached Figure Description

[0010] The embodiments of the present invention will be further described below with reference to the accompanying drawings, wherein:

[0011] Figure 1 shows a schematic equation for the synthesis of α-gal polysaccharides (e.g., Galα1-3Galβ1-4GlcNAc-R) by reacting a desired precursor (e.g., Galβ1-4GlcNAc-R) with a Gal-containing moiety (e.g., UDP-Gal) in the presence of an enzyme (e.g., α1-3GT) (Macher et al. (2008)).

[0012] Figures 2A (top), 2B (middle), and 2C (bottom) illustrate the procedural indicators identified in the DOE screening of ligand fusion proteins from the first cell line. Results are shown from the control (i.e., the condition producing elevated α-gal), the midpoint (i.e., the midpoint between the control and test conditions for each tested parameter), and the test cases. A series of growth profiles were observed. Cases involving pH changes (indicated by asterisks) showed decreased growth.

[0013] Figure 3 shows the total galactosylation (G1F+G2F+G1S1F+G2S1F+G2S2F) of the ligand fusion protein in the first cell line, including control, center point, and DOE test cases. Compared to the control, the center point condition reduced total galactosylation by approximately 20%. The DOE test conditions show a series of total galactosylations, with cases exhibiting pH changes (starred) showing reductions in total galactosylation up to approximately 40%.

[0014] Figure 4A shows the total galactosylation (G1F+G2F+G1S1F+G2S1F+G2S2F) of three cell lines, Figure 4B shows the final viability of the three cell lines, and Figure 4C shows the volumetric viable cell concentration integral (IVCC) of the three cell lines, each adapted to express a different product. The first cell line expresses a ligand fusion protein, the second cell line expresses a complex antibody, and the third cell line expresses a complex bispecific antibody. In each case, control processes and process levers with and without pH changes were compared. In two of the three cell lines, process levers reduced total galactosylation by >10%. In all tested cell lines, pH changes, together with process levers, further reduced galactosylation, ranging from 10% to 30% in all cell lines.

[0015] Figure 5 shows how the relative abundance of α-gal glycans is correlated with total galactosylation in the three cell lines. Samples were treated with sialidase and analyzed using HILIC-MS. The process lever of reducing total galactosylation also reduced the abundance of α-gal. Detailed Implementation

[0016] The abbreviations used in this article have their conventional meanings in the fields of chemistry and biology.

[0017] Throughout the description and claims of this specification, the words “comprising” and “including” and variations thereof mean “including, but not limited to”, and they are not intended to exclude (nor exclude) other parts, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular includes the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification shall be understood to consider both the plural and the singular unless the context requires otherwise.

[0018] Features, integers, properties, compounds, chemical parts, or groups described in connection with a particular aspect, embodiment, or example of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process disclosed thereby may be combined in any combination, except for at least some mutually exclusive combinations of such features and / or steps. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel feature or any novel combination of features disclosed in this specification (including any appended claims, abstract, and drawings), or to any novel step or any novel combination of steps of any method or process disclosed thereby.

[0019] Readers should note that all documents and files submitted concurrently with or prior to this specification and made publicly available together with this specification are incorporated herein by reference.

[0020] To avoid any doubt, it is hereby declared that the information disclosed above under the heading "Background Art" in this specification is related to the present invention and should be understood as part of the disclosure of the present invention.

[0021] All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification (including definitions) shall prevail.

[0022] definition

[0023] The terms used in this specification generally have their common meaning in the art, both in the context of this disclosure and in the specific context in which each term is used. Some terms are discussed below or elsewhere in the specification to provide additional guidance to practitioners in describing the compositions and methods of this disclosure and how they are made and used.

[0024] As used herein, the use of the word "a" or "one" in conjunction with the term "comprising" in the claims and / or specification may mean "a" or "one," but is also consistent with the meaning of "a" or "one or more," "at least one" or "one" and "a" or "more than one."

[0025] The terms “comprising,” “including,” “having,” “possessing,” “may,” “containing,” and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words and do not exclude the possibility of additional actions or structures. This disclosure also contemplates other embodiments that “comprising,” “consisting of,” and “substantially constitute” the embodiments or elements presented herein, whether or not explicitly stated.

[0026] The term "about" or "approximately" refers to an acceptable range of error for a particular value, as determined by one of ordinary skill in the art, where the acceptable range of error will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean 3 or more standard deviations. Alternatively, "about" may represent a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within a certain order of magnitude of a value, preferably within 5 times, more preferably within 2 times.

[0027] The terms "cell culture medium" and "culture medium" refer to nutrient solutions used for growing mammalian cells, which typically provide at least one component from one or more of the following categories:

[0028] 1) Energy source, generally existing in the form of carbohydrates (such as glucose);

[0029] 2) All essential amino acids, and generally a basic group consisting of twenty amino acids plus cysteine;

[0030] 3) The requirement is for low concentrations of vitamins and / or other organic compounds;

[0031] 4) Free fatty acids; and

[0032] 5) Trace elements, which are defined as inorganic compounds or naturally occurring elements, and the required amount is usually at a very low concentration, generally in the micromolar range.

[0033] The nutrient solution may optionally be supplemented with one or more ingredients from any of the following categories:

[0034] 1) Hormones and other growth factors, such as insulin, transferrin, and epidermal growth factor;

[0035] 2) Salts and buffers, such as calcium, magnesium, and phosphates;

[0036] 3) Nucleosides and bases, such as adenosine, thymidine, and hypoxanthine; and

[0037] 4) Proteins and tissue hydrolysates.

[0038] "Culturing" cells refers to bringing cells into contact with a cell culture medium under conditions suitable for cell survival and / or growth and / or proliferation.

[0039] "Batch culture" refers to a culture in which all components for cell culture (including cells and all culture nutrients) are supplied to the culture bioreactor at the beginning of the culture process.

[0040] As used herein, “feed-batch cell culture” refers to batch culture in which cells and culture medium are initially supplied to the culture bioreactor, and additional culture nutrients are continuously or discontinuously supplied to the culture during the culture process, with or without periodic cell and / or product harvesting before the culture is terminated.

[0041] "Perfusion culture," sometimes called continuous culture, is the cultivation of cells by confining them in a culture medium through methods such as filtration, encapsulation, or anchoring to microcarriers, and by continuously, gradually, or intermittently (or any combination thereof) introducing the culture medium and removing it from the culture bioreactor.

[0042] As used herein, the term "cell" refers to animal cells, mammalian cells, cultured cells, host cells, recombinant cells, and recombinant host cells. Such cells are typically cell lines obtained from or derived from mammalian tissues that are capable of growing and surviving when placed in a culture medium containing appropriate nutrients and / or growth factors.

[0043] As used herein, the term "cell line" includes references to reproducible eukaryotic cell cultures. The eukaryotic cells in such cell lines may be selected from any cells as defined herein.

