WO2023040792A1

WO2023040792A1 - Preparation method for erythropoietin

Info

Publication number: WO2023040792A1
Application number: PCT/CN2022/118301
Authority: WO
Inventors: 朱建伟; 江华; 谢跃庆; 王振玉; 范宝庆; 韩雷
Original assignee: 杰科（天津）生物医药有限公司; 杰库(上海)生物医药研究有限公司; 美国杰科实验室有限公司
Priority date: 2021-09-14
Filing date: 2022-09-13
Publication date: 2023-03-23

Abstract

A preparation method for erythropoietin, specifically, a protein separation method. The protein is in contact with two or more cation exchangers, wherein one of the cation exchangers is a fine cation exchanger.

Description

A preparation method of erythropoiesis-stimulating protein

technical field

The present application relates to the field of biomedicine, in particular to a preparation method of erythropoiesis-stimulating protein.

Background technique

The key bottleneck restricting the clinical application of long-acting erythropoietin (EPO) is the production process, especially the purification process. On the one hand, the existing EPO production process may use a large amount of organic reagents for reverse phase chromatography, which requires the construction of an explosion-proof working environment, and there are major safety risks; on the other hand, the chromatography steps may be imperfect, and only some products that meet the standards are obtained. . There is an urgent need in this field to develop a safe and efficient chromatographic technology for purification to obtain quality-qualified erythropoiesis-stimulating protein analogs or functional derivatives.

However, there are two major quality index risks in the existing erythropoiesis-stimulating protein production process. It is difficult to obtain the target protein with fixed glycoforms and the batch-to-batch consistency is poor and the host protein exceeds the standard. Therefore, this application can be abandoned until the reaction On the basis of the phase chromatography process, a sequence combination of various chromatographic techniques and filtration methods have been developed to purify proteins, and obtain erythropoiesis-stimulating protein analogs or functional derivatives with fixed glycoforms, especially long-acting The erythropoiesis-stimulating protein can produce a drug with a purity ≥ 98%, HCP less than 100ppm, and a qualified ratio of glycoform and sialic acid.

Contents of the invention

The present application provides a protein separation method, such as a purification and/or preparation method of an erythropoiesis-stimulating protein analog or a derivative thereof. The protein separation method of the present application can reduce impurities such as host protein (HCP), host DNA (HCD), aggregates, endotoxins, high isoelectric point (PI) charge variants, low PI charge variants, low molecular weight impurities , and/or viruses, etc. The method of the present application can obtain products qualified in charge-to-mass ratio, sialic acid content, and/or glycoform.

In one aspect, the present application provides a protein separation method, which involves contacting the protein with two or more cation exchangers, wherein one of the cation exchangers is a fine cation exchanger.

In some embodiments, the protein comprises erythropoiesis-stimulating protein, a variant thereof, or a functionally active fragment thereof.

In some embodiments, the protein comprises glycosylation modifications.

In some embodiments, the protein comprises glycan structures bound to N-glycosylation sites.

In some embodiments, the glycan structure comprises FA4G4L2S4.

In some embodiments, the ratio of the FA4G4L2S4 structure is more than 15%.

In some embodiments, the glycan structure further comprises FA4G4L1S4.

In some embodiments, the ratio of FA4G4L1S4 is above 20%.

In some embodiments, the glycan structure further comprises FA4G4S4.

In some embodiments, the ratio of FA4G4S4 is above 10%.

In some embodiments, the glycan structure comprises Neu5Gc, and the molar ratio of Neu5Gc is 0.5% or less.

In some embodiments, the protein comprises the amino acid sequence shown in SEQ ID NO.1 or a functionally active fragment thereof.

In some embodiments, the protein comprises an N-glycosylation site selected from the group consisting of N24, N30, N38, N83, and N88.

In some embodiments, the protein is expressed by CHO cells.

In some embodiments, the CHO cells comprise CHO-S cells.

In some embodiments, the fine cation exchanger comprises a cation exchanger having a particle size of about 30 microns or less.

In some embodiments, the fine cation exchanger comprises a cation exchanger having a dispersion of about 3% or less.

In some embodiments, the degree of dispersion is the ratio of the standard deviation of particle size to the average value of particle size.

In some embodiments, the fine cation exchanger comprises Source 30s.

In some embodiments, the cation exchanger other than the fine cation exchanger comprises Capto S impact.

In some embodiments, the protein is contacted with the other cation exchanger before contacting the fine cation exchanger.

In some embodiments, the method further comprises contacting the protein with a mixed mode exchanger.

In some embodiments, the protein is contacted with the fine cation exchanger before contacting the mixed mode exchanger.

In some embodiments, the method further comprises contacting the protein with two or more of the mixed mode exchangers.

In some embodiments, the protein is first contacted with the fine cation exchanger and then contacted with two or more of the mixed mode exchangers.

In some embodiments, the mixed mode exchanger comprises the group consisting of: Hydroxyapatite Type II and CaptoMMC Impres.

In some embodiments, the method further comprises contacting the protein with an anion exchanger.

In some embodiments, the protein is contacted with the anion exchanger before contacting the cation exchanger.

In some embodiments, the anion exchanger comprises the group consisting of: Capto Q and Q.sepharose.HP.

In some embodiments, wherein the protein is contacted with a buffer selected from the group consisting of Tris buffer, arginine solution, phosphate solution, citrate solution and NaCl solution while in contact with the exchanger.

In some embodiments, comprising (1) contacting the protein with the anion exchanger; (2) contacting the protein obtained in the above step with the other cation exchanger, (3) contacting the protein obtained in the above step The protein is contacted with the fine cation exchanger, (4) the protein obtained in the above steps is contacted with the mixed mode exchanger.

In some embodiments, comprising (1) contacting the protein with the anion exchanger; (2) contacting the protein obtained in the above step with the other cation exchanger, (3) contacting the protein obtained in the above step The protein is contacted with the fine cation exchanger, and (4) the protein obtained in the above steps is contacted with two or more of the mixed mode exchangers.

On the other hand, the present application provides an isolated protein, which is separated by the protein separation method as described in the present application.

In some embodiments, the host protein content of the isolated protein of the present application is about 500 ng/mg or less.

In some embodiments, the host protein content of the isolated protein of the present application is about 100 ng/mg or less.

In another aspect, the present application provides a pharmaceutical composition, which comprises the protein described in the present application and a pharmaceutically acceptable adjuvant.

In another aspect, the present application provides the use of the protein described in the present application and/or the pharmaceutical composition described in the present application in the preparation of a medicament for treating anemia.

In some embodiments, the anemia includes renal anemia, multiple myeloma anemia and/or cancerous anemia in the use of the protein described in the application and/or the pharmaceutical composition described in the application in the preparation of a medicament.

In another aspect, the present application provides a method for prolonging the half-life of erythropoiesis-stimulating protein, which includes the following steps: administering the protein described in the present application and/or the pharmaceutical composition described in the present application to a subject in need.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the specification of the present application are only exemplary and not restrictive.

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "glycan structure" generally refers to polysaccharides or oligosaccharides, ie polymeric compounds that yield multiple monosaccharides after acid hydrolysis. Glycosylation-modified proteins may comprise covalent coupling to One or more glycan structures that are side groups of a polypeptide chain. For example, it can be a FA4G4L2S4 glycan structure linked to the N-glycosylation site of erythropoiesis stimulating protein, it can be a FA4G4L1S4 glycan structure linked to the N-glycosylation site of erythropoiesis stimulating protein, it can be FA4G4S4 glycan structure The sugar structure is linked to the N-glycosylation site of erythropoiesis-stimulating protein, which may be the glycan structure Neu5Gc is linked to the N-glycosylation site of erythropoiesis-stimulating protein. Glycan structures are classified into biantennary, triantennary and tetraantennary structures according to the number of their branches (antennas). The glycan structure is composed of various monosaccharides, including fucose (Fuc or F for short), N-acetylglucosamine (GlcNAc, Gn or A for short), galactose ( Galactose, referred to as Gal or G), lactose (Lactose, referred to as Lac or L), mannose (Mannose, referred to as Man or M), N-acetylneuraminic acid (sialic acid, N-Acetylneuraminic, referred to as NANA, Neu5Ac or S) And/or N-glycolylneuraminic (NGNA, Neu5Glc or Neu5Gc for short). For example, the term "FA4G4L2S4" refers to a four-antennary glycan structure containing fucosylation, 4 N-acetylglucosamine, 4 galactose, 2 lactose, and 4 sialic acids, where F represents Modification, A stands for N-acetylglucosamine, G stands for galactose, L stands for lactose, S stands for sialic acid, the number after A represents the number of N-acetylglucosamine on a glycan structure, and the number after G The number represents the number of galactose on a glycan structure, the number after L represents the number of lactose on a glycan structure, and the number after S represents the number of sialic acid on a glycan structure; The term "FA4G4L1S4" refers to a four-antennary glycan structure containing fucosylation, 4 N-acetylglucosamine, 4 galactose, 1 lactose, and 4 sialic acids, wherein F represents a fucose modification, A stands for N-acetylglucosamine, G stands for galactose, L stands for lactose, S stands for sialic acid, the number after A stands for the number of N-acetylglucosamine on a glycan structure, and the number after G stands for The number of galactose on a glycan structure, the number after L represents the number of lactose on a glycan structure, and the number after S represents the number of sialic acid on a glycan structure; the term " FA4G4S4" refers to a four-antenna glycan structure containing fucosylation, 4 N-acetylglucosamines, 4 galactoses, and 4 sialic acids, where F represents fucose modification, and A represents N-acetylglucosamine Sugar amine, G represents galactose, S represents sialic acid, the number after A represents the number of N-acetylglucosamine on a glycan structure, and the number after G represents the number of galactose on a glycan structure The number, the number after S represents the number of sialic acid on a glycan structure; the term "Neu5Gc" refers to N-glycolylneuraminic acid.

