A method for purifying esproglifenzoe alpha and use thereof

Abstract

The application provides a purification method of insulin peptide alpha and application thereof, and belongs to the technical field of biological medicine. The purification method provided by the application sequentially adopts affinity chromatography, hydrophobic chromatography and a composite chromatography column for purification. The column body of the composite chromatography column is sequentially laid with an anion exchange medium and a reversed-phase chromatography medium from top to bottom. Through optimization of screening and elution conditions of each chromatography medium, the content of HCP (less than 0.01%) and the components of charge isomers in the target protein can be effectively removed, and the residual amount of other related impurities (such as endotoxin, residual host cell DNA, Protein A protein shed by the affinity chromatography filler and the like) can also be effectively controlled within a safe range. The total yield of the target protein obtained through separation and purification is high and the target protein has good biological activity. The purification method has good repeatability and stability, can be used in large-scale purification process of insulin peptide alpha, and can be used for insulin peptide alpha drug production.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a purification method for esopragutin α and its application. Background Technology

[0002] Esupagglutide α is a human, long-acting glucagon-like peptide-1 (GLP-1) receptor agonist used as monotherapy for type 2 diabetes with poor glycemic control after diet and exercise interventions, and as a combination therapy for type 2 diabetes with poor glycemic control after metformin treatment. Its structure is a fusion protein of GLP-1 and IgG2-Fc. Specifically, esopagglutide α is produced by linking a human GLP-1 peptide to the Fc fragment of a human IgG2 antibody, forming a stable fusion protein structure. Currently, the genetically engineered production of esopagglutide α typically uses expression processes such as Saccharomyces cerevisiae or Escherichia coli. The resulting fermentation broth or cell culture medium containing the target product is extremely complex, containing not only esopagglutide α but also a large amount of host cell protein (HCP), host cell DNA, and esopagglutide α-related impurities, such as truncated forms and aggregates. To ensure the safety and efficacy of esopagglutide α in pharmaceutical applications, its downstream purification process is crucial. However, esupagglutide α, as a fusion polypeptide, is prone to chemical degradation and physical aggregation during purification. While existing multi-step chromatography processes can achieve a certain level of purity, they are cumbersome. For example, patent CN110526982A discloses a purification method for a human glucagon-like peptide-1 analog fusion protein. The sample first undergoes a crude purification step using Protein A affinity chromatography to effectively remove impurities such as HCP and endotoxins while maintaining a high yield of the target protein. Then, anion exchange chromatography and hydrophobic chromatography are used for further fine purification to effectively remove charge isomers, residual HCP, and other trace impurities from the sample, controlling the content of each impurity within the safe range for drug use, while maintaining a high yield and activity of the target protein. The method provided by this patent involves many intermediate processes, resulting in an excessively long purification time, significantly reducing production efficiency and increasing production costs.

[0003] Therefore, how to avoid intermolecular aggregation during the purification process, shorten the preparation process and time, reduce purification costs, reduce waste liquid discharge, improve product quality and yield, and facilitate industrial production are problems that enterprises and R&D personnel urgently need to solve. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a purification method for esopagraglutide α and its application. This method can effectively remove charge isomers, residual HCP, and other trace impurities from the harvested liquid obtained from the culture of stable expression cell lines obtained through induced expression screening, and control the content of each impurity within the safe range for drug use, while maintaining a high yield and activity of the target protein esopagraglutide α.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The primary objective of this invention is to provide a method for purifying esopagratide α:

[0007] S1. Clarification and filtration: The cell culture harvest medium containing esopagglutinin α is centrifuged and filtered through a filter membrane, and then the virus is inactivated.

[0008] S2. The clarified filtrate obtained from S1 with virus inactivation is subjected to affinity chromatography to obtain the first eluent.

[0009] S3. Perform hydrophobic chromatography on the first eluent obtained in S2 to obtain the second eluent;

[0010] S4. The second eluent obtained in S3 is subjected to composite chromatography column treatment to obtain high-purity target esopagraglutide α. The composite chromatography column is lined with anion exchange medium and reversed-phase chromatography medium from top to bottom. The mass ratio of the anion exchange medium to the reversed-phase chromatography medium is (1~2):1. The anion exchange medium is a polystyrene-divinylbenzene copolymer sphere with a particle size of 30~50μm. The reversed-phase chromatography medium is selected from either C4 or C8 with a particle size of 10~20μm.

[0011] Furthermore, the clarification filtration specifically employs a two-stage depth filtration membrane pack and a sterilization filter to clarify and filter the harvested liquid.