[0044] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells and their progeny from which exogenous nucleic acids may be subsequently introduced to create recombinant cells. These host cells may also be modified (i.e., engineered) to alter or delete the expression of certain endogenous host cell products (e.g., endogenous virus-like particles or endogenous host cell proteins). Host cells include “transformers” and “transformed cells,” which include primary transformed cells and progeny derived from said primary transformed cells, regardless of passage number. Progeny do not need to be identical to the nucleic acid contents of the parent cells but may contain mutations. This document includes mutant progeny with the same function or biological activity as screened or selected in the original transformed cells. Introducing exogenous nucleic acids (e.g., by transfection) into these host cells creates recombinant cells derived from the original “host cell,” “host cell line,” or “host cell line.” The terms “host cell,” “host cell line,” and “host cell culture” may also refer to such recombinant cells and their progeny. The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells and their progeny in which exogenous nucleic acids have been introduced to enable the expression of a desired recombinant product. The recombinant product expressed by such cells can be a recombinant protein, recombinant viral particle, or recombinant viral vector.

[0045] The term "mammalian host cell" or "mammalian cell" refers to cell lines derived from mammals that are capable of growth and survival when placed in a monolayer culture or a suspension culture in a medium containing appropriate nutrients and growth factors. The essential growth factors for a particular cell line can be readily determined empirically without excessive experimentation, as described in *Mammalian Cell Culture* (Mather, JP, ed., Plenum Press, NY 1984) and *Barnes & Sato*, (1980) *Cell*, 22:649. Typically, cells are able to express and secrete large amounts of specific proteins (e.g., glycoproteins) into the culture medium. Examples of suitable mammalian host cells within the context of this disclosure may include Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980); dp12.CHO cells (EP 307,247, published March 15, 1989); CHO-K1 (ATCC, CCL-61); young hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 1980); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL1442); and mouse mammary tumors (MMT 060562, ATCC CCL51). In some embodiments, the mammalian cells include Chinese hamster ovary cells (CHO). In some embodiments, the cells contain polynucleotides encoding a polypeptide. In another embodiment, the cells transiently express the polypeptide or stably express the polypeptide. In a further embodiment, cells stably expressing the polypeptide contain polynucleotides integrated into the cell's genome at a targeted location. In yet another further embodiment, cells stably expressing the polypeptide contain polynucleotides integrated into the cell's genome at a random location.

[0046] The “growth phase” in cell culture refers to the exponential (logarithmic) cell growth phase in which cells typically divide rapidly. For example, the duration for which cells remain in the growth phase can vary depending on cell type, cell growth rate, and / or culture conditions. In some embodiments, cells are cultured for a period during this phase, typically between 1 and 4 days, under conditions that maximize cell growth. The determination of the host cell growth cycle can be tailored to a specific host cell without excessive experimentation. “The period of time for maximizing cell growth and such conditions” refers to those culture conditions determined to be optimal for cell growth and division for a particular cell line. In some embodiments, during the growth phase, cells are cultured in a nutrient medium containing necessary additives, typically at approximately 30 to 40°C in a humid, controlled atmosphere to achieve optimal growth for the specific cell line. In some embodiments, cells are maintained in the growth phase for a period between approximately one and four days, typically between two and three days.

[0047] The “production phase” of a cell culture refers to the period when cell growth reaches / has reached a plateau. Logarithmic cell growth typically decreases before or during this phase, and protein production takes over. During the production phase, logarithmic cell growth has ceased, and protein production is dominant. During this period, culture medium is typically replenished to support continued protein production and obtain the desired glycoprotein product. Feed-and-perfusion cell culture processes replenish cell culture medium or provide fresh medium during this period to achieve and / or maintain the desired cell density, viability, and / or recombinant protein product titers. Production phases can be performed on a large scale.

[0048] As used herein, the term "activity" in relation to protein activity refers to any activity of a protein, including but not limited to enzyme activity, ligand binding, drug transport, ion transport, protein localization, receptor binding, and / or structural activity. This activity can be modulated by reducing or eliminating the expression of the protein, for example, by reducing or eliminating it, thereby reducing or eliminating the presence of the protein. This activity can also be modulated by altering the nucleic acid sequence encoding the protein, for example, by reducing or eliminating it, so that the resulting modified protein exhibits reduced or eliminated activity relative to the wild-type protein.

[0049] The term “expression” as used in this article, whether in its noun or verb form, refers to transcription and translation occurring within a host cell. The expression level of a product gene in a host cell can be determined based on the amount of corresponding mRNA present in the cell or the amount of protein encoded by the product gene produced by the cell. For example, mRNA transcribed from a product gene is ideally quantified by northern hybridization. (Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3–7.57 (Cold Spring Harbor Laboratory Press, 1989)). Proteins encoded by product genes can be quantified by various methods, such as by measuring the protein’s biological activity or by employing assays unrelated to that activity, such as Western blotting or radioimmunoassay using antibodies capable of reacting with the protein. (Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1–18.88 (Cold Spring Harbor Laboratory Press, 1989)). When referring to reducing and / or eliminating the expression of one or more endogenous products relative to the expression of an unmodified cell, such reduction and / or elimination includes reducing and / or eliminating the active endogenous product, even in the presence of mRNA encoding all or part of the endogenous product or in the presence of an endogenous product translated from such mRNA.

[0050] As used herein, "polypeptide" generally refers to peptides and proteins having more than about ten amino acids. Polypeptides may be homologous to host cells or preferably exogenous, meaning that these polypeptides are heterologous to the host cells utilized, i.e., foreign, such as human proteins produced by Chinese hamster ovary cells or yeast polypeptides produced by mammalian cells. In some embodiments, mammalian polypeptides (polypeptides originally derived from mammalian organisms) are used, more preferably those polypeptides that are secreted directly into the culture medium.

[0051] The term "protein" means an amino acid sequence with a chain length sufficient to produce higher levels of tertiary and / or quaternary structures. This is to distinguish it from "peptides" or other small molecular weight drugs that do not possess such structures. Typically, proteins as defined herein will have a molecular weight of at least about 15 to 20 kD, preferably at least about 20 kD. Examples of proteins covered in this definition include host cell proteins as well as all mammalian proteins, particularly therapeutic and diagnostic proteins, such as therapeutic and diagnostic antibodies, and are generally proteins containing one or more disulfide bonds, including multi-chain polypeptides containing one or more interchain and / or intrachain disulfide bonds.

[0052] The term "glycoprotein" refers to a protein containing oligosaccharide chains covalently linked to amino acid side chains. During a process known as glycosylation, oligosaccharides can be linked to proteins in co-translation or post-translational modifications. Exemplary glycoproteins include antibodies that typically have N-linked oligosaccharides on each heavy chain.

[0053] The term "antibody" is used in the broadest sense herein and encompasses a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies consisting of a single heavy chain sequence and a single light chain sequence, including such paired multimers), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity. Therapeutic antibodies are antibodies that can be used to treat diseases.

[0054] As used herein, “antibody fragment,” “antigen-binding portion” of an antibody (or simply “antibody portion”), or “antigen-binding fragment” of an antibody refers to a molecule other than the intact antibody that contains the antigen-binding portion of the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0055] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0056] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some embodiments, the antibody is the IgG1 isotype. In some embodiments, the antibody is the IgG2 isotype. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody, based on the amino acid sequence of its constant domain, can be classified into one of two types, called Kappa (κ) and Lambda (λ).

[0057] As used herein, the term "titer" refers to the total amount of recombinant expressed antibody produced from a cell culture divided by a given volume of culture medium. Titers are typically expressed in milligrams of antibody per milliliter or liter of culture medium (mg / ml or mg / L). In some embodiments, titers are expressed in grams of antibody per liter of culture medium (g / L). Titers can be expressed or assessed based on relative measurements, such as the percentage increase in titer compared to protein products obtained under different culture conditions.