In this application, the term "N-glycosylation site" generally refers to a site on a glycosylated protein that contains asparagine or arginine for covalently linking glycan structures, such as N-glycosylation The sylation site may be an asparagine residue used to covalently link the glycan structure to the glycosylated protein. For example, the N-glycosylation site can be asparagine ( Asparagines, referred to as Asn or N) residues.

In this application, the term "combination" between the glycan structure and the N-glycosylation site generally refers to the connection between the glycan structure and the N-glycosylation site on the glycosylation-modified protein. physical or chemical interaction. For example, binding can be direct or indirect linking or attachment, indirect can be through linking or attachment of another biomolecule or compound, direct can be covalent (such as by chemical coupling) or non-covalent ( such as ionic interactions, hydrophobic interactions, hydrogen bonds, etc.) binding or attachment. For example, the binding can be the covalent link between the glycan structure and the N-glycosylation site on the glycosylated protein, or the asparagus between the monosaccharide of the glycan structure and the N-glycosylation site. The free -NH2 group of the amide residue is linked covalently.

In the present application, the "ratio" of the glycan structure in the glycosylation-modified erythropoiesis-stimulating protein usually refers to the ratio of the glycan structure in the glycosylation-modified erythropoiesis-stimulating protein. The molar ratio of the protein, the ratio can be analyzed by mass spectrometry after enzymatically digesting the glycosylation-modified erythropoiesis-stimulating protein, and an exemplary mass spectrometry analysis method can include mass spectrometry combined with HPLC. For example, in the glycosylation-modified erythropoiesis-stimulating protein, the ratio of the FA4G4L2S4 structure can be more than 15%, the ratio of the FA4G4L1S4 can be more than 20%, and the ratio of the FA4G4S4 can be more than 10%. , the molar ratio of Neu5Gc may be 0.5% or less.

In this application, the term "molar ratio" usually refers to the release of all glycan structures after the glycosidase hydrolyzes the glycoprotein, wherein the molar ratio of a specific glycan structure = its molar number/the molar number of all glycan structures in the protein Calculate and give. For example, the molar ratio can be the number of moles of FA4G4L2S4 structure/the number of moles of all glycan structures in the glycosylation-modified erythropoiesis-stimulating protein, which can be the number of moles of FA4G4L1S4/all the glycan structures in the glycosylation-modified erythropoiesis-stimulating protein The number of moles of glycan structures can be the number of moles of FA4G4S4/the number of moles of all glycan structures in glycosylation-modified ESP, which can be the number of moles of Neu5Gc/glycosylation-modified ESP The number of moles of all glycan structures in .

In this application, the term "protein" generally refers to a polymer of amino acid residues that is not limited to a minimum length. Polypeptides, peptides, oligopeptides, dimers, multimers and the like are included within this definition. Intact proteins and fragments thereof are also included in this definition. The term also includes post-expression modified forms of proteins including, but not limited to, glycosylation, acetylation, phosphorylation, and the like. For example, in the present application, protein may refer to glycosylation-modified erythropoiesis-stimulating protein and fragments thereof.

In this application, the cell "CHO-S cell" generally refers to a CHO-S Chinese hamster ovary cell into which nucleic acid encoding a heterologous polypeptide can be introduced, for example, by transfection. The CHO-S cells include variant progeny that have the same function or biological activity as those screened out from the original transfected cells.

In this application, the term "erythropoiesis-stimulating protein" and its abbreviation "EPO" generally refer to any erythropoiesis-stimulating protein polypeptide, including but not limited to, recombinantly produced erythropoiesis-stimulating protein polypeptide, synthetic Produced erythropoiesis-stimulating protein polypeptides, native EPO polypeptides, erythropoiesis-stimulating protein polypeptides extracted from cells and tissues including but not limited to kidney, liver, urine and blood. For example, the erythropoiesis-stimulating protein can be an erythropoiesis-stimulating protein having the amino acid sequence of SEQ ID NO:1. The term "erythropoiesis-stimulating protein" also refers to a variant of the protein of SEQ ID NO: 1, wherein one or more amino acid residues are changed, deleted or inserted, and which have the same biological activity as the unmodified protein, e.g. Reported in EP 1 064 951 or US 6,583,272. The biological activity produced after the erythropoiesis-stimulating protein binds to the EPO receptor can include: compared with the individuals of the non-injected group or the control group, administering the erythropoiesis-stimulating protein to the subject by injection causes the bone marrow cell to increase network Production of erythrocytes and red blood cells.

In this application, the "glycosylation modification" generally means that carbohydrate moieties can be attached to one or more amino positions in a protein or polypeptide. Typically, glycosylated proteins or polypeptides contain one or more amino acid residues, such as arginine or asparagine, to link carbohydrate moieties. For example, the glycosylation modification can be an N-linked glycoprotein. An N-linked glycoprotein may comprise a glycan structure bound to an N-glycosylation site, eg, a glycan structure linked to an N-glycosylation site of an asparagine residue in the protein. Sugars found on glycosylated proteins (glycoproteins) include the following groups: glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and/or sialic acid. For example, the glycosylation modification of erythropoiesis-stimulating protein can be FA4G4L2S4-containing glycan structure linked to the N-glycosylation site of erythropoiesis-stimulating protein, can be FA4G4L1S4-containing glycan structure linked to erythropoiesis-stimulating protein The N-glycosylation site can be the N-glycosylation site containing the FA4G4S4 glycan structure connected to the erythropoiesis-stimulating protein, and can be the N-glycosylation site containing the Neu5Gc-containing glycan structure connected to the erythropoiesis stimulating protein Kylation site.

In the present application, the ratio of the FA4G4L2S4 structure is "above" 15%, which generally means that the ratio of the FA4G4L2S4 glycan structure may be at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, At least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96 %, at least 97%, at least 98%, or at least 99% of the glycosylated modified erythropoiesis-stimulating protein. For example, the glycosylation-modified erythropoiesis-stimulating protein may comprise a ratio of FA4G4L2S4 glycan structures of at least 18.17%.

In the present application, the ratio of the FA4G4L12S4 structure is "above" 20%, which generally means that the ratio of the FA4G4L1S4 glycan structure may be at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, At least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80% %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% glycosylated modified erythropoiesis stimulating protein. For example, the glycosylation-modified erythropoiesis-stimulating protein may comprise a ratio of FA4G4L1S4 glycan structures of at least 21.58%.

In the present application, the ratio of the FA4G4S4 structure is "above" 10%, which generally means that the ratio of the FA4G4S4 glycan structure may be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, At least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60% %, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% glycosylated modified erythropoiesis stimulating protein. For example, the glycosylation-modified erythropoiesis-stimulating protein may comprise a ratio of FA4G4S4 glycan structures of at least 14.02%.

The molar ratio of Neu5Gc described in the present application is "less than" 0.5%, which generally means that the molar ratio of Neu5Gc can be at most 0.5%, at most 0.4%, at most 0.3%, at most 0.2%, at most 0.1%, at most 0.05%, At most 0.02%, at most 0.01%, or 0% glycosylated modified erythropoiesis-stimulating protein. For example, it may be a glycosylation-modified erythropoiesis-stimulating protein whose glycan structure does not include Neu5Gc.

In this application, "prolonging" the half-life of erythropoiesis-stimulating protein generally refers to the increased resistance of the glycosylated-modified erythropoiesis-stimulating protein of the present application to proteases, which can lead to the glycosylation-modified erythropoiesis-stimulating protein The half-life of the protein is increased in vitro (eg, during production, purification, and storage) or in vivo (eg, after administration to a subject) compared to EPO without a glycosylated form or glycosylated EPO with other glycan structures. In this application, the term "half-life" or its abbreviation "T _1/2 ", generally refers to the time used to quantify the time it takes for half the dose of a drug to be excreted by a subject. For example, after administering the glycosylation-modified erythropoiesis-stimulating protein of the present application to a subject, it exhibits an increase in half-life. Compared with unmodified EPO or glycosylated EPO with other glycan structures, the glycosylated EPO of the present application The increased half-life of the glycosylated erythropoiesis-stimulating protein may be increased by at least about or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20% %, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more. For example, compared with unmodified EPO or glycosylated EPO with other glycan structures, the half-life of the glycosylated modified erythropoiesis-stimulating protein of the present application can be increased by at least about or at least 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more times.

In this application, the terms "host cell", "host cell line" and "host cell culture" are used interchangeably and generally refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. For example, a host cell can include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not have the exact same nucleic acid content as the parental cell, for example may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell.