[0012] Furthermore, in step S2, the affinity chromatography uses MabSelect SuRe packing material, specifically including the following steps:

[0013] (1) Column equilibration: First wash the affinity chromatography column with 0.1 mol / L~0.15 mol / L sodium hydroxide solution, and then equilibrate with the first equilibration buffer; the first equilibration buffer is 40~50 mmol / L sodium acetate-acetic acid buffer, 100~150 mmol / L sodium chloride solution, and the pH is 7.30~7.50;

[0014] (2) Sample loading: Load the clarified filtrate into the equilibrated affinity chromatography column at a flow rate of 1-5 mL / min; the sample loading amount of the clarified filtrate is 14.5-26 mg / mL;

[0015] (3) Washing: Elution of unbound material with 4-5 column volumes of first wash solution; the first wash solution is 40-50 mmol / L sodium acetate-acetic acid buffer, 100-150 mmol / L sodium chloride solution, pH 4.95-5.15;

[0016] (4) Elution: The target protein was eluted with 2-3 column volumes of pH 3.65-3.80 40mM-50mM sodium acetate-acetic acid buffer and collected to obtain the first eluent.

[0017] Furthermore, in step S3, the hydrophobic chromatography uses Phenyl Sepharose 6 FastFlow as the packing material, specifically including the following steps:

[0018] (1) Column equilibration: First wash the hydrophobic chromatography column with 0.1 mol / L to 0.15 mol / L sodium hydroxide solution, and then equilibrate with the second equilibration buffer; the second equilibration buffer is 40 to 50 mmol / L Tris-HCl buffer with a pH of 7.90 to 8.10;

[0019] (2) Loading: Load the first eluent into the equilibrated affinity chromatography column, control the flow rate at 1~5 mL / min, control the conductivity of the loaded sample at 1~6 mS / cm; the loading amount of the first eluent is 25~160 mg / mL;

[0020] (3) Elution: Elute the target protein with 2-3 column volumes of the second equilibration solution and collect it to obtain the second eluent.

[0021] Furthermore, the particle size of the Phenyl Sepharose 6 Fast Flow is 45~165μm.

[0022] Further, in step S4, the purification process of the composite chromatography column includes the following steps:

[0023] (1) Column equilibration: The composite chromatography column is washed with mobile phase A and then equilibrated with mobile phase B;

[0024] (2) Loading: Load the second eluent into the balanced composite chromatography column at a flow rate of 1-5 mL / min; the loading amount of the second eluent is 5%-7% of the mass of the medium in the composite chromatography column.

[0025] (3) Elution: Wash and desalt with 3-4 column volumes of mobile phase B, and collect the eluent;

[0026] (4) Desalting: Elute with 1-2 column volumes of mobile phase C to remove residual salt ions in the composite double-layer chromatography column, and discard the eluent;

[0027] (5) Replace the second eluent in step (2) with the eluent obtained from step (3), and repeat steps (2) to (4) until the sample is completely desalted. Its conductivity should not be greater than 300 μs / cm.

[0028] The mobile phase A is a 20-30 mM ammonium formate aqueous solution.

[0029] The mobile phase B is prepared by dissolving 20 mM ammonium formate aqueous solution in acetonitrile:water at a volume ratio of 80:20, and adjusting the pH to 7.4-7.6 with ammonia water.

[0030] The mobile phase C is a 0.5~1M ammonium formate aqueous solution.

[0031] Furthermore, the anion exchange medium is NanoGel-50Q with a particle size of 50 μm.

[0032] Furthermore, the reversed-phase chromatographic medium is C8 with a particle size of 10-15 μm and a pore size of 300 Å.

[0033] Furthermore, the cell culture harvest medium for esopagglutide α is obtained by constructing a pKN012 prokaryotic expression vector based on the esopagglutide α expression sequence, transfecting it into CHO-K1 host cells, and then culturing a stable expression cell line obtained through induction expression screening.

[0034] A second objective of this invention is to provide a method for producing pharmaceutical-grade esopaglutide α product, comprising the purification method provided by this invention.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] (1) This invention provides a purification method for esopagratide α, which uses affinity chromatography, hydrophobic chromatography and a composite chromatography column for purification in sequence. The composite chromatography column is lined with anion exchange medium and reversed-phase chromatography medium from top to bottom. By screening each chromatography medium and optimizing the elution conditions, not only can HCP (content < 0.01%) and charge isomers in the target protein be effectively removed, but the residual amount of other related impurities (such as endotoxin, residual host cell DNA, Protein A protein shed from affinity chromatography packing material, etc.) can also be effectively controlled within a safe range. Moreover, the total yield of the purified target protein is high and it has good biological activity.

[0037] (2) The purification method for esopagraglutide α provided has good reproducibility and stability and can be used in the large-scale purification process of esopagraglutide α.