[0058] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or generated during the production of a monoclonal antibody formulation, such variants are typically present in small quantities), the individual antibodies comprising this group are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used in the currently disclosed subject matter can be prepared using a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0059] A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or to a non-human antibody derived from a complete library of human antibodies or other antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0060] "Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from a non-human complementarity-determining region (CDR) and amino acid residues from a human frame region (FR). In some respects, humanized antibodies will substantially contain at least one, typically both, variable domains, wherein all or substantially all CDRs correspond to the CDRs of a non-human antibody, and all or substantially all FRs correspond to the FRs of a human antibody. Humanized antibodies may optionally contain at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have already undergone humanization.

[0061] As used herein, the term “recombinant protein” generally refers to peptides and proteins encoded by “heterologous” (i.e., foreign to the host cell used) nucleic acids, such as nucleic acids encoding human antibodies introduced into non-human host cells, including antibodies.

[0062] As used herein (unless the context requires otherwise), the term “specified level” refers to a pH level below approximately 7.1 as listed in condition (a).

[0063] The following abbreviations are used in this article:

[0064]

[0065] Methods for producing glycoproteins

[0066] In one aspect, the present invention provides a method for producing a glycoprotein having a reduced α-Gal content, the method comprising: culturing a eukaryotic cell line containing a polynucleotide encoding a polypeptide portion of the glycoprotein under conditions suitable for glycoprotein production, wherein the conditions suitable for production include one or more of the following: (a) a pH below about 7.1; (b) an N-acetylglucosamine (GlcNAc) concentration in the culture of at least about 10 mM; (c) a zinc concentration in the culture of at least about 400 µM; (d) a uridine concentration in the culture of less than about 15 mM; and (e) a manganese concentration in the culture of less than about 400 nM.

[0067] In the implementation, (a) the pH is from about 7.1 to about 6.7. In the implementation, (a) the pH is from about 7.1 to about 6.8. In the implementation, (a) the pH is from about 7.1 to about 6.9. The pH may be about 6.9.

[0068] In the implementation scheme, the eukaryotic cell line is cultured under fed-batch culture conditions, wherein condition (a) includes shifting the pH from a higher pH to a pH below about 7.1. The higher pH may be from about 7.2 to about 7.4. The higher pH may be about 7.2.

[0069] In one implementation, the pH change can be performed at least 2 days after the start of culture. In another implementation, the pH change can be performed at least 4 days after the start of culture. In yet another implementation, the pH change can be performed at least 6 days after the start of culture.

[0070] In one implementation, (a) includes at least the last 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days of maintaining pH at a specified level suitable for production. In another implementation, (a) includes at least the last 5, 6, 7, or 8 days of maintaining pH at a specified level suitable for production. In yet another implementation, (a) includes at least the last 6 days of maintaining pH at a specified level suitable for production.

[0071] In the implementation scheme, (a) includes maintaining the pH at a specified level suitable for production for no more than the last 4, 5, 6, 7, 8, 9, 10, 11, or 12 days. In the implementation scheme, (a) includes maintaining the pH at a specified level suitable for production for no more than the last 5, 6, 7, or 8 days. In the implementation scheme, (a) includes maintaining the pH at a specified level suitable for production for no more than the last 6, 7, or 8 days.

[0072] Enzymes typically exhibit optimal activity within a specific pH range for a given enzyme. Without wishing to be bound by theory, it is believed that lowering the pH to approximately 7.1 increases the activity of galactosidases (e.g., CHO galactosidase exhibits optimal activity at lower pH). Therefore, as described in this disclosure, lowering the pH or reducing the pH should decrease galactosylation and thus decrease α-Gal levels.

[0073] In the embodiments, (b) the concentration of GlcNAc in the culture is about 10 mM to about 50 mM. In the embodiments, the concentration of GlcNAc in the culture is about 10 mM to about 25 mM. In the embodiments, the concentration of GlcNAc in the culture is about 10 mM to about 20 mM. In the embodiments, the concentration of GlcNAc in the culture is about 10 mM to about 18 mM. It is not intended to be theoretically constrained, but it is believed that supplementing cell culture medium with GlcNAc reduces galactosyltransferase activity. Specifically, the enzyme UDP-glucose 4-epimerase / UDP-galactose 4-epimerase (GALE) converts UDP-glucose to UDP-Gal and UDP-GlcNAc to UDP-GalNAc. It is believed that supplementing with GlcNAc increases the amount of UDP-GlcNAc, which acts as a competitive substrate for GALE and thus reduces the production of UDP-Gal. Lower UDP-Gal levels are expected to reduce α-Gal levels.

[0074] In one embodiment, the zinc concentration in (c) culture is from about 400 µM to about 2,500 µM. In another embodiment, the zinc concentration in (c) culture is from about 800 µM to about 2,000 µM. In yet another embodiment, the zinc concentration in (c) culture is from about 1,200 µM to about 1,800 µM. In yet another embodiment, the zinc concentration in (c) culture is from about 1,200 µM to about 1,600 µM. It is not intended to be theoretically constrained, but rather that zinc (e.g., Zn) is considered to be... 2+ It acts as an inhibitor of galactosyltransferase, resulting in reduced galactosyltransferase activity when the concentration of zinc in the culture increases above a given level.

[0075] In the implementation scheme, the uridine concentration in the (d) culture is from about 0 mM to about 0.2 mM. In the implementation scheme, the uridine concentration in the (d) culture is from about 0 mM to about 0.15 mM. In the implementation scheme, the uridine concentration in the (d) culture is from about 0 mM to about 0.1 mM. In the implementation scheme, the uridine concentration in the (d) culture is about 0 mM. It is not intended to be theoretically constrained, but it is believed that reducing the amount of uridine can lead to a reduction in the amount of UDP-Gal produced. In particular, uridine is a precursor to uridine triphosphate (UTP) required for the conversion of Gal-1P to UDP-Gal, which provides a source of α-Gal epitopes in the production of α-Gal-containing glycans. By reducing the amount of UDP-Gal, the amount of galactosylated glycans will also decrease. Therefore, it is believed that reducing the amount of uridine leads to a decrease in galactosyltransferase activity.

[0076] In the embodiment, (e) the manganese concentration in the culture is from about 0 nm to about 400 nm. In the embodiment, (e) the manganese concentration in the culture is from about 0 nm to about 200 nm. In the embodiment, (e) the manganese concentration in the culture is about 0 nm. It is not intended to be theoretically constrained, but it is believed that manganese (e.g., Mn) 2+ ) is a cofactor for galactosyltransferase activity, which causes the activity of manganese (e.g., Mn) in the culture to increase. 2+ The decrease in the concentration of ) resulted in a decrease in the activity of galactosyltransferase.

[0077] In the implementation scheme, suitable conditions for production include one or more of (a) and (b), (c), (d), (e) and (f).

[0078] In the implementation scheme, the conditions suitable for production include (d) and (e). In the implementation scheme, the conditions suitable for production further include one or more of (a), (b) and (c).

[0079] In the implementation scheme, suitable conditions for production include (a), (b), and (c), and optionally (d) and / or (e).

[0080] In the implementation plan, the conditions suitable for production include (a), (b), (c), (d), and (e).