In this application, the term "impurity" generally refers to a substance different from the desired polypeptide product. For example, impurities can include, but are not limited to: host cell material, such as a host cell protein (HCP); nucleic acid; another polypeptide; endotoxin; In some examples, the impurity can be HCP from, for example, but not limited to, bacterial cells. In some examples, the impurity can be HCP from mammalian cells, such as CHO cells.

In this application, the term "sequence" generally refers to chromatographic steps in a specific sequence. For example, a first chromatography step, then a second chromatography step, then a third chromatography step, etc. For example, additional steps may be included between sequential chromatography steps.

In this application, the term "loading amount" generally refers to the amount (eg, milligrams) of a composition that contacts a certain volume (eg, milliliters) of a chromatography material. In some examples, loading can be expressed in mg/mL. For example, loading can be expressed by the amount of lysozyme that a certain volume of chromatography material contacts.

Detailed description of the invention

In one aspect, the present application provides a method for isolating a target protein. In another aspect, the present application provides a method for purifying the target protein from a composition comprising the target protein and impurities. For example, the protein of interest may comprise erythropoiesis-stimulating protein, its variants or the above-mentioned functionally active fragments.

For example, proteins of the present application may contain glycosylation modifications. For example, the glycosylated erythropoiesis-stimulating protein of the present application may contain a glycan structure, and the glycan structure may contain one or more FA4G4L2S4 glycan structures, and the ratio of the FA4G4L2S4 structure may be more than 15%. The glycan structure may contain one or more FA4G4L1S4 structures, and the ratio of the FA4G4L1S4 may be more than 20%. The glycan structure may contain one or more FA4G4S4 structures, and the ratio of the FA4G4S4 may be 10% or more. The glycan structure may contain Neu5Gc, and the molar ratio of Neu5Gc may be 0.5% or less.

For example, the glycan structure may comprise one or more FA4G4L2S4 structures and one or more FA4G4L1S4 structures, the ratio of the FA4G4L2S4 structures may be 15% or more and the ratio of the FA4G4L1S4 structures may be 20% or more. The glycan structure may comprise one or more FA4G4L2S4 structures and one or more FA4G4S4 structures, and the ratio of the FA4G4L2S4 structures may be 15% or more and the ratio of the FA4G4S4 may be 10% or more. The glycan structure may comprise one or more FA4G4L1S4 structures and one or more FA4G4S4 structures, the ratio of the FA4G4L1S4 may be 20% or more and the ratio of the FA4G4S4 may be 10% or more. The glycan structure may comprise one or more FA4G4L2S4 structures and one or more Neu5Gc, and the molar ratio of the FA4G4L2S4 may be 15% or more and the Neu5Gc may be 0.5% or less. The glycan structure may comprise one or more FA4G4L1S4 structures and one or more Neu5Gc, and the molar ratio of the FA4G4L1S4 may be 20% or more and the Neu5Gc may be 0.5% or less. The glycan structure may comprise one or more FA4G4L2S4 structures, one or more FA4G4L1S4 structures, one or more FA4G4S4 structures and one or more Neu5Gc, the ratio of the FA4G4L2S4 structures may be 15% or more, and the FA4G4L1S4 The ratio of FA4G4S4 may be 20% or more, the ratio of FA4G4S4 may be 10% or more, and the molar ratio of Neu5Gc may be 0.5% or less.

In another aspect, the present application provides a glycan structure of a glycosylation-modified erythropoiesis-stimulating protein. The FA4G4L2S4 structure can bind to the N-glycosylation sites: N24, N30, N38, N83 and N88 of the glycosylation-modified erythropoiesis-stimulating protein.

On the other hand, the glycosylation-modified erythropoiesis-stimulating protein of the present application and Darbepoetin were administered to human blood leukemia cell TF-1 to observe the cell proliferation effect, and the affinity of the erythropoiesis-stimulating protein of the present application may be lower than that of Darbepoetin. The glycosylated modified erythropoiesis-stimulating protein and Darbepoetin of the present application were administered to immunodeficiency mouse CD-1, and the erythropoiesis-stimulating protein of the present application was effective for mouse hemoglobin content, red blood cell level, hematocrit, And/or the effect of increasing the level of reticulocytes can be stronger than that of Darbepoetin. Meanwhile, the in vivo elimination half-life of the erythropoiesis-stimulating protein of the present application can be about 30% longer than that of Darbepoetin.

In another aspect, the present application provides a method for preparing a glycosylation-modified erythropoiesis-stimulating protein, comprising culturing the glycosylation-modified erythropoiesis-stimulating protein under the condition of expressing the glycosylation-modified erythropoiesis-stimulating protein Dylation of nucleic acid molecules of erythropoiesis-stimulating protein in CHO-S cells. The erythropoiesis-stimulating protein encoding an amino acid sequence such as SEQ ID NO: 1 is inserted into an expression vector using a vector suitable for maintenance in mammalian host cells using standard techniques. Such vectors typically contain the following elements suitable for use in mammalian host cells: a promoter and other "upstream" regulatory elements, an origin of replication, a ribosome binding site, a transcription termination site, a polylinker site and a selectable marker. The vector may also contain elements that also permit proliferation and maintenance in prokaryotic host cells. For example, suitable cells or cell lines include any cell or cell line of mammalian origin, including human origin, including Chinese hamster ovary cells, CHO-S cells. A nucleic acid molecule comprising a sequence encoding an erythropoiesis-stimulating protein having an amino acid sequence such as SEQ ID NO: 1 is introduced into host cells using standard transformation or transfection techniques.

For example, the method of the present application may comprise contacting the protein with two or more cation exchangers, one of which may be a fine cation exchanger. For example, the method of the present application may comprise two or more cation exchange chromatography steps, wherein one of the cation exchange chromatography steps may comprise a fine cation exchange chromatography step. For example, the fine cation exchange chromatography step of the present application may comprise contacting a mixture comprising the protein of interest and impurities with a fine cation exchanger.

For example, the cation exchanger of the present application may comprise a negatively charged solid-phase chromatographic material and have free ions for exchange with cations in an aqueous solution (such as a composition comprising a protein of interest and an impurity), which is passed through ( over) or through (through) the solid phase. For example, the cation exchanger can be a membrane, a monolith or a resin. For example, the cation exchanger can be a resin. For example, cation exchangers may contain carboxylic acid functional groups or sulfonic acid functional groups, such as sulfonate, carboxylic acid, carboxymethylsulfonic acid, sulfoisobutyl, sulfoethyl, carboxyl, sulfopropyl, sulfonyl, sulfo Oxyethyl or orthophosphate, etc. For example, the cation exchanger can be a cation exchange chromatography column. For example, the cation exchanger can be a cation exchange chromatography membrane. Examples of cation exchangers known in the art may include, but are not limited to, Capto S impact. For example, cation exchange chromatography can be performed in "bind and elute" mode. For example, cation exchange chromatography can be performed in "flow-through" mode. For example, a cation exchanger can be in a column. For example, the cation exchanger can be in the membrane.

For example, the finely divided cation exchangers of the present application may comprise cation exchangers having a particle size of about 30 microns or less. For example, the fine cation exchangers of the present application may comprise particle sizes of about 30 microns or less, about 25 microns or less, about 20 microns or less, about 15 microns or less, about 10 microns or less, about A cation exchanger of 5 microns or less, about 2 microns or less, or about 1 micron or less.

For example, the fine cation exchangers of the present application may comprise cation exchangers having a dispersion of about 3% or less. For example, the degree of dispersion may be the ratio (CV) of the standard deviation of the particle size to the mean value of the particle size. For example, the fine cation exchangers of the present application may comprise a dispersion of about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, or about 0.1% or less cation exchanger. For example, the fine cation exchanger of the present application may comprise a cation exchanger comprising a loading of about 80 mg lysozyme or higher per milliliter of said fine cation exchanger. For example, the fine cation exchanger of the present application may comprise a loading of about 80 mg lysozyme or higher, about 90 mg lysozyme or higher, about 100 mg lysozyme or higher, about 200 mg lysozyme per milliliter of said fine cation exchanger. enzyme or higher, about 300 mg lysozyme or higher, or about 500 mg lysozyme or higher cation exchanger. For example, the fine cation exchanger of the present application may comprise a cation exchanger having a pressure of about 1 MPa or less at a flow rate of 1800 cm/h. For example, the fine cation exchanger of the present application may comprise a cation exchange fluid having a pressure of about 1 MPa or less, about 0.5 MPa or less, about 0.2 MPa or less, or about 0.1 MPa or less at a flow rate of 1800 cm/h. agent. For example, the matrix of the fine cation exchanger of the present application can be porous cross-linked polystyrene microspheres. Examples of fine cation exchangers known in the art may include, but are not limited to, Source 30s.

For example, after the protein or the mixture containing the protein of interest has been contacted with a cation exchanger other than the fine cation exchanger, it may be directly contacted with the fine cation exchanger. For example, the protein or the mixture comprising the protein of interest may be subjected to one or more further chromatography steps after contact with a cation exchanger other than the fine cation exchanger and before contact with the fine cation exchanger. Chromatographic steps known in the art may comprise hydrophobic interaction (HIC) chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, ceramic hydroxyapatite (CHT) chromatography , hydrophilic interaction liquid chromatography (HILIC), etc.