[0038] (3) The purification method of esopagglutide α provided by the present invention is conducive to the linear scale-up of the esopagglutide α purification process to adapt to large-scale production. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention are described in detail below. In these embodiments, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0040] In this invention, the amino acid sequence of esopagglutide α (code: GLP-1-IgG2-Fc) consists of three parts: human GLP-1 sequence, linker sequence, and Fc sequence of human immunoglobulin G2 subtype (IgG2).

[0041] The source of the cell culture harvest medium containing esopagglutide α in this invention is: the pKN012 prokaryotic expression vector constructed from the esopagglutide α expression sequence, transfected into CHO-K1 host cells, and the harvest medium obtained by culturing stable expression cell lines after induction expression screening.

[0042] The host cell used in the construction of engineered cell line A for the production of esupragglutide α was Chinese hamster ovary cell line CHO-K1. The initial cell line was purchased by Beijing Konosun Technology Co., Ltd. from the American Center for Type Culture Collection (ATCC, catalog number CCL-61, batch number 58995535). Konosun subjected the wild-type adherent CHO-K1 cells to serum-free and suspension acclimation culture to obtain CHO-K1 host cells that could be cultured in suspension using chemically defined media.

[0043] Expression vector construction: The synthesized target gene GLP-1-IgG2 was inserted into the stable expression vector pKN012 using genetic engineering methods to obtain pKN012-GLP-1-IgG2. Specifically, the amino acid sequence of esopagraglutide α was codon optimized and nucleotide sequence synthesized according to the expression system of Chinese hamster ovary cells (both completed by Genewiz Biotechnology Co., Ltd.), and inserted into the pKN009 transient expression vector through HindIII and EcoRI restriction sites to form the pKN009-GLP-1-IgG2 plasmid. The pKN009-GLP-1-IgG2 plasmid was double-digested with HindIII and EcoRI, and the GLP-1-IgG2-Fc fragment was recovered and ligated into the linearized stable expression vector pKN012 (obtained by recovering the pKN012 vector double-digested with HindIII and EcoRI). The vector was transformed into E. coli DH5α competent cells, plated on Amp medium plates, and single colonies were picked for amplification, plasmid extraction, enzyme digestion verification, and sequencing verification (sequencing by Genewiz Biotechnology Co., Ltd.) to obtain the stable expression vector pKN012-GLP-1-IgG2.

[0044] Monoclonal cell line screening: After linearizing the expression plasmid vector pKN012-GLP-1-IgG2, CHO-K1 host cells were transfected using electroporation transfection technology. Cell populations expressing and secreting GLP-1-IgG2 protein were obtained through pressure screening. The cell population was subjected to two rounds of limiting dilution for monoclonalization, and three high-expression preferred monoclonal cell lines were selected using U-shaped 96-well plates and photographic imaging to ensure single-cell representation. Cell growth, expression levels, bioactivity of the expressed products, and pharmacodynamics were evaluated on the three preferred monoclonal cell lines. Cell line stability assessment confirmed that clone A was the engineered cell line for esopagraglutide α.

[0045] Seed cell culture:

[0046] 1) Open the biosafety cabinet, turn on the ultraviolet light, and irradiate for more than 30 minutes.

[0047] 2) Preheating the culture medium: Turn on the water bath and check the water level. When the temperature rises to 37°C, put the complete culture medium into the water bath for preheating.

[0048] 3) Resuscitate seed cells; the density after resuscitation should be 1~5×10⁻⁶. 5 cells / mL, volume 20ml.

[0049] 4) Viability Recovery: After the seed cells are revived, passage them for two or more generations according to their post-revival state until the cells are in good condition, with a viability of 95% or higher, and meet the required number of cells for batch culture. If the seed cells are transferred from another source and are in the logarithmic growth phase, and meet the required number of cells for inoculation, this step can be omitted.

[0050] Batch culture inoculation:

[0051] According to the pre-set experimental protocol, the inoculation density was 0.5~1×10⁻⁶. 5 The inoculation density should be 30 ml (cells / ml or the inoculation density recommended by the culture medium manufacturer), with a viability of ≥95%. Inoculate into a 125 ml shake flask. After inoculation, place the shake flask in a cell culture shaker with shaking at 37.0℃, 120 rpm, and 8% CO2 concentration. The day of inoculation is designated as day 0 (D0). Taking HyClone's Actipro basal culture with CellBoost 7a / 7b as an example, according to their recommended culture protocol, add 3% / 0.3% of the inoculation volume of 7a / 7b on days 3, 5, 7, 9, 11, and 13. CellBoost 7b is a highly concentrated, highly alkaline, low-dosage supplement, added for more precise dosage and to extend its shelf life after preparation.