[0081] In the implementation plan, the glycoprotein is a therapeutic glycoprotein.

[0082] In the implementation scheme, the polypeptide portion of the glycoprotein is a recombinant polypeptide.

[0083] In the implementation scheme, the glycoprotein is a recombinant glycoprotein selected from fusion proteins (e.g., ligand fusion proteins), antibodies, antigens, enzymes, or vaccines.

[0084] In the implementation plan, the antibody is a multispecific antibody or its antigen-binding fragment.

[0085] In the implementation scheme, the antibody consists of a single heavy chain sequence and a single light chain sequence or an antigen-binding fragment thereof.

[0086] In the implementation plan, antibodies include chimeric antibodies, human antibodies, or humanized antibodies.

[0087] In the implementation plan, antibodies include monoclonal antibodies.

[0088] In an embodiment, the method further includes separating the glycoprotein. The glycoprotein can be separated by any suitable method. For example, separation may include contacting the glycoprotein with a reagent having an affinity for the glycoprotein, and optionally washing and / or eluting the glycoprotein from the reagent having an affinity for the glycoprotein. The affinity reagent may be provided as part of a stationary phase. Examples of reagents having an affinity for glycoproteins include HILIC stationary phases or lectins. In the case that the glycoprotein is a specific class of glycoprotein, the reagent having an affinity for the glycoprotein may be selected to have an affinity for that specific class. For example, in the case that the glycoprotein is an antibody, the affinity reagent may be selected from protein G, protein A, protein A / G, protein L, or a combination of one or more thereof.

[0089] In the implementation scheme, the eukaryotic cell line is an animal cell line. In the implementation scheme, the eukaryotic cell line is a mammalian cell line. For example, the mammalian cell line may be selected from the Chinese hamster ovary (CHO) cell line, the young hamster kidney (BHK) cell line, the mouse support cell line, the Madin-Darby canine kidney (MDCK) cell line, the Buffalo rat liver (BRL) cell line, or the mouse mammary tumor cell line or its derivatives.

[0090] In the implementation scheme, the mammalian cell line is a modified mammalian cell line. For example, the modified mammalian cell line may be selected from CHO / DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980), dp12.CHO (EP 307,247 published on March 15, 1989), CHO-K1 (ATCC, CCL-61), BHK ATCC CCL 10, mouse supporting cells (TM4, Mather, Biol. Reprod., 23:243-2511980), MDCK ATCC CCL 34, HEK 293, BRL 3A ATCC CRL 1442, MMT 060562 ATCC CCL51, or derivatives thereof.

[0091] In the implementation scheme, the cell line is a CHO cell line or a derivative thereof. For example, the cell line may be a CHO K1 cell line, a CHO K1SV cell line, a DG44 cell line, a DUKXB-11 cell line, a CHOK1S cell line, or a CHO K1M cell line, or a derivative thereof.

[0092] In the embodiments, the polynucleotide encoding the polypeptide portion of the glycoprotein is an extrachromosomal polynucleotide or an integrated polynucleotide incorporated into the chromosome of the cell line. The polynucleotide encoding the polypeptide portion of the glycoprotein can be an extrachromosomal polynucleotide. The polynucleotide encoding the polypeptide portion of the glycoprotein can be integrated into the chromosome of the cell line. In embodiments where the polynucleotide is an integrated polynucleotide, the integrated polynucleotide can be randomly integrated or targeted integrated.

[0093] In the implementation plan, the eukaryotic cell line is cultured in a cell culture medium.

[0094] In the implementation plan, eukaryotic cell lines are cultured under batch or fed-batch culture conditions or perfusion culture conditions (continuous or semi-continuous perfusion). Fed-batch culture conditions can be enhanced batch culture conditions.

[0095] In the implementation scheme, eukaryotic cell lines are cultured under perfusion culture conditions. Perfusion culture conditions can be semi-continuous or continuous perfusion. As those skilled in the art will understand, when the pH is at a specified level during perfusion culture, the pH can remain at that level throughout the entire period of perfusion culture, rather than shifting the pH from a higher pH to a lower pH during perfusion culture.

[0096] In the implementation scheme, reducing α-Gal content includes reducing α-Gal content by at least about 20% compared to a corresponding control method for producing glycoproteins, wherein the control method does not include any of (a), (b), (c), (d), or (e).

[0097] In one implementation, the α-Gal content is reduced by at least about 50%. In another implementation, the α-Gal content is reduced by at least about 75%.

[0098] In the implementation scheme, the reduction in α-Gal content is calculated after determining the level of α-Gal polysaccharide using the hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) protocol disclosed herein.

[0099] In the implementation plan, the recombinant polypeptide is an antibody, antigen, enzyme, gene vector, or vaccine.

[0100] In the implementation plan, the recombinant polypeptide is a therapeutic antibody.

[0101] In the implementation scheme, the therapeutic antibody is selected from anti-HER receptor family antibodies (such as anti-HER1 (EGFR), anti-HER2, anti-HER3, and anti-HER4); anti-CD protein antibodies (such as anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD20, anti-CD21, anti-CD22, anti-CD25, anti-CD33, anti-CD34, anti-CD38, and anti-CD52); anti-IL-8 antibodies; anti-VEGF antibodies; anti-CD40 antibodies, anti-CD11a antibodies; anti-CD18 antibodies; anti-IgE antibodies; anti-Apo-2 receptor antibodies; anti-tissue factor (TF) antibodies; anti-cell adhesion molecules (such as LFA-1, Mol, p150,95, VLA-4, ICAM-1, and VCAM); anti-human α4β7 integrin antibodies; anti-human αvβ8 integrin antibodies; and anti-αvβ3 antibodies (including their α, β, or subunits) (e.g., anti-CD11a and anti-CD18). Or anti-CD11b antibody); anti-EGFR antibody; anti-Fc receptor antibody; anti-carcinoembryonic antigen (CEA) antibody; anti-human renal cell carcinoma antibody; anti-human colorectal tumor antibody; anti-human melanoma antibody R24 against GD3 ganglioside; anti-human squamous cell carcinoma; antibody against breast epithelial cells; antibody binding to colon cancer cells; anti-EpCAM antibody; anti-GpIIb / IIIa antibody; anti-RSV antibody; anti-CMV antibody; anti-HIV antibody; anti-hepatitis antibody; anti-CA 125 antibody; anti-human 17-1A antibody; and anti-human leukocyte antigen (HLA) antibody, and anti-HLA DR antibody, anti-growth factor (such as vascular endothelial growth factor) (anti-VEGF) or fragments; anti-IgE; anti-blood group antigen; anti-flk2 / flt3 receptor; and anti-obesity (OB) antibody. Receptors; anti-amyloid antibodies, anti-α-synuclein (e.g., plasciizumab), anti-amyloid β, anti-growth hormone (GH) (including human growth hormone (hGH) and bovine growth hormone (bGH)); anti-growth hormone releasing factor; anti-parathyroid hormone; anti-thyroid-stimulating hormone; anti-lipoprotein; anti-α-1-antitrypsin; anti-insulin A chain; anti-insulin B chain; anti-proinsulin; anti-follicle-stimulating hormone; anti-calcitonin; anti-luteinizing hormone; anti-glucagon; anticoagulation factors (such as factor VIIIC, tissue factor, or von Willebrands factor); anticoagulation factors (such as protein C); anti-atrial natriuretic factor; anti-pulmonary surfactant; anti-plasminogen activator (such as urokinase or tissue-type plasminogen activator (t-PA)); bombazine; thrombin; anti-tumor necrosis factor-α and-β; anti-neprilysin;RANTES (regulation of normal T cell expression and secretion activation); anti-human macrophage inflammatory protein (MIP-1-α); anti-serum albumin (such as human serum albumin (HSA)); anti-Müllerian inhibitory substance; anti-relaxin A chain; anti-relaxin B chain; anti-relaxinogen; anti-mouse gonadotropin-related peptide; anti-DNase; anti-inhibin; anti-activin; anti-hormone or growth factor receptor; anti-protein A or D; anti-rheumatoid factor; anti-neurotrophic factors (such as bone-derived neurotrophic factor (BDNF), neurotrophic factor-3,-4,-5 or-6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factor (such as NGF-β)); platelet-derived growth factor (PDGF); fibroblast growth factor (such as aFGF and bFGF); epidermal growth factor (EGF); anti-transforming growth factor (TGF) (such as TGF-α) And TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5); anti-insulin-like growth factor-I and-II (IGF-I and IGF-II); anti-des(1-3)-IGF-I (brain IGF-I); insulin-like growth factor binding protein (IGFBP); anti-erythropoietin (EPO); anti-thrombopoietin (TPO); anti-bone-inducing factor; anti-immunotoxin; anti-bone morphogenetic protein (BMP); anti-interferon (such as interferon-α,-β, and-γ); anti-colony-stimulating factor (CSF) (e.g., M-CSF, GM-CSF, and G-CSF); anti-interleukin (IL) (e.g., IL-1 to IL-10); superoxide dismutase; anti-T cell receptor; anti-surface membrane protein; anti-degeneration accelerator factor (DAF); anti-viral antigen (such as AIDS). (part of the capsule); antitransporter; antihoming receptor; antiaddressin; antiregulatory protein; antiimmunoadhesin; and therapeutic antibodies against biologically active fragments or variants of any of the peptides listed above.