For example, hydrophobic interaction chromatography can be used to separate biomolecules based on their hydrophobicity. In certain embodiments, HIC chromatography can be performed in a "bind and elute" mode. In some embodiments, HIC chromatography can be performed in "flow-through" mode. In some of the embodiments described above, the HIC chromatography material may be in a column. In some of the embodiments described above, the HIC chromatography material may be in a membrane.

For example, the functional groups of a hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ) chromatography material can include positively charged crystalline calcium ion pairs (C-sites) and six Clusters of negatively charged oxygen atoms (P-sites). Proteins can be adsorbed to hydroxyapatite at low concentrations (eg, 10-25 mM) of phosphate buffered saline. Proteins can be eluted using an increasing phosphate gradient for selective elution. In some embodiments, the hydroxyapatite chromatography material can be a resin. In some of the embodiments described above, the hydroxyapatite chromatography material may be a column. Examples of hydroxyapatite chromatography materials known in the art include, but are not limited to, CHT ceramic hydroxyapatite type I support, CHT ceramic hydroxyapatite type II support. In some embodiments, hydroxyapatite chromatography can be performed in a "bind and elute" mode. In some embodiments, hydroxyapatite chromatography can be performed in "flow-through" mode.

For example, the methods of the present application may also comprise contacting the protein with a mixed mode exchanger. For example, mixed mode materials may contain functional groups capable of one or more of the following functionalities: anion exchange, cation exchange, hydrogen bonding, π-π bond interactions, hydrophilic interactions, and hydrophobic interactions. In certain embodiments, mixed mode materials may contain functional groups capable of anion exchange and hydrophobic interactions. In certain embodiments, mixed mode materials may contain functional groups capable of cation exchange and hydrophobic interactions. In certain embodiments, mixed mode materials may comprise the following groups: Hydroxyapatite Type II and CaptoMMC Impres. In certain embodiments, the first mixed-mode chromatography can be performed in a "bind and elute" mode. In certain embodiments, elution may be a gradient elution. In certain embodiments, the first mixed-mode chromatography can be performed in "flow-through" mode. In certain embodiments described above, the first mixed mode material can be in a column. In certain embodiments described above, the first mixed mode material can be in the film.

For example, the protein or mixture comprising the protein of interest may be contacted directly with the mixed mode exchanger after contacting the fine cation exchanger. For example, the protein or mixture comprising the protein of interest may be contacted directly with the mixed mode exchanger before being contacted with the fine cation exchanger. For example, the protein or mixture comprising the protein of interest may be subjected to one or more additional chromatography steps after contact with the fine cation exchanger and before contact with the mixed mode exchanger. Chromatographic steps known in the art may comprise hydrophobic interaction (HIC) chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, ceramic hydroxyapatite (CHT) chromatography , hydrophilic interaction liquid chromatography (HILIC), etc.

For example, the methods of the present application may further comprise contacting the protein with an anion exchanger. For example, the anion exchanger may be a positively charged solid-phase exchange material with free ions for exchange with anions in an aqueous solution (such as a composition comprising a protein of interest and impurities) that can pass through (over) Or through (through) the solid phase. In some embodiments of any of the methods described herein, the anion exchange material can be a membrane, monolith, or resin. In one embodiment, the anion exchange material may be a resin. In some embodiments, the anion exchange material may contain primary, secondary, tertiary, or quaternary ammonium ion functional groups, polyamine functional groups, or diethylaminoethyl functional groups. Examples of anion exchange materials are known in the art and include, but are not limited to, Capto Q and Q.sepharose.HP. In some embodiments, anion exchange chromatography can be performed in a "bind and elute" mode. In some embodiments, anion exchange chromatography can be performed in "flow-through" mode. In some of the embodiments described above, the anion exchange chromatography material may be in a column. In some of the embodiments described above, the anion exchange chromatography material can be a membrane.

For example, after the protein or the mixture comprising the protein of interest has been contacted with the anion exchanger, it can be directly contacted with the cation exchanger. For example, the protein or mixture comprising the protein of interest may be directly contacted with the cation exchanger before being contacted with the anion exchanger. For example, the protein or the mixture comprising the protein of interest may be subjected to one or more additional chromatography steps after contacting with the anion exchanger and before contacting with the cation exchanger. Chromatographic steps known in the art may comprise hydrophobic interaction (HIC) chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, affinity chromatography, ceramic hydroxyapatite (CHT) chromatography , hydrophilic interaction liquid chromatography (HILIC), etc.

For example, the methods of the present application include the use of buffers. Various buffers can be used during the purification of proteins, depending on eg the desired pH of the buffer, the desired conductivity of the buffer, the characteristics of the protein to be purified and the method of purification. For example, the buffer can be a loading buffer, an equilibration buffer or an elution buffer. For example, one or more of the loading buffer, equilibration buffer and/or wash buffer may be the same. For example, loading buffer, equilibration buffer and/or wash buffer can be different. For example, a buffer may contain salt. In certain embodiments, the buffer may comprise sodium chloride, sodium acetate, Tris HCl, Tris acetate, sodium phosphate, potassium phosphate, sodium citrate, potassium citrate, arginine, arginine HCl, or mixture. In certain embodiments, the buffer is a sodium chloride buffer. For example, the buffer may be selected from the group consisting of Tris buffer, arginine solution, phosphate solution, citrate solution and NaCl solution. Conductivity generally refers to the ability of an aqueous solution to conduct an electric current between two electrodes, and the conductivity of a solution can be changed by changing the concentration of ions in the solution.

For example, the methods provided herein can remove impurities such as host protein (HCP), host DNA (HCD), aggregates, endotoxins, high isoelectric point (PI) charge variants, low PI Charge variants, low molecular weight impurities, and/or viruses, etc. For example, the presence or reduction in the level of an impurity can be determined by comparing the amount of the impurity in the mixture recovered from the purification step to the amount of the impurity in the mixture prior to the purification step.

For example, the amount of host protein (HCP) in the composition recovered from one or more purification steps of the present application can be reduced by more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, Any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, inclusive of values in between any range of .

For example, the amount of host DNA (HCD) in the composition recovered from one or more purification steps of the present application can be reduced by more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, Any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, inclusive of values in between any range of .

For example, the amount of aggregates in a composition recovered from one or more purification steps of the present application can be reduced by more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, including any range between these values .

For example, the amount of endotoxin in the composition recovered from one or more purification steps of the present application can be reduced by more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, including any range between these values .

For example, the amount of higher isoelectric point (PI) charge variants in compositions recovered from one or more purification steps of the present application can be reduced by more than about 5%, 10%, 15%, 20%, 25%, 30% %, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, Any range between these values is included.

For example, the amount of low PI charge isomers in compositions recovered from one or more purification steps of the present application can be reduced by more than about 5%, 10%, 15%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, inclusive of any of these values any range in between.

For example, the amount of low molecular weight impurities in the composition recovered from one or more purification steps of the present application can be reduced by more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% , any of 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, including any value in between scope.

In another aspect, the method of the present application may comprise: (1) contacting the mixture comprising the target protein and impurities with a cation exchanger, (2) contacting the mixture obtained in the above steps with a fine cation exchanger.

In another aspect, the method of the present application may include: (1) contacting the mixture containing the protein of interest and impurities with a cation exchanger, (2) contacting the mixture obtained in the above steps with a fine cation exchanger, (3) contacting the above-mentioned The mixture obtained in step is brought into contact with said mixed mode exchanger.

In another aspect, the method of the present application may comprise: (1) contacting the mixture comprising the protein of interest and impurities with an anion exchanger; (2) contacting the mixture obtained in the above steps with a cation exchanger, (3) contacting the mixture obtained in the above steps The mixture obtained is contacted with a fine cation exchanger.

In another aspect, the method of the present application may comprise: (1) contacting the mixture comprising the protein of interest and impurities with an anion exchanger; (2) contacting the mixture obtained in the above steps with a cation exchanger, (3) contacting the mixture obtained in the above steps The mixture obtained is contacted with a fine cation exchanger, (4) the mixture obtained in the above step is brought into contact with a mixed mode exchanger.

In another aspect, the method of the present application may comprise: (1) contacting the mixture comprising the protein of interest and impurities with an anion exchanger; (2) contacting the mixture obtained in the above steps with a cation exchanger, (3) contacting the mixture obtained in the above steps The obtained mixture is contacted with a fine cation exchanger, and (4) the mixture obtained in the above step is contacted with two or more mixed mode exchangers.

In another aspect, the method of the present application may comprise: (1) contacting the mixture comprising the protein of interest and impurities with two or more anion exchangers; (2) contacting the mixture obtained in the above steps with a cation exchanger, (3) The mixture obtained in the above steps is contacted with a fine cation exchanger, (4) the mixture obtained in the above steps is contacted with a mixed mode exchanger.

In another aspect, the method of the present application may comprise: (1) contacting the mixture comprising the protein of interest and impurities with two or more anion exchangers; (2) contacting the mixture obtained in the above steps with a cation exchanger, (3) The mixture obtained in the above steps is contacted with a fine cation exchanger, and (4) the mixture obtained in the above steps is contacted with two or more mixed mode exchangers.

On the other hand, the method of the present application can comprise: (1) make the mixture obtained above-mentioned step contact with Q.Sepharose.HP; (2) make the mixture obtained above-mentioned step contact with CaptoS impact; (3) make above-mentioned step obtain The mixture obtained in the above steps is contacted with Source30S; (4) the mixture obtained in the above steps is contacted with filler CHT (hydroxyapatite, II); (5) the mixture obtained in the above steps is contacted with CaptoMMC.