[0052] Sampling and counting: On the day of replenishment, take 500 μl of sample from each shake flask into an EP tube before replenishment. Part of the sample is used for cell counting (see the standard cell counting management procedure for details). The remaining sample (≥300 μl is recommended) is centrifuged and used for biochemical analysis. The test items are glucose and lactate. For D15, IGG is added as a test item.

[0053] End the culture and harvest when the cell viability is below 85% or D15.

[0054] The analytical method of this invention for esopaglutide α is as follows:

[0055] Peptide profiling: 100 µg of esopagglutide α sample was placed in a 1.5 mL centrifuge tube, mixed with an equal volume of RapiGest™, and denatured at 60 °C for 15 min. 10 µL of 500 mM dithiothreitol (DTT) was added to the sample, and the mixture was reacted at 60 °C for 1 h, then cooled to room temperature. 10 µL of 1 M iodoacetamide (IAM) was added to the sample, and the mixture was reacted at room temperature in the dark for 30 min. The sample was then removed, centrifuged at 21,000 g for 10 min, and 100 µL of 50 mM ammonium bicarbonate solution and 4 µg of trypsin were added. The mixture was reacted overnight at 37 °C, and then 1% formic acid (FA) was added to terminate the enzymatic digestion. The sample was centrifuged at 21,000 g for 10 min, and the filtrate was analyzed by LC-MS.

[0056] iCIEF analysis: 10 µL of esopaglutide α sample, 5 µL of marker solution with pI 7.9, 35 µL of 1% methylcellulose, 4 µL of AESlyte UH3-10 amphoteric electrolyte, and water to a final volume of 100 µL.

[0057] Instruments and reagents

[0058] Mass spectrometer: 6500 series Q-TOF (Agilent);

[0059] Chromatography instrument: 1290 Infinity II series liquid chromatography system (Agilent);

[0060] iCIEF Instrument: ProteinSimple Whole-Column Imaging Isoelectric Focusing Electrophoresis System

[0061] Column: C18 (2.1 × 150 mm, 1.7 µm)

[0062] MabPacRP (2.1× 50 mm, 4 µm)

[0063] Analysis parameters

[0064] 1) Liquid phase conditions

[0065] Mobile phase: A: 0.1% FA-water; B: 0.1% FA-acetonitrile; Analytical flow rate: 0.2 mL / min; Injection volume: 20 µL. Gradient: 0-3 min 4%B, 3-60 min 4%-27%B, 60-85 min 27%-40%B, 85-95 min 40%-90%B, 95-95.1 min 90%-4%B, 95.1-100 min 4%B.

[0066] 2) Mass spectrometry conditions

[0067] Detection mode: ESI+; Spray voltage: 3.8 kV; Capillary temperature: 320 °C; Evaporation temperature: 350 °C; Sheath gas: 35; Auxiliary gas: 10; Resolution: Level 1 60,000@m / z 200; Level 2 15,000@m / z 200; Precursor ion scan range: m / z 350-200; AGC: Level 1 300%; Level 2 100%; (n)CE: 30%.

[0068] 3) iCIEF conditions

[0069] 1 min 1000 V; 1 min 2000 V; 10 min 3000 V. Injection volume: 17 µL.

[0070] The detection of process-related residual substances and the determination of biological activity can be carried out by referring to the bioanalytical methods in the pharmacopoeia.

[0071] The affinity chromatography column used in this invention has the following dimensions: column volume 30 mL, column diameter 25 mm, and column length 105 mm.

[0072] The dimensions of the hydrophobic chromatography column are as follows: column volume 20 mL, column diameter 20 mm, column length 90 mm.

[0073] The composite chromatography column of this invention is prepared by filling it with 12, 20, or 30 mL SPE empty column tubes. The filling sequence is as follows: first, fill the empty column with reversed-phase medium, cover with a sieve plate, then fill with anion exchange medium, and cover with a sieve plate again. The dimensions of the 12 mL SPE column tube are: column diameter 18 mm, column length 80 mm; the dimensions of the 20 mL SPE column tube are: column diameter 20 mm, column length 90 mm; and the dimensions of the 30 mL SPE column tube are: column diameter 25 mm, column length 105 mm.

[0074] The purification method for esopragutin α provided by this invention is as follows:

[0075] S1. Clarification and filtration: The cell culture harvest medium containing esopagglutinin α is centrifuged and filtered through a filter membrane, and then the virus is inactivated.