[0102] In the implementation plan, the therapeutic antibody is selected from AVASTIN® (bevacizumab), HERCEPTIN® (trastuzumab), LUCENTIS® (ranizumab), RAPTIVA® (efalizumab), RITUXAN® (rituximab), ACTEMRA® (tocilizumab-anti-IL-6 receptor), XOLAIR® (omalizumab), OCREVUS® (ozeralimumab-anti-CD20 antibody), PERJETA® (pertuzumab-HER dimer inhibitor (HDI)), TECENTRIQ® (anti-PD-L1 antibody), LUNSUMIO® or COLUMVI™ (anti-CD20 x anti-CD3 bispecific antibody), VABYSMO® (anti-VEGF-A x anti-angiogenic-2 bispecific antibody), anti-CD79b antibody, anti-OX40 ligand, antioxidant LDL (oxLDL), anti-amyloid β (e.g., erlotinib), anti-CD4 (MTRX1011A), anti-EGFL7 (EGF-like domain 7), anti-IL13, apotumab (anti-DR5-targeting pro-apoptotic receptor agonist (PARA)), anti-BR3 (CD268), anti-BLyS receptor 3, anti-BAFF-R (BAFF receptor), anti-TIGIT (anti-T-cell immune receptor containing immunoglobulin (Ig) and immune receptor tyrosine inhibitory motif domains), ateritumab (anti-ST2, IL-33 receptor), anti-β7 integrin subunit, anti-αvβ8 integrin antibody, dasizumab (anti-CD40), GA101 (oxotuzumab-anti-CD20 monoclonal antibody), MetMAb (anti-MET receptor tyrosine kinase), civastab (anti-Fc receptor homolog 5 (FcRH5) X anti-CD3 bispecific antibody), anti-neuropiliin-1 (NRP1) and rhuMAb IFNα.

[0103] Glycoprotein

[0104] On the other hand, glycoproteins that can be obtained or obtained by the methods of this disclosure are provided.

[0105] The methods disclosed herein relate to the production of glycoproteins (such as antibodies and fusion proteins; or conjugates thereof). In the case of an antibody, the antibody may be, but is not limited to, a monospecific antibody (e.g., an antibody consisting of a single heavy chain sequence and a single light chain sequence, including such paired multimers), a multispecific antibody, and an antigen-binding fragment thereof.

[0106] Multispecific antibodies

[0107] Antibodies can be multispecific antibodies, such as bispecific antibodies. A “multispecific antibody” is a monoclonal antibody that has binding specificity for at least two distinct sites (i.e., different epitopes on different antigens) (i.e., bispecific) or binding specificity for different epitopes on the same antigen (i.e., biepitaxy). Multispecific antibodies may have three or more binding specificities. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments, as described herein.

[0108] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and engineered “mortar and pestle structures” (see, for example, U.S. Patent No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be prepared by: engineering electrostatic manipulation effects for the preparation of antibody Fc-heterodimer molecules (see, for example, WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using common light chain techniques to avoid light chain mismatch problems (see, for example, WO 98 / 50431); using “dibody antibody” techniques for the preparation of bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448). (1993)); and the use of single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and the preparation of trispecific antibodies as described in Tutt et al., J. Immunol. 147:60 (1991).

[0109] This document also includes engineered antibodies having three or more antigen-binding sites, including, for example, “octopus antibodies” or DVD-Ig (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other non-limiting examples of multispecific antibodies having three or more antigen-binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792 and WO 2013 / 026831. Bispecific antibodies or their antigen-binding fragments also include “dual-acting FAbs” or “DAFs” (see, for example, US 2008 / 0069820 and WO 2015 / 095539).

[0110] Multispecific antibodies can also be provided in an asymmetric form, wherein there is domain interchange in one or more binding arms having the same antigen specificity, i.e., by exchanging the VH / VL domain (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), the CH1 / CL domain (see, e.g., WO 2009 / 080253), or the complete Fab arm (see, e.g., WO 2009 / 080251, WO 2016 / 016299, also see Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). Multispecific antibodies may include cross-Fab fragments. The terms "cross-Fab fragment," "xFab fragment," or "crossover Fab fragment" refer to Fab fragments in which the variable or constant regions of the heavy and light chains are exchanged. Cross-Fab fragments comprise polypeptide chains consisting of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), as well as polypeptide chains consisting of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interfaces to guide correct Fab pairing. See, for example, WO 2016 / 172485.

[0111] Various other molecular forms of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol. Immunol. 67 (2015) 95-106).

[0112] This article also includes a specific type of multispecific antibody, namely a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell (e.g., a tumor cell) and an activation-invariant component of the T cell receptor (TCR) complex (such as CD3), for retargeting T cells to kill the target cell.