On the other hand, the method of the present application can comprise: (1) make the mixture obtained above-mentioned step contact with Q.Sepharose.HP; (2) make the mixture obtained above-mentioned step contact with CaptoS impact; (3) make above-mentioned step obtain (4) contact the mixture obtained in the above steps with CaptoMMC; (5) contact the mixture obtained in the above steps with filler CHT (hydroxyapatite, II).

On the other hand, the method of the present application can comprise: (1) make the mixture that comprises object protein and impurity contact with CaptoQ; (2) make the mixture that above-mentioned step obtains contact with Q.Sepharose.HP; (3) make above-mentioned steps The mixture obtained is contacted with CaptoS impact; (4) the mixture obtained in the above steps is contacted with Source30S; (5) the mixture obtained in the above steps is contacted with filler CHT (hydroxyapatite, II).

On the other hand, the method of the present application can comprise: (1) make the mixture that comprises object protein and impurity contact with CaptoQ; (2) make the mixture that above-mentioned step obtains contact with Q.Sepharose.HP; (3) make above-mentioned steps The mixture obtained is contacted with CaptoS impact; (4) the mixture obtained in the above steps is contacted with Source30S; (5) the mixture obtained in the above steps is contacted with CaptoMMC.

On the other hand, the method of the present application can comprise: (1) make the mixture that comprises object protein and impurity contact with CaptoQ; (2) make the mixture that above-mentioned step obtains contact with Q.Sepharose.HP; (3) make above-mentioned steps The mixture obtained is contacted with CaptoS impact; (4) the mixture obtained in the above steps is contacted with Source30S; (5) the mixture obtained in the above steps is contacted with filler CHT (hydroxyapatite, II); (6) the above steps are obtained The mixture is contacted with CaptoMMC.

In some embodiments, the protein of interest can also be further purified by virus filtration. Viral filtration can be the removal of viral contaminants in a polypeptide purification feed stream. Examples of viral filtration may include, for example, ultrafiltration and microfiltration. In some embodiments, the polypeptide can be purified using a parvovirus filter.

In some embodiments, the protein of interest can also be concentrated after chromatography. Examples of concentration methods are known in the art and may include, but are not limited to, eg ultrafiltration and diafiltration. In some embodiments, the concentration of the target protein after concentration can be about any one of 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, or 0.5 mg/mL.

In some embodiments of any of the methods described herein, the method can further comprise combining the purified protein of interest of the purification method with a pharmaceutically acceptable carrier. In some embodiments, the protein of interest can be formulated into a pharmaceutical formulation by ultrafiltration/diafiltration.

In certain embodiments, the methods provided herein can produce a composition comprising a protein of interest that can be greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% pure. Any of %, 90%, 95%. In certain embodiments, the protein of interest in the composition may be greater than any of about 96%, 97%, 98%, 99%, 99.5%, or 99.9% pure.

For example, the compositions provided herein can have reduced amounts of impurities such as host proteins (HCP), host DNA (HCD), aggregates, endotoxins, high isoelectric point (PI) charge variants, low PI charge variants , low molecular weight impurities, and/or viruses, etc. For example, the presence or reduction in the level of an impurity can be determined by comparing the amount of the impurity in the mixture recovered from the purification step to the amount of the impurity in the mixture prior to the purification step.

In certain embodiments, compositions comprising a protein of interest purified according to any of the methods described herein are provided.

In certain embodiments, the protein of interest in the composition can be more than about any one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% pure kind. In certain embodiments, the purity of the egg of interest in the composition may be greater than any of about 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

In one aspect, the present application provides a pharmaceutical composition, which includes a therapeutically effective amount of the glycosylation-modified erythropoiesis-stimulating protein and pharmaceutically acceptable adjuvants such as diluents, carriers, solubilizers, emulsifiers, Preservatives and/or Auxiliaries. The pharmaceutical composition is suitable for a dosing regimen of less than three times per week. The composition may be in liquid or lyophilized form and it contains diluents (Tris, citrate, acetate or phosphate buffers) of varying pH and ionic strength, solubilizers such as Tween or polysorbate esters, carriers such as human serum albumin or gelatin, preservatives such as thimerosal, parabens or benzyl alcohol, antioxidants such as ascorbic acid or sodium metabisulfite, and other ingredients such as lysine or glycine. The choice of a particular composition will depend on many factors, including the condition being treated, the route of administration and the desired pharmacokinetic parameters. For a more extensive review of ingredients suitable for use in pharmaceutical compositions, see Remington's Pharmaceutical Sciences, 18th Edition, edited by A.R. Gennaro, Mack, Easton, PA (1980). For example, the glycosylated modified erythropoiesis-stimulating protein described herein is formulated in liquid form with isotonic sodium chloride/sodium citrate buffer containing human albumin and optionally benzyl alcohol as a preservative. The composition may contain analogues having 1, 2, 3, 4 or more additional sugar chains. For example, the pharmaceutical composition of the present application can be administered by subcutaneous or intravenous injection. The final route of administration chosen will depend on many factors and can be determined by one skilled in the art.

In one aspect, the present application provides an application for treating anemia. The glycosylation-modified erythropoiesis-stimulating protein provided in this application has a unique glycosylation form. When it binds to the EPO receptor, it can cause a change in the conformation of the EPO receptor, thereby activating multiple downstream signaling pathways and causing red blood cells. Proliferation and differentiation The glycosylated modified erythropoiesis-stimulating protein is administered to a subject in need of treatment to stimulate erythropoiesis, increase hemoglobin content, red blood cell level, hematocrit, reticulocyte level, and alleviate anemia, such as kidney Anemia of chronic kidney disease, multiple myeloma and/or cancerous anemia, such as renal anemia caused by chronic kidney disease and uremia, multiple myeloma anemia and cancerous anemia caused by chemotherapy, etc.

In one aspect, the present application provides a glycosylation-modified erythropoiesis-stimulating protein and/or a pharmaceutical composition for treating anemia. The glycosylated modified erythropoiesis-stimulating protein is administered to a subject in need of treatment to stimulate erythropoiesis, increase hemoglobin content, red blood cell level, hematocrit, reticulocyte level, and alleviate anemia, such as renal anemia, Anemia in multiple myeloma and/or anemia in cancer.

In one aspect, the present application provides a glycosylation-modified erythropoiesis-stimulating protein and/or a pharmaceutical composition, and its application in the preparation of a drug for treating anemia. The glycosylated modified erythropoiesis-stimulating protein is administered to a subject in need of treatment to stimulate erythropoiesis, increase hemoglobin content, red blood cell level, hematocrit, reticulocyte level, and relieve anemia, such as renal anemia, Anemia in multiple myeloma and/or anemia in cancer.

In one aspect, the present application provides a method for prolonging the half-life of erythropoiesis-stimulating protein. After administering the glycosylated-modified erythropoiesis-stimulating protein described in the present application to a subject in need of treatment, the glycosylated-modified The clearance rate of erythropoiesis-stimulating protein in the subjects was significantly reduced, and the half-life in vivo was prolonged.

Not intending to be limited by any theory, the following examples are only for explaining the methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1

The expression method of long-acting erythropoiesis-stimulating protein is divided into the following steps: working cell bank cells are cultured in shake flasks, through a series of inoculation, cultivation and expansion of culture volume until sufficient biomass is used to inoculate bioreactors. Product expression was promoted by supplementation of feed medium as well as glucose.

Glycosylation-modified erythropoiesis-stimulating protein, for example, contains 165 amino acids and contains 5 N glycosylation sites (N24, N30, N38, N83, N88) as shown in SEQ ID NO:1 sugar protein:

The corresponding nucleic acid sequences were transfected into parental CHO-S cells via expression plasmids. Through pressurized selection, high-yielding clones were screened. Through culturing and purification, the glycosylation-modified erythropoiesis-stimulating protein culture of the present application is obtained.

Example 2

The present application provides a sequential combination of various chromatographic techniques to finally obtain a protein stock solution with qualified quality such as charge-to-mass ratio, sialic acid content, and/or qualified glycoform. The chromatographic process of the present application may comprise the following chromatographic steps:

Q.Sepharose.HP (anion exchange) purification parameters: Sterilization: 0.5～1.0M NaOH, 2CV, soaking for 30～60mins; Regeneration: 1M NaCl, 2CV; Equilibrium: 20mM～100mM Tris, pH 7.0～8.5, 5CV; Sample: source post CaptoQ ultrafiltration sample after liquid replacement, pH 7.0-8.50, conductance less than 10ms/cm; wash 1: 20mM-100mM Tris, pH7.0-8.5, 5CV; wash 2: 2-5CV linear elution from 20mM～100mM Tris, pH7.0～8.5 to 10m～20mM Arginine, 5～20mM NaPO ₄ , 5～20mM citrate, pH3.0～4.0; wash 3: 10m～20mM Arginine, 5～20mM NaPO ₄ , 5～20mM citrate, pH3.0～4.0, 5～10CV; wash 4: 2～5CV linear prewash from 10m～20mM Arginine, 5～20mM NaPO ₄ , 5～20mM citrate, pH3.0～4.0 to 20mM～100mM Tris, pH 7.0～8.5; washing 5: 20mM～100mM Tris, pH 7.0～8.5, 5CV; elution: 200～500mM NaCl, 20mM～100mM Tris, pH 7.0～8.5, one-step elution, 3CV; principle of peak collection: after the peak Start to collect 5mAu, stop collecting 5mAu after the peak, combine the collected solution, add 5% Tween-80 mother solution to the eluted target protein solution to a final concentration of 0.01% ~ 1% Tween-80; the temperature of the purification buffer in this step is 2 ~ At 8°C, add 5% Tween-80 mother solution to the eluted target protein solution to a final concentration of 0.01%-1% Tween-80.