[0076] S2. The clarified filtrate obtained from S1 with virus inactivation is subjected to affinity chromatography to obtain the first eluent; wherein the affinity chromatography uses MabSelect SuRe packing material, and specifically includes the following steps:

[0077] (1) Column equilibration: First wash the affinity chromatography column with 0.1 mol / L~0.15 mol / L sodium hydroxide solution, and then equilibrate with the first equilibration buffer; the first equilibration buffer is 40~50 mmol / L sodium acetate-acetic acid buffer, 100~150 mmol / L sodium chloride solution, and the pH is 7.30~7.50;

[0078] (2) Sample loading: Load the clarified filtrate into the equilibrated affinity chromatography column at a flow rate of 1-2 mL / min; the sample loading amount of the clarified filtrate is 14.5-26 mg / mL;

[0079] (3) Washing: Elution of unbound material with 4-5 column volumes of first wash solution; the first wash solution is 40-50 mmol / L sodium acetate-acetic acid buffer, 100-150 mmol / L sodium chloride solution, pH 4.95-5.15;

[0080] (4) Elution: The target protein was eluted with 2-3 column volumes of pH 3.65-3.80 40mM-50mM sodium acetate-acetic acid buffer and collected to obtain the first eluent.

[0081] S3. Perform hydrophobic chromatography on the first eluent obtained in S2 to obtain the second eluent. The hydrophobic chromatography uses Phenyl Sepharose 6 Fast Flow packing material and includes the following steps:

[0082] (1) Column equilibration: First wash the hydrophobic chromatography column with 0.1 mol / L to 0.15 mol / L sodium hydroxide solution, and then equilibrate with the second equilibration buffer; the second equilibration buffer is 40 to 50 mmol / L Tris-HCl buffer with a pH of 7.90 to 8.10;

[0083] (2) Loading: Load the first eluent into the equilibrated affinity chromatography column, control the flow rate at 1~5 mL / min, control the conductivity of the loaded sample at 1~6 mS / cm; the loading amount of the first eluent is 25~160 mg / mL;

[0084] (3) Elution: Elute the target protein with 2-3 column volumes of the second equilibration solution and collect it to obtain the second eluent.

[0085] S4. The second eluent obtained in S3 is subjected to composite chromatography column treatment to obtain high-purity target esopagraglutide α. The composite chromatography column is lined from top to bottom with anion exchange medium and reversed-phase chromatography medium, and the mass ratio of the anion exchange medium to the reversed-phase medium is (1~2):1. The anion exchange medium is a polystyrene-divinylbenzene copolymer sphere with a particle size of 30-50 μm. The reversed-phase chromatography medium is selected from either C4 or C8 and has a particle size of 10-20 μm. The purification process of the composite chromatography column includes the following steps:

[0086] (1) Column equilibration: The composite chromatography column is washed with mobile phase A and then equilibrated with mobile phase B;

[0087] (2) Sample loading: Load the second eluent into the balanced composite chromatography column at a flow rate of 1-5 mL / min; the amount of the second eluent loaded is 5%-7% of the mass of the medium in the composite chromatography column.

[0088] (3) Elution: Wash and desalt with 3-4 column volumes of mobile phase B, and collect the eluent;

[0089] (4) Desalting: Elute with 1-2 column volumes of mobile phase C to remove residual salt ions in the composite chromatography column, and discard the eluent;

[0090] (5) Replace the second eluent in step (2) with the eluent obtained from step (3), and repeat steps (2) to (4) until the sample is completely desalted. Its conductivity should not be greater than 300 μs / cm.

[0091] The mobile phase A is a 20-30 mM ammonium formate aqueous solution; the mobile phase B is prepared by dissolving a 20 mM ammonium formate aqueous solution in acetonitrile:water at a volume ratio of 80:20, adjusting the pH to 7.4-7.6 with ammonia; and the mobile phase C is a 0.5-1 M ammonium formate aqueous solution.

[0092] To investigate the characteristics of purifying esopagraglutide α using affinity chromatography and hydrophobic chromatography columns, this invention examined the effect of different hydrophobic chromatography column media on the enrichment of esopagraglutide α.

[0093] (1) To investigate the effect of different hydrophobic chromatography columns on the enrichment of esopaglutide α, the experimental and control groups were designed as follows:

[0094] Experimental group 1: The hydrophobic chromatography column medium was Octyl Sepharose 4Fast Flow (octyl agarose gel 4FF), with a particle size of 45-165 μm and a column volume of 20 mL;

[0095] Experimental group 2: The hydrophobic chromatography column medium was Butyl Sepharose 4 Fast Flow (butyl agarose gel), and the column volume was 20 mL;

[0096] Experimental group 3: The hydrophobic chromatography column medium was Phenyl Sepharose 6 Fast Flow (phenyl agarose gel 4FF), with a particle size of 45-165 μm and a column volume of 20 mL;

[0097] Experimental group 4: The hydrophobic chromatography column medium was Phenyl Sepharose 6 Fast Flow (phenyl agarose gel 4FF), with a particle size of 45-165 μm and a column volume of 12 mL;

[0098] Experimental group 5: The hydrophobic chromatography column medium was Phenyl Sepharose 6 Fast Flow (phenyl agarose gel 4FF), with a particle size of 45-165 μm and a column volume of 30 mL.