[0113] Other non-limiting examples of bispecific antibody forms that can be used for this purpose include, but are not limited to, so-called “BiTE” (bispecific T-cell conjugate) molecules in which two scFv molecules are fused via a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261 and WO 2008 / 119567; Nagorsen and Bäuerle, Exp Cell Res 317, 1255-1260 (2011)); bispecific antibodies (Holliger et al., Prot. Eng. 9, 299-305 (1996)) and their derivatives, such as tandem bispecific antibodies (“TandAb”; Kipriyanov et al., J MolBiol 293, 41-56). (1999)); “DART” (dual affinity retargeting) molecules, which are based on bisomatic antibody forms but characterized by C-terminal disulfide bridges for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomab antibodies, which are fully hybridized mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat. Rev. 36, 458-467 (2010)). The specific T-cell bispecific antibody forms included in this article are described in the following references: WO 2013 / 026833; WO 2013 / 026839; WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0114] antibody fragments

[0115] Glycoproteins can be antibody fragments. For example, but not limited to, antibody fragments can be Fab, Fab', Fab'-SH, or F(ab')2 fragments, particularly Fab fragments. Papain digests an intact antibody to produce two identical antigen-binding fragments called "Fab" fragments. Each "Fab" fragment contains a heavy chain variable domain and a light chain variable domain (VH and VL, respectively), as well as a constant domain (CL) of the light chain and a first constant domain (CH1) of the heavy chain. Therefore, the term "Fab fragment" refers to an antibody fragment comprising a light chain containing the VL and CL domains and a heavy chain containing the VH and CH1 domains. The difference between a "Fab' fragment" and a Fab fragment is that residues are added to the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domain have free thiol groups. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of the Fab fragment and the F(ab')2 fragment containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0116] Antibody fragments can be bisomatic, trisomatic, or tetrasomatic antibodies. A “bisomatic antibody” is an antibody fragment having two antigen-binding sites, and it can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Trisomatic and tetrasomatic antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0117] The antibody fragment can be a single-chain Fab fragment. A “single-chain Fab fragment” or “scFab” is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. Specifically, the linker can be a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab fragments can be further stabilized by generating interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0118] Antibody fragments can be single-chain variable fragments (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the antibody's heavy chain variable domain (VH) and light chain variable domain (VL), linked by a linker. Specifically, the linker can be a short polypeptide of 10 to 25 amino acids, typically rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of the VH to the C-terminus of the VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original antibody. For reviews of scFv fragments, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, Vol. 113, eds. Rosenburg and Moore, (Springer-Verlag, New York), pp. 269–315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458.

[0119] Antibody fragments can be single-domain antibodies. A "single-domain antibody" is an antibody fragment containing all or part of the variable domain of the heavy chain or all or part of the variable domain of the light chain of an antibody. Single-domain antibodies can be human single-domain antibodies (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6248516 B1).

[0120] Antibody fragments can be prepared using various techniques, including but not limited to the proteolytic digestion of intact antibodies.

[0121] Chimeric antibodies and humanized antibodies

[0122] Antibodies can be chimeric antibodies. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). Chimeric antibodies may contain a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, or rabbit) and a human constant region. A chimeric antibody may be a “class-switching” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragment.

[0123] Chimeric antibodies can be humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Humanized antibodies typically contain one or more variable domains, wherein the CDR (or a portion thereof) is derived from the non-human antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Optionally, humanized antibodies may also contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0124] Humanized antibodies and their preparation methods have been reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US patents 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “surface reshaping”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR reorganization”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes the “guided selection” method for FR reorganization).

[0125] Commercial manufacturing

[0126] The methods disclosed herein can be used to produce target molecules on a manufacturing scale. "Manufacturing scale" production of therapeutic proteins or other proteins utilizes cell cultures ranging from about 400 L to about 80,000 L, depending on the protein being produced and the demand. Typically, such manufacturing scale production utilizes cell culture scales from about 400 L to about 25,000 L. Within this range, specific cell culture scales such as about 4,000 L, about 6,000 L, about 8,000 L, about 10,000 L, about 12,000 L, about 14,000 L, about 16,000 L, or about 25,000 L can be utilized.

[0127] The methods disclosed herein can be used to support high-density growth of mammalian cells in suspension cell cultures, and / or to support the expression of recombinant proteins derived from mammalian cells, and / or to produce large quantities of target molecules within a shorter timeframe compared to conventional culture media or media previously used in cell cultures. In some embodiments, the methods disclosed herein can be used to improve the quality of target molecules compared to conventional methods previously used in cell cultures. In some embodiments, the methods disclosed herein can be used for the optimal expression of cell culture products such as peptides, proteins, antibodies (monoclonal, bispecific, trispecific, multispecific, etc.), antibody fragments, etc.

[0128] Antibodies are manufactured using the culture medium disclosed herein.

[0129] Therapeutic antibodies that can be generated in the culture media described in this disclosure include, but are not limited to, anti-HER receptor family antibodies (such as anti-HER1 (EGFR), anti-HER2, anti-HER3, and anti-HER4); anti-CD protein antibodies (such as anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD20, anti-CD21, anti-CD22, anti-CD25, anti-CD33, anti-CD34, anti-CD38, and anti-CD52); anti-IL-8 antibodies; anti-VEGF antibodies; anti-CD40 antibodies, anti-CD11a antibodies; anti-CD18 antibodies; anti-IgE antibodies; anti-Apo-2 receptor antibodies; anti-tissue factor (TF) antibodies; anti-cell adhesion molecules (such as LFA-1, Mol, p150,95, VLA-4, ICAM-1, and VCAM); anti-human α4β7 integrin antibodies; anti-human αvβ8 integrin antibodies; and anti-αvβ3 antibodies (including their α or β components). Anti-CD11a, anti-CD18, or anti-CD11b antibodies (or subunits); anti-EGFR antibodies; anti-Fc receptor antibodies; anti-carcinoembryonic antigen (CEA) antibodies; anti-human renal cell carcinoma antibodies; anti-human colorectal tumor antibodies; anti-human melanoma antibody R24 against GD3 gangliosides; anti-human squamous cell carcinoma; antibodies against mammary epithelial cells; antibodies binding to colon cancer cells; anti-EpCAM antibodies; anti-GpIIb / IIIa antibodies; anti-RSV antibodies; anti-CMV antibodies; anti-HIV antibodies; anti-hepatitis antibodies; anti-CA 125 antibodies; anti-human 17-1A antibodies; and anti-human leukocyte antigen (HLA) antibodies, and anti-HLA DR antibodies; anti-growth factors (such as vascular endothelial growth factor) (anti-VEGF) or fragments; anti-IgE; anti-blood group antigens; anti-flk2 / flt3 receptors; and anti-obesity (OB) receptors.Other exemplary proteins for designing therapeutic antibodies include anti-amyloid antibodies, anti-α-synuclein (e.g., plasciizumab), anti-amyloid β, anti-growth hormone (GH) (including human growth hormone (hGH) and bovine growth hormone (bGH)); growth hormone-releasing factor; parathyroid hormone; thyroid-stimulating hormone; lipoproteins; α-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors (such as factor VIIIC, tissue factor, or von Willebrands factor); anticoagulation factors (such as protein C); atrial natriuretic factor; pulmonary surfactant; plasminogen activator (such as urokinase or tissue-type plasminogen activator (t-PA)); bombazine; thrombin; tumor necrosis factor-α. And -β; enkephalinase; RANTES (regulation of normal T cell expression and secretion activation); human macrophage inflammatory protein (MIP-1-α); serum albumin (such as human serum albumin (HSA)); Müllerian duct inhibitory substance; relaxin A chain; relaxin B chain; pro-relaxin; mouse gonadotropin-related peptide; DNase; inhibin; activin; hormone or growth factor receptor; protein A or D; rheumatoid factor; neurotrophic factors (such as bone-derived neurotrophic factor (BDNF), neurotrophic factor-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factors such as NGF-β); platelet-derived growth factor (PDGF); fibroblast growth factor (such as aFGF and bFGF); epidermal growth factor (EGF); transforming growth factor (TGF) (such as TGF-α) And TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5); insulin-like growth factor-I and-II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I); insulin-like growth factor binding protein (IGFBP); erythropoietin (EPO); thrombopoietin (TPO); bone-inducing factor; immunotoxins; bone morphogenetic protein (BMP); interferons such as interferon-α,-β, and-γ; colony-stimulating factors (CSF) (e.g., M-CSF, GM-CSF, and G-CSF); interleukins (ILs) (e.g., IL-1 to IL-10); superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerators (DAF); viral antigens (such as AIDS, for example). (part of the envelope); transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; and biologically active fragments or variants of any of the polypeptides listed above.Many other antibodies and / or other proteins may be used in accordance with this disclosure, and the above list is not intended to be limiting.