Liquid replacement for ultrafiltration: 10-30KDa, 0.1-0.5m ² , sample source: post Q.HP; concentrate 2-6 times, continuously change the volume of 5 times the liquid; take out the concentrated solution, wash the membrane 1-2 times, and wash the membrane together solution and concentrate; final buffer system: 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 6.0～7.0, final concentration: 0.4～0.6mg/ml, final volume restored to the initial volume.

CaptoS impact purification (cation exchange): Sterilization: 0.5～1.0M NaOH, 2CV, soaking for 30～60mins; Regeneration: 1M NaCl, 2CV; Equilibrium: 5CV, 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～4.5 ;Sample loading: post Q.HP eluent low pH inactivated sample; Washing 1: 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～4.5, 7CV; Washing 2: 50mM M～100mM NaCl, 10mM～ 50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～4.5, 7CV; elution: 10～20CV from: 50mM M～100mM NaCl, 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～4.5 to 50mM M～100mM NaCl, 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 6.5～7.5; peak collection principle: start to collect at 5mAu after the peak, stop collecting at 5mAu after the peak, collect in separate tubes, and combine the components according to the results of IEF and SDS-PAGE.

Liquid replacement by ultrafiltration: ultrafiltration membrane 10-30KDa, 0.1-0.5m ² , sample source: post CaptoS impact sample; concentrate 8-10 times, continuously change liquid 5 times the volume; take out the concentrated solution, wash 1-2 times, and combine Membrane washing solution and concentrated solution; final buffer system: 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 6.0～7.0; final concentration: 0.08～0.15mg/ml.

Source30S (fine cation exchange) purification: sample volume: less than 3mg/ml resin; sterilization: 1.0M NaOH, 2CV, soaking for 30-60mins; regeneration: 1M NaCl, 2CV; balance: 5CV, 10mM～50mM citrate, 10mM～ 50mM sodium phosphate, pH 3.5 ~ 5.0; sample loading: post CaptoS impact sample after ultrafiltration, pH adjusted to 3.5 ~ 5.0 with 0.5M HCl; washing 1: 10mM ~ 50mM citrate, 10mM ~ 50mM sodium phosphate, pH 3.5 ~ 5.010CV; washing 2: 50～100mM NaCl, 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～5.0 3CV; elution: 10～30CV from 50～100mM NaCl, 10mM～50mM citrate, 10mM～50mM sodium phosphate ,pH 3.5～5.0 to 150～300mM NaCl, 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～5.0; peak collection principle: start to collect at 2mAu after the peak, stop collecting at 10mAu after the peak, collect in separate tubes, and detect according to CZE The results were pooled for the samples.

Ultrafiltration liquid change: ultrafiltration membrane: 10 ~ 30KDa, 0.1 ~ 0.5m ² sample source: post Source30S sample; concentrate 2 ~ 4 times, continuously change liquid 5 times volume; take out the concentrated solution, wash the membrane 1-2 times, and combine Membrane wash solution and concentrated solution; final buffer system: 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 6.0～7.0; final concentration controlled at around 0.07-0.10mg/ml; 0.22um filter.

Filler CHT (hydroxyapatite, Ⅱ) (mixed mode) flow through: load: less than 0.5mg/ml resin; sterilization: 1.0M NaOH, 2CV, soak for 30mins; regeneration: 500mM sodium phosphate, 2CV; balance: 10mM ～100sodium phosphate, pH 6.0～7.0, 5CV; sample loading: sample source: post Source30S component combined and changed the sample, the sample concentration before loading is controlled between 0.05-0.30mg/ml, pH6.0-7.0; Washing/elution: 10mM～100sodium phosphate, pH 6.0～7.0, 10CV; principle of peak collection: collect the flow-through solution, start collecting at 5mAu after the peak, stop collecting at 5mAu after the peak, and combine the collected solution; washing: 500mM sodium phosphate, 2CV .

CaptoMMC (mixed mode) flow through: Sterilization: 0.5～1.0M NaOH, 2CV, soaking for 30～60mins; Regeneration: 1M NaCl, 2CV; Balance: 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 6.0～7.0, 5CV ;Sample loading: the source of the sample is CHT flow-through solution, the sample concentration before loading is controlled between 0.005mg/ml-0.10mg/ml, and the pH is 6.0-7.0; pre-washing/elution: 10mM～50mM citrate, 10mM～ 50mM sodium phosphate, pH 6.0-7.0, 5CV, peak collection principle: collect the flow-through liquid, start to collect at 5mAu after the peak, stop collecting at 5mAu after the peak, and combine the collected liquid.

Virus removal nanofiltration: Sample source: #post Capto MMC flow-through liquid pre-filtration membrane: 0.2/0.1um Sartorius pre-filtration membrane, filtered in series mode; the permeate is collected, and the filtration driving force is 2Mpa.

DS stock solution preparation: ultrafiltration membrane: 10-30KDa, 0.1-0.5m ² ; sample source: post Virus filtration; final buffer system: 20mM-100mM sodium phosphate, 100-300mM NaCl, 0.005%-0.01% Tween-80, pH6 .0-7.0; Concentrate about 8-10 times, continuously change the volume of 5-10 times; final concentration: 0.1-0.5mg/ml; 0.22um sterile filtration.

Through the above steps, a target protein stock solution with qualified molecular weight, purity, N-terminal sialic acid content, glycoform and content, HCD (host DNA), endotoxin, biological activity, etc. can be obtained, wherein the content of HCP (host protein) is about Between 100-50～0ug/mg, it has a significantly lower level. The peptide spectrum and sugar spectrum of each batch of protein are consistent with the reference, and the specific test data are as follows:

Example 3

Q.Sepharose.HP (anion exchange) purification parameters and ultrafiltration liquid replacement are basically the same as the above-mentioned embodiment steps;

CaptoS impact purification (cation exchange) and ultrafiltration liquid replacement are basically the same as the above-mentioned embodiment steps;

Source30S (fine cation exchange) purification and ultrafiltration liquid replacement are basically the same as the above-mentioned embodiment steps;

CaptoMMC (mixed mode) flows through, and the steps are basically the same as the steps of the above-mentioned embodiments;

The filler CHT (II) (mixed mode) flows through, and the steps are basically the same as those in the above-mentioned examples;

The steps of virus removal nanofiltration and preparation of DS stock solution are basically the same as those in the above examples.

By changing the order of the mixing modes, a target protein stock solution with qualified molecular weight, purity, N-terminal sialic acid content, glycoform and content, HCD (host DNA), endotoxin, biological activity, etc. can be obtained, among which HCP (host protein ) content is about 100-500ug/mg, with a significantly lower level. The peptide spectrum and sugar spectrum of each batch of protein are consistent with the reference, and the specific test data are as follows:

Example 4

CaptoQ capture (anion exchange): Sterilization: 1.0M NaOH, 2CV, soaking for 30-60mins; Regeneration: 1M NaCl, 2CV; Equilibrium: 20-100mM Tris, pH 7.0-8.5, 5CV; Loading: Fermentation after concentration change The pH of the solution is 7.0-8.5, and the conductivity value is less than 10ms/cm; pre-washing: 20-100mM Tris, pH 7.0-8.5, 5-10CV; elution: 200-500mM NaCl, 20-100mM Tris, pH7.0-8.5, One-step elution, 3~5CV; peak collection principle: start to collect at 10mAu after the peak, stop collecting at 10mAu after the peak, and combine the collected solution; the temperature of the purification buffer in this step is 2~15°C, and the eluted target protein solution is added with 5 % Tween-80 mother liquor to a final concentration of 0.01% to 1% Tween-80.

Liquid replacement for ultrafiltration: Ultrafiltration membrane: 10-30KDa, 0.1-0.5m ² , sample source: post Capt Q; concentrate 5-10 times, continuously change liquid 5-7 times the volume; take out the concentrated solution, wash the membrane 1-2 times For the second time, combine the washing solution and the concentrated solution; final buffer system: 20mM ~ 100mM Tris, 0.01% ~ 1% Tween-80, pH 7.0 ~ 8.5; final concentration: 0.4 ~ 0.8mg/ml; 0.22um sterile filtration.