[0099] The results are shown in Table 1. The hydrophobic chromatography column medium selected was Phenyl Sepharose 6 Fast Flow (phenyl agarose gel 4FF), with a particle size of 45-165 μm and a column volume of 20 mL, which resulted in the lowest impurity content for enriching esopagraglutide α.

[0100] Table 1. Summary of test data.

[0101]

[0102] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0103] Example 1

[0104] 1. Sample pretreatment

[0105] The cell culture harvest medium containing esopaglutide α was clarified by two-stage deep filtration and sterile filtration. First, a 0.8-1 μm glass fiber membrane was used for pre-filtration to remove most of the particulate matter and turbidity. Then, a 0.45 μm or 0.22 μm polyethersulfone (PES) or polyvinylidene fluoride (PVDF) membrane was used for fine filtration to obtain a clear filtrate. Finally, a 0.22 μm sterile filtration membrane was used to filter the clarified liquid to remove microorganisms and obtain a clear filtrate.

[0106] Add 10% (w / w) of Triton X-100 S / D virus inactivation stock solution to the clarified filtrate at a ratio of clarified filtrate to S / D virus inactivation stock solution of 15.67:1 (w / w), bringing the final concentration of Triton X-100 to 0.6% (w / w). Stir at 100 rpm for at least 15 minutes, then incubate at 18°C–26°C for 150 minutes to inactivate the virus.

[0107] 2. Affinity chromatography

[0108] The clarified filtrate from S1, after virus inactivation, was subjected to affinity chromatography to obtain the first eluent. The affinity chromatography used MabSelect SuRe packing material and a column volume of 30 mL. The specific steps included:

[0109] (1) Column equilibration: First wash the affinity chromatography column with 0.15 mol / L sodium hydroxide solution, and then equilibrate with the first equilibration solution; the first equilibration solution is 40 mmol / L sodium acetate-acetic acid buffer, 100~150 mmol / L sodium chloride solution, pH 7.30~7.50;

[0110] (2) Loading: Load the clarified filtrate into the equilibrated affinity chromatography column at a flow rate of 1 mL / min; the loading amount of the clarified filtrate is 5 mg / mL based on the volume of the affinity chromatography column.

[0111] (3) Washing: Elution of unbound material with 4-5 column volumes of first wash solution; the first wash solution is 40 mmol / L sodium acetate-acetic acid buffer, 100 mmol / L sodium chloride solution, pH 5;

[0112] (4) Elution: Elute the target protein with 2-3 column volumes of pH 3.7 40mM sodium acetate-acetic acid buffer and collect the first eluent.

[0113] 3. Hydrophobic Chromatography

[0114] The first eluent obtained from S2 was subjected to hydrophobic chromatography to obtain the second eluent. The hydrophobic chromatography used Phenyl Sepharose 6 Fast Flow packing material with a column volume of 20 mL, and specifically included the following steps:

[0115] (1) Column equilibration: First wash the hydrophobic chromatography column with 1 mol / L sodium hydroxide solution, and then equilibrate with the second equilibration buffer; the second equilibration buffer is 50 mmol / L Tris-HCl buffer with pH 8;

[0116] (2) Loading: Load the first eluent into the hydrophobic chromatography column after equilibration, control the flow rate at 1 mL / min, and control the conductivity of the loaded sample at 1~6 mS / cm; the loading amount of the first eluent is 25 mg / mL; based on the volume of the hydrophobic chromatography column, the loading amount of the first eluent is 3 mg / mL.

[0117] (3) Elution: Elute the target protein with 2-3 column volumes of the second equilibration solution and collect it to obtain the second eluent.

[0118] 4. Purification using a composite chromatography column

[0119] An empty SPE column was packed with reversed-phase high-performance liquid chromatography (RP-HPLC) medium, and further packed with anion exchange medium: NanoGel-50Q, particle size 50 μm. Mobile phase A was prepared as a 20 mM ammonium formate aqueous solution, and mobile phase B was prepared as a 20 mM ammonium formate aqueous solution dissolved in acetonitrile:water at a volume ratio of 80:20. The pH was adjusted to 7.4-7.6 with ammonia. Mobile phase C was a 1 M ammonium formate aqueous solution. The flow rate was controlled at 1 mL / min, and the purification temperature was 20-24 °C. Column activation was performed using mobile phase A (1 column volume), and the column was equilibrated using mobile phase B (2 column volumes). The sample was loaded with a second eluent at a concentration of 5 mg / mL (column volume). Washing and desalting were performed using mobile phase B (4 column volumes), and the peptide toxin was collected by elution using mobile phase C (1 column volume). The flow rate of the mobile phase through the column was controlled by a solid-phase extraction instrument at 1 mL / min. The collected esopaglutide α was analyzed.