[0130] Therapeutic antibodies of particular interest include commercially available therapeutic antibodies or those in clinical development, such as: AVASTIN® (bevacizumab), HERCEPTIN® (trastuzumab), LUCENTIS® (ranizumab), RAPTIVA® (efalizumab), RITUXAN® (rituximab), ACTEMRA® (tocilizumab - anti-IL-6 receptor), XOLAIR® (omalizumab), OCREVUS® (ozrelizumab - anti-CD20 antibody), PERJETA® (pertuzumab - HER dimer inhibitor (HDI)), TECENTRIQ® (anti-PD-L1 antibody), LUNSUMIO® or COLUMVI™ (anti-CD20 X anti-CD3 bispecific antibody), VABYSMO® (anti-VEGF-A X anti-angiogenic-2 bispecific antibody), anti-CD79b antibody, anti-OX40 ligand, and antioxidant LDL. (oxLDL), anti-amyloid β (e.g., tertinex), anti-CD4 (MTRX1011A), anti-EGFL7 (EGF-like domain 7), anti-IL13, apotumab (anti-DR5-targeting pro-apoptotic receptor agonist (PARA)), anti-BR3 (CD268), anti-BLyS receptor 3, anti-BAFF-R (BAFF receptor), anti-TIGIT (anti-T-cell immune receptor containing immunoglobulin (Ig) and immune receptor tyrosine inhibitory motif domains), atelizumab (anti-ST2, IL-33 receptor), anti-β7 integrin subunit, anti-αvβ8 integrin antibody, dasizumab (anti-CD40), GA101 (oxutuzumab-anti-CD20 monoclonal antibody), MetMAb (anti-MET receptor tyrosine kinase), civastastatab (anti-Fc receptor homolog 5 (FcRH5) X anti-CD3) Bispecific antibodies), anti-neuropiliin-1 (NRP1), rhuMAb IFN α, etc. Many other antibodies and / or other proteins may be used according to this disclosure, and the above list is not intended to be limiting.

[0131] Example

[0132] The following examples will provide a more detailed understanding of this disclosure. However, they should not be construed as limiting the scope of this disclosure. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or variations thereof will be suggested to those skilled in the art and will be included within the spirit and limits of this application and within the scope of the appended claims.

[0133] Materials and methods

[0134] Recombinant CHO cell lines expressing three different therapeutic proteins were used. For small-scale studies, cells were thawed and expanded to inoculate production cultures in miniaturized systems (Sartorius Ambr® 15 or Ambr® 250 systems (Sartorius, Edgewood, NY)). Temperature, pH, and dissolved oxygen (DO) in the bioreactor were controlled using their respective Sartorius ambr15 and Sartorius ambr250 software. All feed batches of production cultures were maintained at 35°C, 7.20, and 30% (air saturation); and at 48 hours, the temperature setpoint was shifted to 33°C and maintained thereafter. Concentrated nutrient feeds were added to the production cultures at 24, 96, and 192 hours post-inoculation. The cultures were maintained for a total duration of 12 days.

[0135] In various test cases, the pH was adjusted to 6.90 after 48 or 144 hours of incubation. Depending on the test conditions, uridine, N-acetylglucosamine (GlcNAc), manganese, and zinc were excluded from the basal medium or added to the culture at inoculation.

[0136] During each production run, all cultures were sampled multiple times, and VCD, activity pH, dissolved oxygen, glucose, lactate, and ammonium levels were analyzed using a BioProfileFLEX2 (Nova Biomedical, Waltham, MA). Culture supernatants were frozen at -80°C until analysis of product titers and glycan distribution. Samples were screened using a high-throughput HILIC glycan method to select those for presentation to the α-gal method for further characterization.

[0137] Example 1: The α-Gal method for determining glycoform levels

[0138] α-Gal represents a non-human glycosylation mode. The levels of α-Gal-containing glycans were determined by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) analysis of glycans treated with sialidase. In this analysis, the glycans were first treated with sialidase to remove sialic acid, and then the glycans were enzymatically released from the protein by treatment with PNGase F. The released glycans were then labeled with a procaine-based IPC fluorophore (InstantPC, Agilent Technologies) and separated by hydrophilic interaction liquid chromatography. The relative quantification of the labeled glycans was achieved by integrating the glycan fluorescence signal, and the identification of the separated glycans was determined by mass spectrometry.

[0139] Although the above methods are used in this disclosure, there are also methods known to those skilled in the art for determining glycoforms, such as the method proposed in L. Zhang et al., “Glycan analysis of therapeutic glycoproteins”, MABS, 2016, Vol. 8, No. 2, 205–215.

[0140] Example 2: Efficient process parameters for α-Gal levels in ligand fusion proteins, complex antibodies, and complex bispecific antibodies.

[0141] α-Gal-containing glycans possess terminal Gala1-3Gal sequences and are expressed via α1-3-galactosyltransferase catalysis using UDP-Gal and glycoproteins as substrates (Figure 1). Since galactosylated glycans are the desired substrates for α-gal epitope addition, reducing the abundance of total galactosylation should also reduce the abundance of α-gal-containing glycans.

[0142] Screening studies were used to identify process levers that influence galactosylation and thus affect α-gallic acid-containing glycans. The experimental design and statistical methods used for analysis are detailed in “Design of Experiments” (Douglas Montgomery, 8th ed., 2012, John Wiley & Sons). The statistical software JMP 16 was used for both experimental design and analysis. A 16-case quarter-factor screening design was used, including additional centerpoint cases, with the cell lines producing the ligand fusion protein. This design was capable of assessing major effects and some interactions.

[0143] The pH conditions tested included: no transition, with pH maintained at 7.20 throughout the entire period of continuous production culture, or pH maintained at 7.20 from the start of production inoculation until 48 hours, at which point pH transitioned to 6.90 for the remainder of continuous culture. Culture medium additives were also screened: GlcNAc in the range of 0 to 18 mM, zinc in the range of 111 to 800 µM, uridine in the range of 0 to 0.32 mM, manganese in the range of 0 to 850 nM, and copper in the range of 0.001 to 0.003 mM. Compared to the experimental control cases, the center-point cases reduced total galactosylation by approximately 20%, and the DOE test cases, including pH transitions, showed the largest change in galactosylation, up to approximately 40%. As shown in the parameter effect assessment in Table 1, a low pH transition to 6.90 at 48 hours post-inoculation exhibited the greatest effect, reducing total galactosylation by 18% compared to a constant pH of 7.20. Increased GlcNAc and zinc concentrations also reduced total galactosylation. The interaction of uridine and manganese together also affected galactosylation.

