Process for the preparation of erythropoietin-fc fusion proteins

Abstract

The application provides a purification process and a preparation method of an erythropoietin-Fc (EPO-Fc) fusion protein. The purification process effectively reduces the generation of aggregates in the purification process, improves the process stability, sample yield and purity, and reduces the process complexity and cost, and is suitable for industrial production of the EPO-Fc fusion protein.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to the purification and production process of erythropoietin-Fc fusion protein (EPO-Fc, particularly recombinant human EPO-Fc (rhEPO-Fc)). Background Technology

[0002] Human erythropoietin-Fc fusion protein (EPO-Fc) is a long-acting EPO drug that fuses the Fc region of human IgG with erythropoietin (EPO) via a linker sequence, thereby significantly prolonging the drug's half-life in serum, improving therapeutic efficacy, and reducing the frequency of dosing for patients. Sequences of such fusion proteins are disclosed in, for example, US patents US6900292B2, US7030226B2, and US7250493B2. These patents disclose methods for preparing EPO-Fc fusion proteins, which mainly include eukaryotic fermentation and downstream purification. During eukaryotic fermentation, EPO-Fc fusion proteins are produced by culturing engineered cells. Subsequently, in the downstream purification stage, the filtrate after fermentation is first pre-purified using affinity chromatography to remove most impurities; then, the sample is concentrated using ultrafiltration, and the buffer solution is replaced to form the final stock solution, which can be used for in vivo animal studies.

[0003] Although the above purification methods have achieved certain results in animal experiments, their application to human trials and commercial-scale production has revealed significant limitations: 1) Safety risks: Existing purification processes lack effective virus removal steps, which may lead to viral contamination in the final product, posing a potential safety risk to humans; 2) Insufficient purity: Due to limitations in purification technology, existing methods struggle to obtain high-purity EPO-Fc fusion protein samples; the presence of impurities may trigger immunogenic reactions in humans, reducing drug efficacy and increasing the risk of adverse reactions; 3) Low yield: During purification, a large amount of precipitation is easily generated, resulting in a low yield of the target protein EPO-Fc, which not only increases production costs but also limits the large-scale production of the product; 4) Challenges in industrial production: Existing purification processes are mainly suitable for small-scale experiments or animal studies, and their efficiency, stability, and economic viability are all significantly insufficient for large-scale commercial production.

[0004] Given the aforementioned limitations of existing technologies, there is an urgent need to develop a more stable, efficient, and industrially viable method for preparing EPO-Fc (especially rhEPO-Fc). Summary of the Invention

[0005] To address the aforementioned issues, this application provides a purification process and preparation method for EPO-Fc (especially rhEPO-Fc) fusion proteins, aiming to reduce the generation of aggregates during purification, improve process stability, increase sample yield and purity, and reduce process complexity and cost.

[0006] In one aspect, this application provides a method for purifying the EPO-Fc fusion protein, the method comprising:

[0007] 1) Affinity chromatography was performed on the fermentation broth;

[0008] 2) Incubation with acidic buffer solution to inactivate the virus; and

[0009] 3) Perform ion chromatography and hydrophobic chromatography on the incubated solution to obtain the purified product;

[0010] Optionally, hydroxyapatite chromatography may be performed before or after ion chromatography and / or hydrophobic chromatography.

[0011] In one embodiment, the affinity chromatography is Protein A chromatography.

[0012] In one embodiment, the Protein A chromatography is performed using, for example but not limited to, the following packing materials: Mabselect series packing materials, NMab series packing materials, UniMab series packing materials, UNOsphere SUPrA series packing materials, ProSepUltra Plus packing materials, Eshmuno A packing materials, Sepromax A series packing materials, etc. In a preferred embodiment, the Protein A chromatography is performed using Mabselect SURE packing materials or NMab Pro packing materials.

[0013] In one embodiment, the elution buffer used in affinity chromatography is an amino acid buffer with a pH of 2.5-3.5. The amino acid buffer includes, but is not limited to, glycine buffer, arginine buffer, proline buffer, histidine buffer, etc., such as a 20-100 mM glycine buffer with a pH of 2.5-3.5.

[0014] In one embodiment, the acidic buffer used to inactivate the virus is selected from amino acid buffers, such as glycine buffer, arginine buffer, proline buffer, histidine buffer, etc.

[0015] In one embodiment, the pH of the acidic buffer solution is 3.8-4.5.

[0016] Optionally, the pH of the sample is adjusted after incubation in an acidic buffer. Preferably, it is adjusted to a neutral pH, such as pH 6.5-7.5.

[0017] In one embodiment, the ion chromatography is anion chromatography or cation chromatography.

[0018] In one embodiment, the anion chromatography is performed using anion exchange packing material, which includes anion exchangers containing diethylaminoethyl, quaternary aminoethyl, quaternary ammonium, triethylaminoethyl, aminoethyl, etc., preferably anion exchangers containing quaternary ammonium.

[0019] In one embodiment, the anion exchanger may be a strong anion exchanger or a weak anion exchanger, preferably a strong anion exchanger.

[0020] In one embodiment, the cation chromatography is performed using a cation exchange packing material, which includes a cation exchanger containing sulfonic acid groups, carboxymethyl groups, sulfopropyl groups, sulfomethyl groups, phosphate groups, carboxyl groups, etc., preferably a cation exchanger containing sulfopropyl groups.

[0021] In one embodiment, the cation exchanger may be a strong cation exchanger or a weak cation exchanger, preferably a strong cation exchanger.