[0120] The SPE column has a volume of 20 mL, and the anion exchange medium is NanoGel 50Q 2 g; the C8 packing material is 2 g, with a particle size of 10 μm and a pore size of 300 Å.

[0121] Example 2

[0122] It is basically the same as Example 1, except that:

[0123] The SPE column has a volume of 20 mL, 2 g of anion exchange medium, and 1 g of C8 packing material.

[0124] Example 3

[0125] It is basically the same as Example 1, except that:

[0126] The SPE column has a volume of 20 mL, 1.5 g of anion exchange medium, and 1 g of C8 packing material.

[0127] Example 4

[0128] It is basically the same as Example 1, except that:

[0129] The SPE column has a volume of 12 mL, 2 g of anion exchange medium, and 2 g of C8 packing material.

[0130] Example 5

[0131] It is basically the same as Example 1, except that:

[0132] The SPE column has a volume of 12 mL, 2 g of anion exchange medium, and 1 g of C8 packing material.

[0133] Example 6

[0134] It is basically the same as Example 1, except that:

[0135] The SPE column has a volume of 12 mL, 1.5 g of anion exchange medium, and 1 g of C8 packing material.

[0136] Example 7

[0137] It is basically the same as Example 1, except that:

[0138] The SPE column has a volume of 30 mL, 2 g of anion exchange medium, and 2 g of C8 packing material.

[0139] Example 8

[0140] It is basically the same as Example 1, except that:

[0141] The SPE column has a volume of 30 mL, 2 g of anion exchange medium, and 1 g of C8 packing material.

[0142] Example 9

[0143] It is basically the same as Example 1, except that:

[0144] The SPE column has a volume of 30 mL, 1.5 g of anion exchange medium, and 1 g of C8 packing material.

[0145] Example 10

[0146] It is basically the same as Example 1, except that:

[0147] The SPE column has a volume of 20 mL, 1.5 g of anion exchange medium, and 1 g of C4 packing material.

[0148] Example 11

[0149] It is basically the same as Example 1, except that:

[0150] The SPE column has a volume of 20 mL, 2 g of anion exchange medium, and 1 g of C4 packing material.

[0151] Example 12

[0152] It is basically the same as Example 1, except that:

[0153] The SPE column has a volume of 20 mL, anion exchange medium of 2 g, and C4 packing material of 2 g.

[0154] Example 13

[0155] It is basically the same as Example 1, except that:

[0156] The SPE column has a volume of 20 mL, 3 g of anion exchange medium, and 2 g of C4 packing material.

[0157] Example 14

[0158] It is basically the same as Example 1, except that:

[0159] The pore size of C8 packing is 60 Å.

[0160] Example 15

[0161] It is basically the same as Example 1, except that:

[0162] The pore size of C8 packing is 100 Å.

[0163] The conductivity of the purified esopagraglutide α was tested. The conductivity values ​​of esopagraglutide α obtained in Examples 1-3 and 6-9 were 200-300 μS / cm, while the conductivity values ​​of esopagraglutide α obtained in Examples 4-5 and 10-15 were all greater than 300 μS / cm. The yield analysis results of Examples 1-15 are shown in Table 2, and the detection analysis results of esopagraglutide α are shown in Table 3. As can be seen from Tables 2 and 3, the esopagraglutide α obtained in Examples 1 and 2 had relatively high purity and yield.

[0164] Table 2. Summary of Yields

[0165]

[0166] Table 3. Summary of test results for each embodiment.

[0167]

[0168] For any points not covered above, existing technologies shall apply.