The filler CHT (hydroxyapatite, II) (mixed mode) flows through, and the steps are basically the same as those in the above examples;

Through the above steps, the target protein stock solution with qualified molecular weight, purity, N-terminal sialic acid content, glycoform and content, HCD (host DNA), endotoxin, biological activity, etc. can be obtained, wherein the content of HCP (host protein) is about Between 100 and 500ug/mg, it has a significantly lower level. The peptide spectrum and sugar spectrum of each batch of protein are consistent with the reference, and the specific test data are as follows:

Example 5

This application provides a sequential combination of various chromatographic techniques to finally obtain a protein stock solution with qualified quality such as charge-to-mass ratio, sialic acid content, and/or qualified glycoform. The chromatographic process of the present application may comprise the following chromatographic steps:

CaptoQ capture (anion exchange) and ultrafiltration liquid replacement are basically the same as the steps of the above-mentioned examples;

By changing the composition of the mixing mode, a target protein stock solution with qualified molecular weight, purity, N-terminal sialic acid content, glycoform and content, HCD (host DNA), endotoxin, biological activity, etc. can be obtained, and the content of HCP (host protein) About 100-500ug/mg, with a significantly lower level. The peptide spectrum and sugar spectrum of each batch of protein are consistent with the reference, and the specific test data are as follows:

Example 6

CaptoQ Capture: Sterilization: 1.0M NaOH, 2CV, soaking for 30-60mins; Regeneration: 1M NaCl, 2CV; Equilibrium: 20-100mM Tris, pH 7.0-8.5, 5CV; ～8.5, conductance value is less than 10ms/cm; prewash: 20～100mM Tris, pH 7.0～8.5, 5～10CV; elution: 200～500mM NaCl, 20～100mM Tris, pH7.0～8.5, one-step elution, 3-5CV; peak collection principle: start to collect at 10mAu after the peak, stop collecting at 10mAu after the peak, and combine the collected solution; the temperature of the purification buffer in this step is 2-15°C, and the eluted target protein solution is added with 5% Tween-80 The final concentration of the mother liquor is 0.01% to 1% Tween-80.

Ultrafiltration fluid replacement 1: Ultrafiltration membrane: 10～30KDa, 0.1～0.5m ² , sample source: post Capt Q; concentrate 5～10 times, continuously change fluid volume 5～7 times; take out the concentrated solution and wash the membrane 1 ~2 times, combine washing liquid and concentrate; final buffer system: 20mM ~ 100mM Tris, 0.01% ~ 1% Tween-80, pH 7.0 ~ 8.5; final concentration: 0.4 ~ 0.8mg/ml; 0.22um sterile filtration (Sartorius).

Q.Sepharose.HP purification parameters: Sterilization: 1.0M NaOH, 2CV, soaking for 30mins; Regeneration: 1M NaCl, 2CV; Equilibrium: 20mM～100mM Tris, pH 7.0～8.5, 5CV; Loading: Source post CaptoQ ultrafiltration Sample after solution, pH 7.0-8.50, conductance less than 10ms/cm; wash 1: 20mM-100mM Tris, pH7.0-8.5, 5CV; wash 2: 2-5CV linear elution from 20mM-100mM Tris, pH7.0 ～8.5 to 10m～20mM Arginine (arginine), 5～20mM NaPO ₄ , 5～20mM citrate (citrate), pH3.0～4.0; wash 3: 10m～20mM Arginine, 5～20mM NaPO ₄ , 5 ～20mM citrate, pH3.0～4.0, 5～10CV; washing 4: 2～5CV linear prewash from 10m～20mM Arginine, 5～20mM NaPO ₄ , 5～20mM citrate, pH3.0～4.0 to 20mM～100mM Tris , pH 7.0-8.5; washing 5: 20mM-100mM Tris, pH 7.0-8.5, 5CV; elution: 200-500mM NaCl, 20mM-100mM Tris, pH 7.0-8.5, one-step elution, 3CV; principle of peak collection: 5mAu after the peak began to be collected, and the collection of 5mAu after the peak was stopped. The collected solutions were combined, and the eluted target protein solution was added with 5% Tween-80 mother solution to a final concentration of 0.01% to 1% Tween-80; the temperature of the purification buffer in this step was Add 5% Tween-80 mother solution to the eluted target protein solution at 2-8°C to a final concentration of 0.01%-1% Tween-80.

Ultrafiltration fluid change 2: 10～30KDa, 0.1～0.5m ² , sample source: post Q.HP; concentrate 2～6 times, continuously change fluid volume 5 times; take out concentrated solution, wash membrane 1～2 times, combine Membrane wash solution and concentrated solution; final buffer system: 10mM～50mM citrate, 10mM～50mM sodium phosphate (sodium phosphate), pH 6.0～7.0, final concentration: 0.4～0.6mg/ml, and the final volume was restored to the initial volume.

CaptoS impact purification: Sterilization: 1.0M NaOH, 2CV, soaking for 30mins; Regeneration: 1M NaCl, 2CV; Equilibrium: 5CV, 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～4.5; Loading: post Q.HP Sample after low pH inactivation of eluent; wash 1: 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～4.5, 7CV; wash 2: 50mM M～100mM NaCl, 10mM～50mM citrate, 10mM～50mM sodium phosphate , pH 3.5～4.5, 7CV; Elution: 10～20CV from: 50mM M～100mM NaCl, 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～4.5 to 50mM M～100mM NaCl, 10mM～50mM citrate, 10mM ～50mM sodium phosphate, pH 6.5～7.5; peak collection principle: start to collect at 5mAu after the peak, stop collecting at 5mAu after the peak, collect in separate bottles, the collection volume is to be determined, 10L/bottle before the peak, 2.5L/bottle after the peak; Principle of pooling samples: after the results of IEF and SDS-PAGE, all components were combined.

Ultrafiltration fluid change 3: Ultrafiltration membrane: 10～30KDa, 0.1～0.5m ² , sample source: post CaptoS impact sample; concentrate 8～10 times, continuously change fluid 5 times volume; take out the concentrated solution, wash 1-2 For the second time, combine the washing solution and the concentrated solution; final buffer system: 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 6.0～7.0; final concentration: 0.08～0.15mg/ml.

Source30S purification: sample volume: less than 3mg/ml resin; sterilization: 1.0M NaOH, 2CV, soaking for 30mins; regeneration: 1M NaCl, 2CV; balance: 5CV, 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～ 5.0; sample loading: post CaptoS impact sample after ultrafiltration, pH adjusted to 3.5-5.0 with 0.5M HCl; rinse 1: 10mM-50mM citrate, 10mM-50mM sodium phosphate, pH 3.5-5.0 10CV; rinse 2: 50 ～100mM NaCl, 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～5.0 3CV; elution: 10～30CV from 50～100mM NaCl, 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～5.0 to 150 ～300mM NaCl, 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 3.5～5.0; peak collection principle: start to collect at 2mAu after the peak, stop at 10mAu after the peak, collect in separate bottles, the collection volume is to be determined, 1.25L/bottle, About 20 bottles.

Ultrafiltration liquid replacement 4: Ultrafiltration membrane: 10～30KDa, 0.1～0.5m ² Sample source: post Source30S sample; concentrate 2～4 times, continuously change liquid 5 times volume; take out the concentrated solution, wash the membrane 1-2 times , combined wash solution and concentrated solution; final buffer system: 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 6.0～7.0; final concentration controlled at about 0.07-0.10mg/ml; 0.22um filter (Sartorius).

Filler CHT (hydroxyapatite, type II) flow through: load: less than 0.5mg/ml resin; sterilization: 1.0M NaOH, 2CV, soak for 30mins; regeneration: 500mM sodium phosphate, 2CV; balance: 10mM～100sodium phosphate ,pH 6.0～7.0, 5CV; sample loading: sample source: post Source30S component combined and changed the sample, the sample concentration before loading is controlled between 0.05-0.30mg/ml, pH6.0-7.0; pre-washing/washing Detachment: 10mM～100sodium phosphate, pH 6.0～7.0, 10CV; principle of peak collection: collect the flow-through solution, start to collect at 5mAu after the peak, stop collecting at 5mAu after the peak, and combine the collected solution; washing: 500mM sodium phosphate, 2CV.

Capto MMC Impres flowthrough: Sterilization: 1.0M NaOH, 2CV, soaking for 30mins; Regeneration: 1M NaCl, 2CV; Equilibrium: 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 6.0～7.0, 5CV; Loading: sample source It is CHT flow-through solution, the sample concentration before loading is controlled between 0.005mg/ml-0.10mg/ml, pH is 6.0-7.0; pre-washing/elution: 10mM～50mM citrate, 10mM～50mM sodium phosphate, pH 6.0 ～7.0, 5CV, the principle of peak collection: collect the flow-through liquid, start to collect 5mAu after the peak, stop collecting 5mAu after the peak, and combine the collected liquid.

Virus filtration: Sample source: #post Capto MMC flow-through liquid pre-filtration membrane: 0.2/0.1um Sartorius pre-filtration membrane, using series mode filtration; collect permeate, filtration driving force is 2Mpa.

DS stock solution preparation: ultrafiltration membrane: 10-30KDa, 0.1-0.5m ² . Sample source: post Virus filtration; final buffer system: 20mM-100mM sodium phosphate, 100-300mM NaCl, 0.005%-0.01% Tween-80, pH6 .0- 7.0; Concentrate about 8-10 times, continuously change the volume of 10 times; Final concentration: 0.1-0.5mg/ml; 0.22um sterile filtration.