[0144] In subsequent studies, process levers were combined using three cell lines, each producing a ligand fusion protein (cell line 1), a complex antibody (cell line 2), and a complex bispecific antibody (cell line 3), respectively. Three conditions were tested for each cell line: (1) control conditions, a constant pH setpoint of 7.20 (no pH change), 0 mM GlcNAc, 111 µM zinc, 1 µM copper, and 0.32 mM uridine; (2) a process lever combination of 18 mM GlcNAc, 1600 µM zinc, 3 mM copper, and 0 mM uridine, with no pH change; and (3) a process lever combination of 18 mM GlcNAc, 1600 µM zinc, 3 mM copper, and 0 mM uridine, with the pH changing from 7.20 to 6.90 at 144 hours of culture. In all three cell lines tested, process levers with and without low pH changes reduced galactosylation. In all cases, pH shifts further reduced galactosylation, demonstrating that these levers have an effect across a variety of cell lines and molecular forms (Figure 3).

[0145] Table 1 - Parameter evaluation from DOE analysis of total glycosylation. It shows that implementing pH changes, adding GlcNAc, zinc or manganese, and removing uridine reduced total glycosylation.

[0146]

[0147] Sample characterization by the HILIC-MS assay showed that the total galactosylation level was correlated with the relative abundance of α-gal polysaccharides (Figure 4). The test cases using the process lever showed a reduction in α-gal compared to their respective control cases.

Claims

1. A method for producing a glycoprotein with reduced α-Gal content, the method comprising: A eukaryotic cell line containing a polynucleotide encoding a polypeptide portion of the glycoprotein is cultured under conditions suitable for the production of the glycoprotein. The conditions suitable for production include one or more of the following: (a) pH below approximately 7.1; (b) The concentration of GlcNAc in the culture is at least about 10 mM; (c) The zinc concentration in the culture is at least about 400 µM; (d) The concentration of uridine in the culture is less than about 15 mM; and (e) The manganese concentration in the culture is less than about 400 nM.

2. The method according to claim 1, wherein (a) the pH is from about 7.1 to about 6.7; Optionally, the pH is approximately 6.

9.

3. The method according to claim 1 or claim 2, wherein the eukaryotic cell line is cultured under fed-batch culture conditions, wherein condition (a) includes changing the pH from a higher pH to a pH below about 7.

1.

4. The method according to any of the preceding claims, wherein (a) comprises maintaining the pH at the specified level for at least the last 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days of the conditions suitable for production; Optionally, (a) includes maintaining the pH at the specified level for at least the last 4, 5, 6, 7 or 8 days of conditions suitable for production.

5. The method according to any of the preceding claims, wherein (a) comprises maintaining the pH at a specified level for no more than the last 4, 5, 6, 7, 8, 9, 10, 11 or 12 days of the conditions suitable for production; Optionally, (a) includes maintaining the pH at a specified level for no more than the last 5, 6, 7 or 8 days under conditions suitable for production.

6. The method according to any of the preceding claims, wherein (b) the concentration of GlcNAc in the culture is from about 10 mM to about 50 mM; Optionally, the concentration of GlcNAc in the culture is from about 10 mM to about 20 mM.

7. The method according to any of the preceding claims, wherein (c) the zinc concentration in the culture is from about 400 µM to about 2,500 µM; Optionally, the zinc concentration in the culture is from about 800 µM to about 2,000 µM; Further optionally, the zinc concentration in the culture is from about 1,200 µM to about 1,800 µM.

8. The method according to any of the preceding claims, wherein (d) the concentration of uridine in the culture is from about 0 mM to about 0.2 mM; Optionally, the concentration of uridine in the culture is from about 0 mM to about 0.1 mM; Further optionally, the concentration of uridine in the culture is about 0 mM.

9. The method according to any of the preceding claims, wherein (e) the manganese concentration in the culture is from about 0 nm to about 400 nm; Optionally, the manganese concentration in the culture is from about 0 nm to about 200 nm; Further optionally, the manganese concentration in the culture is approximately 0 nm.

10. The method according to any of the preceding claims, wherein the conditions suitable for production include one or more of (a) and (b), (c), (d) and (e).

11. The method according to any of the preceding claims, wherein the conditions suitable for production include (d) and (e); Optionally, it may further include one or more of (a), (b) and (c).

12. The method according to any of the preceding claims, wherein the glycoprotein is a therapeutic glycoprotein.

13. The method according to any of the preceding claims, wherein the polypeptide portion of the glycoprotein is a recombinant polypeptide.

14. The method according to any of the preceding claims, wherein the glycoprotein is a recombinant glycoprotein selected from fusion proteins, antibodies, antigens, enzymes, or vaccines.

15. The method of claim 14, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

16. The method of claim 14 or claim 15, wherein the antibody comprises a single heavy chain sequence and a single light chain sequence or an antigen-binding fragment thereof.

17. The method according to any one of claims 14 to 16, wherein the antibody comprises a chimeric antibody, a human antibody, or a humanized antibody.

18. The method according to any one of claims 14 to 17, wherein the antibody comprises a monoclonal antibody.

19. The method according to any of the preceding claims, further comprising isolating the glycoprotein.

20. The method according to any of the preceding claims, wherein the eukaryotic cell line is cultured in a cell culture medium.

21. The method according to any of the preceding claims, wherein the eukaryotic cell line is an animal cell line.

22. The method according to any preceding claim, wherein the eukaryotic cell line is a mammalian cell line; Optionally, the mammalian cell line is a modified mammalian cell line.

23. The method according to any of the preceding claims, wherein the cell line is a CHO cell line or a derivative thereof.

24. The method according to any preceding claim, wherein the polynucleotide encoding the polypeptide portion of the glycoprotein is an extrachromosomal polynucleotide or an integrated polynucleotide integrated into the chromosome of the cell line.

25. The method of claim 24, wherein the integrated polynucleotide is randomly integrated or targeted integrated.

26. The method according to any of the preceding claims, wherein the eukaryotic cell line is cultured under batch or fed-batch culture conditions or perfusion culture conditions (using continuous or semi-continuous perfusion).

27. The method of claim 24, wherein the eukaryotic cell line is cultured under fed-batch culture conditions; Optionally, the fed-batch culture conditions described therein are enhanced fed-batch culture conditions.

28. The method according to any one of claims 1, 2, or 6 to 24, wherein the eukaryotic cell line is cultured under perfusion culture conditions; Optionally, the perfusion culture conditions are semi-continuous perfusion or continuous perfusion.

29. The method according to any of the preceding claims, wherein the reduced α-Gal content comprises a reduction of at least about 20% in α-Gal content compared to a corresponding control method for producing the glycoprotein, wherein the control method does not include any of (a), (b), (c), (d), or (e).

30. The method of claim 27, wherein the reduction in α-Gal content is at least about 50%; Optionally, the reduction in the α-Gal content is at least about 75%.

31. The method according to claim 27 or 28, wherein the reduction in α-Gal content is calculated after determining the level of α-Gal polysaccharide using the hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) protocol disclosed herein.

32. A glycoprotein that is obtained or can be obtained by any one of claims 1 to 29.

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