[0169] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for purifying Eptiozilatide alpha, characterized in that, Includes the following steps: S1. Clarification and filtration: The cell culture harvested from CHO-K1 cells expressing esopagglutinin α is centrifuged and filtered through a filter membrane, and then the virus is inactivated. S2. The clarified filtrate obtained from S1 with virus inactivation is subjected to affinity chromatography to obtain the first eluent; the affinity chromatography uses MabSelect SuRe packing material and specifically includes the following steps: (1) Column equilibration: First wash the affinity chromatography column with 0.1 mol / L~0.15 mol / L sodium hydroxide solution, and then equilibrate with the first equilibration buffer; the first equilibration buffer is 40~50 mmol / L sodium acetate-acetic acid buffer, 100~150 mmol / L sodium chloride solution, and the pH is 7.30~7.50; (2) Sample loading: Load the clarified filtrate into the equilibrated affinity chromatography column at a flow rate of 1-2 mL / min; the amount of the clarified filtrate loaded is 4-6 mg / mL based on the volume of the affinity chromatography column. (3) Washing: Elution of unbound material with 4-5 column volumes of first wash solution; the first wash solution is 40-50 mmol / L sodium acetate-acetic acid buffer, 100-150 mmol / L sodium chloride solution, pH 4.95-5.15; (4) Elution: Elute the target protein with 2-3 column volumes of pH 3.65-3.80 40mM-50mM sodium acetate-acetic acid buffer and collect the first eluent. S3. Perform hydrophobic chromatography on the first eluent obtained in S2 to obtain the second eluent; the hydrophobic chromatography uses Phenyl Sepharose 6 Fast Flow packing material and specifically includes the following steps: (1) Column equilibration: First wash the hydrophobic chromatography column with 1 mol / L~1.2 mol / L sodium hydroxide solution, and then equilibrate with the second equilibration buffer; the second equilibration buffer is 40~50 mmol / L Tris-HCl buffer with a pH of 7.90~8.10; (2) Loading: Load the first eluent into the hydrophobic chromatography column after equilibration, control the flow rate at 1~5 mL / min, and control the conductivity of the loaded sample at 1~6 mS / cm; based on the volume of the hydrophobic chromatography column, the loading amount of the first eluent is 3~8 mg / mL. (3) Elution: Elute the target protein with 2-3 column volumes of the second equilibration buffer and collect the eluent to obtain the second eluent; S4. The second eluent obtained in S3 is subjected to composite chromatography column treatment to obtain high-purity target esopagraglutide α. The composite chromatography column is lined with anion exchange medium and reversed-phase chromatography medium from top to bottom. The anion exchange medium is polystyrene-divinylbenzene copolymer spheres with a particle size of 50 μm. The reversed-phase chromatography medium is C8 with a particle size of 10 μm and a pore size of 300 Å. The volume of the composite chromatography column is 20 mL, the mass of the anion exchange medium is 2 g, and the mass of the reversed-phase chromatography medium is 2 g. Alternatively, the volume of the composite chromatography column is 20 mL, the mass of the anion exchange medium is 2 g, and the mass of the reversed-phase chromatography medium is 1 g. Alternatively, the volume of the composite chromatography column is 30 mL, the mass of the anion exchange medium is 1.5 g, and the mass of the reversed-phase chromatography medium is 1 g. The purification process of the composite chromatography column includes the following steps: (1) Column equilibration: The composite chromatography column is washed with mobile phase A and then equilibrated with mobile phase B; (2) Sample loading: Load the second eluent into the equilibrated composite chromatography column at a flow rate of 1-5 mL / min; the amount of the second eluent loaded is 5%-7% of the mass of the medium in the composite chromatography column; (3) Elution: Wash and desalt with 3-4 column volumes of mobile phase B, and collect the eluent; (4) Desalting: Elute with 1-2 column volumes of mobile phase C to remove residual salt ions in the composite chromatography column, and discard the eluent; (5) Replace the second eluent in step (2) with the eluent obtained from step (3), and repeat steps (2) to (4) until the sample is completely desalted. Its conductivity should not be greater than 300 μs / cm. The mobile phase A is a 20-30 mM ammonium formate aqueous solution. The mobile phase B is prepared by dissolving 20 mM ammonium formate aqueous solution in acetonitrile:water at a volume ratio of 80:20, and adjusting the pH to 7.4-7.6 with ammonia water. The mobile phase C is a 0.5~1M ammonium formate aqueous solution.

2. The purification method according to claim 1, characterized in that, The clarification filtration specifically employs a two-stage deep filtration membrane and a sterilization-grade membrane to clarify and filter the harvest liquid. The two-stage deep filtration membranes are a glass fiber membrane and an organic membrane, respectively.

3. The purification method according to claim 1, characterized in that, The particle size of the Phenyl Sepharose 6 Fast Flow is 45~165μm.

4. The purification method according to claim 1, characterized in that, The cell culture harvest medium for esopagglutide α is obtained by constructing a pKN012 prokaryotic expression vector based on the esopagglutide α expression sequence, transfecting it into CHO-K1 host cells, and then culturing stable expression cell lines obtained through induction expression screening.

5. A method for producing pharmaceutical-grade esopaglutide α product, characterized in that, The purification method comprising any one of claims 1-4.

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