Through the above steps, the protein stock solution can be obtained, and the yield is 0.2-100mg/L; the SEC-HPLC purity is 98-100%; the endotoxin, HCD content and the like are all within the quality requirement range. The content of glycoforms and components, sialic acid content, peptide spectrum, and sugar spectrum are basically consistent with those of the reference product. The HCP content ranges from 10ng/mg to 500ng/mg, and can continue to reduce the HCP to maintain the qualified samples below 10-100ng/mg. The peptide spectrum and sugar spectrum of each batch of protein are consistent with the reference, and the specific test data are as follows:

Example 7

The chromatographic process may comprise the following chromatographic steps:

Through the above steps, the protein with the same charge variant composition as the glycosylation-modified erythropoiesis-stimulating protein standard product cannot be obtained, and the charge variant impurities with high PI are relatively more. At the same time, the glycoform is consistent with the standard product, but the content and ratio are different. Endotoxin less than 2Eu/mg, HCD less than 10pg/mg process-related impurities are within the quality requirements. The content of HCP is about 500ng/mg, which is quite different from 100ng/mg.

The chromatographic process may comprise the following chromatographic steps:

Filler CHT (II) (mixed mode) flows through, and the steps are basically the same as those in the above examples.

Through the above steps, the target protein stock solution with qualified endotoxin and HCD contents can be obtained. The protein stock solution glycoform and the content of each component, peptide map, and sialic acid content are basically consistent with the standard product. But the content of HCP is 600.52ng/mg. The peptide spectrum and sugar spectrum of the prepared protein are consistent with the reference, and the specific test data are as follows:

Example 8

The chromatographic process may comprise the following chromatographic steps:

Through the above steps, the protein stock solution can be obtained, and the yield is 0.2-100mg/L; the SEC-HPLC purity is 98-100%; the endotoxin, HCD content and the like are all within the quality requirement range. The content of glycoforms and components, peptide map, and sialic acid content are basically the same as those of the standard product. The HCP content ranges from 50ng/mg to 500ng/mg.

The chromatographic process may comprise the following chromatographic steps:

Through the above steps, a protein stock solution with a yield of 0.2-100 mg/L, a SEC-HPLC purity of 98-100%, and contents of endotoxin and HCD all within the range of quality requirements can be obtained. The content of glycoforms and components, peptide map, and sialic acid content are basically the same as those of the standard product. The content of HCP is 50ng/mg～500ng/mg. The peptide spectrum and sugar spectrum of each batch of protein are consistent with the reference, and the specific test data are as follows:

Example 9

The chromatographic process may comprise the following chromatographic steps:

Through the above steps, a protein stock solution with a yield of 0.2-100 mg/L, a SEC-HPLC purity of 98-100%, and contents of endotoxin and HCD all within the range of quality requirements can be obtained. The content of glycoforms and components, peptide map, and sialic acid content are basically the same as those of the standard product. The content of HCP is about 5ng/mg～200ng/mg. The peptide spectrum and sugar spectrum of the prepared protein are consistent with the reference, and the specific test data are as follows:

Example 10

The application also provides comparative examples for comparison

For example, choose ordinary cation exchange media for purification, such as SP.Sepharoe.FF and Capto S Impact.

10-1 Purification route: Capto Q capture—Q.Sepharose.HP purification—SP.Sepharoe.FF purification—Capto S Impact purification fine purification—CHT flow through—Capto MMC flow through—original solution. where the result looks like this:

批次batch	料液体积(L)Feed liquid volume (L)	产率(mg/L)Yield (mg/L)	SEC-HPLC(％)SEC-HPLC(%)	HCP(ng/mg)HCP (ng/mg)
Lot#20190802Lot#20190802	4L4L	0.10mg/L0.10mg/L	99.68％99.68%	150ng/mg150ng/mg

Using this process, due to the lack of effective typing means for erythropoiesis-stimulating proteins with different sialic acid contents in the protein of this application, the yield of the product of this process is greatly reduced. Compared with the refined filler Source 30S, the yield of this comparison scheme is lost About 70%. At the same time, HCP residues cannot be effectively removed, resulting in higher residues.

10-2 Purification route: Capto Q capture—Q.Sepharose.HP purification—Capto S Impact purification—Capto S Impact fine purification—CHT flow through—Capto MMC flow through—original solution. where the result looks like this:

批次batch	料液体积(L)Feed liquid volume (L)	产率(mg/L)Yield (mg/L)	SEC-HPLC(％)SEC-HPLC(%)	HCP(ng/mg)HCP (ng/mg)
Lot#20190901Lot#20190901	4L4L	0.12mg/L0.12mg/L	99.54％99.54%	120ng/mg120ng/mg

Wherein the comparison scheme productive rate and HCP residual result are similar to 10-1.

The application provides other comparative examples for comparison at the same time

Among them, the results of schemes 10-3 to 10-6 are summarized as follows:

The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of this application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims

A protein separation method, which involves contacting the protein with two or more cation exchangers, wherein one of the cation exchangers is a fine cation exchanger.
The method according to claim 1, wherein said protein comprises erythropoiesis-stimulating protein, a variant thereof or a functionally active fragment thereof.
The method of any one of claims 1-2, wherein the protein comprises a glycosylation modification.
The method of any one of claims 1-3, said protein comprising a glycan structure bound to an N-glycosylation site.
The method of claim 4, said glycan structure comprising FA4G4L2S4.
The method according to claim 5, wherein the ratio of the FA4G4L2S4 structure is above 15%.
The method of any one of claims 4-6, wherein the glycan structure further comprises FA4G4L1S4.
The method according to claim 7, wherein the ratio of said FA4G4L1S4 is 20% or more.
The method of any one of claims 4-8, wherein the glycan structure further comprises FA4G4S4.
The method of claim 9, wherein the ratio of FA4G4S4 is 10% or more.
The method according to any one of claims 4-10, wherein the glycan structure comprises Neu5Gc, and the molar ratio of Neu5Gc is 0.5% or less.
The method according to any one of claims 1-11, wherein the protein comprises the amino acid sequence shown in SEQ ID NO.1 or a functionally active fragment thereof.
The method of any one of claims 1-12, said protein comprising an N-glycosylation site selected from the group consisting of N24, N30, N38, N83 and N88.
The method according to any one of claims 1-13, wherein the protein is expressed by CHO cells.
The method of claim 14, said CHO cells comprising CHO-S cells.
The method of any one of claims 1-15, the finely divided cation exchanger comprising a cation exchanger having a particle size of about 30 microns or less.
The method of any one of claims 1-16, the finely divided cation exchanger comprising a cation exchanger having a dispersion of about 3% or less.
The method according to claim 17, said degree of dispersion is the ratio of the standard deviation of the particle size to the average value of the particle size.
The method according to any one of claims 1-18, said fine cation exchanger comprising Source 30s.
The method according to any one of claims 1-19, other cation exchangers other than the fine cation exchangers comprise Capto S impact.
The method according to claim 20, wherein said protein is first contacted with said other cation exchanger, and then contacted with said fine cation exchanger.
The method of any one of claims 1-21, further comprising contacting the protein with a mixed mode exchanger.
The method of claim 22, wherein said protein is first contacted with said fine cation exchanger and then contacted with said mixed mode exchanger.
The method of any one of claims 22-23, further comprising contacting the protein with two or more of the mixed mode exchangers.
The method according to any one of claims 22-24, wherein the protein is first contacted with the fine cation exchanger, and then contacted with two or more of the mixed mode exchangers.
The method of any one of claims 22-25, said mixed mode exchanger comprising the group consisting of: Hydroxyapatite Type II and CaptoMMC Impres.
The method of any one of claims 1-26, further comprising contacting the protein with an anion exchanger.
The method of claim 27, wherein said protein is first contacted with said anion exchanger and then contacted with said cation exchanger.
The method according to any one of claims 27-28, said anion exchanger comprises the following groups: Capto Q and Q.sepharose.HP.
The method according to any one of claims 1-29, wherein the buffer is selected from the group consisting of Tris buffer, arginine solution, phosphate solution, citrate solution and NaCl solution.
The method according to any one of claims 27-30, comprising (1) contacting the protein with the anion exchanger; (2) contacting the protein obtained in the above steps with the other cation exchanger , (3) contacting the protein obtained in the above steps with the fine cation exchanger, (4) contacting the protein obtained in the above steps with the mixed mode exchanger.
The method according to any one of claims 27-31, comprising (1) contacting the protein with the anion exchanger; (2) contacting the protein obtained in the above steps with the other cation exchanger , (3) contacting the protein obtained in the above steps with the fine cation exchanger, (4) contacting the protein obtained in the above steps with two or more of the mixed mode exchangers.
An isolated protein obtained by being isolated by the method according to any one of claims 1-32.
The protein of claim 33, wherein the host protein content is about 500 ng/mg or less.
The protein of any one of claims 33-34, wherein the host protein content is about 100 ng/mg or less.
A pharmaceutical composition comprising the protein of any one of claims 33-35 and a pharmaceutically acceptable adjuvant.
Use of the protein according to any one of claims 33-35 and/or the pharmaceutical composition according to claim 36 in the preparation of a medicament for treating anemia.
The use according to claim 37, wherein the anemia comprises renal anemia, multiple myeloma anemia and/or cancerous anemia.
The method for prolonging the half-life of erythropoiesis-stimulating protein, comprising the following steps: administering the protein according to any one of claims 33-35 and/or the pharmaceutical composition according to claim 36 to a subject in need.