[0022] In one embodiment, the hydrophobic chromatography is performed using a hydrophobic chromatography packing material, which includes a hydrophobic chromatography medium containing phenyl, octyl, butyl, butyl sulfide, butyl nitrogen, polyethylene glycol, and polyether, preferably a hydrophobic chromatography medium containing butyl or phenyl (e.g., phenyl glycidyl ether).

[0023] In one embodiment, the ion chromatography and hydrophobic chromatography can be performed individually or using a combination of hydrophobic and ion chromatography.

[0024] In a specific embodiment, ion chromatography may be performed first, followed by hydrophobic chromatography.

[0025] In one embodiment, the ion chromatography and hydrophobic chromatography are a combination of anion chromatography and hydrophobic chromatography.

[0026] In one embodiment, the ion chromatography and hydrophobic chromatography are separate cation chromatography and hydrophobic chromatography processes. In one embodiment, the packing material for the cation chromatography comprises a sulfopropyl cation exchanger; the packing material for the hydrophobic chromatography comprises a butyl hydrophobic chromatography medium.

[0027] On the other hand, this application provides a method for preparing an EPO-Fc fusion protein, the method comprising:

[0028] 1) Fermenting the host cell;

[0029] 2) Perform affinity chromatography on the fermentation broth;

[0030] 3) Incubate with acidic buffer to inactivate the virus; and

[0031] 4) Perform ion chromatography and hydrophobic chromatography on the incubated solution to obtain the purified product;

[0032] Optionally, hydroxyapatite chromatography may be performed before or after ion chromatography and / or hydrophobic chromatography.

[0033] Beneficial effects:

[0034] This invention provides a purification process and preparation method suitable for EPO-Fc (especially rhEPO-Fc), enabling efficient purification of EPO-Fc with relatively higher purity and yield, and with simple operation, while also improving process stability. Furthermore, samples produced according to the process of this invention meet the requirements of the Chinese Pharmacopoeia and can be used to prepare pharmaceutical products for human use. Attached Figure Description

[0035] Figure 1 The results of purity determination of the samples in Example 1 and the comparative example of this application using size exclusion high-performance chromatography (SEC-HPLC) are shown. The top figure is an overall view, with the sample loading amounts for the two samples being substantially the same; the bottom figure is a magnified view. Blue represents the results of the samples obtained according to the purification process of this application, and red represents the results of the samples obtained according to the purification process used in the comparative example. It can be seen that the purification process according to the embodiments of this application reduces aggregates and degradation substances, thereby improving product quality.

[0036] Figure 2 The SDS-PAGE images of samples from an embodiment of the purification process according to Example 1 of this application are shown. Lane 1: Sample under non-reducing conditions; M: Molecular weight marker; Lane 2: Sample under reducing conditions.

[0037] Figure 3 The results of purity determination of samples in Example 2 and the comparative example using size exclusion high-performance chromatography (SEC-HPLC) are shown. Blue represents the results of samples obtained by the purification process of Example 2, and red represents the results of samples obtained by the comparative process. It can be seen that the purification process according to the embodiments of this application effectively reduces degradation substances while keeping the aggregates essentially unchanged, which is beneficial for maintaining effectiveness. Detailed Implementation

[0038] The following embodiments are given as exemplary embodiments of the present application and are not intended to limit the scope of the present application.

[0039] In one aspect, this application provides a method for purifying the EPO-Fc fusion protein, the method comprising:

[0040] 1) Affinity chromatography was performed on the fermentation broth;

[0041] 2) Incubation with acidic buffer solution to inactivate the virus; and

[0042] 3) Perform ion chromatography and hydrophobic chromatography on the incubated solution to obtain the purified product;

[0043] Optionally, hydroxyapatite chromatography may be performed before or after ion chromatography and / or hydrophobic chromatography.

[0044] In one embodiment, the fermentation broth is pretreated. For cases where the EPO-Fc fusion protein is secreted extracellularly, the fermentation broth can be filtered or centrifuged to remove intact cells and cell debris. Furthermore, dialysis, ultrafiltration, or other procedures can be performed to change the medium, ensuring the sample is in a suitable buffer environment and concentration range.

[0045] In some embodiments, the filtration includes hollow fiber filtration, depth filtration, ultrafiltration, etc., as long as it can achieve the purpose of removing intact cells and cell debris.

[0046] In one embodiment, the affinity chromatography is Protein A chromatography.

[0047] In one embodiment, the Protein A chromatography is performed using, for example but not limited to, the following packing materials: Mabselect series packing materials, NMab series packing materials, UniMab series packing materials, UNOsphere SUPrA series packing materials, ProSepUltra Plus packing materials, Eshmuno A packing materials, Sepromax A series packing materials, etc. In a preferred embodiment, the Protein A chromatography is performed using Mabselect SURE packing materials or NMab Pro packing materials.

[0048] The sample used for loading can be adjusted according to the affinity chromatography method used. For example, the sample used for affinity chromatography can be in a buffer solution with pH 6.0-8.0 containing 50-200 mM NaCl, such as a phosphate buffer solution with pH 6.0-8.0 containing 50-200 mM NaCl. In a preferred embodiment, the fermentation broth can be filtered and then directly loaded into the sample for affinity chromatography.

[0049] In a preferred embodiment, the affinity chromatography includes equilibration, loading, optional equilibration, optional elution, and elution; for example, it includes equilibration, loading, equilibration, first elution, second elution, and elution.

[0050] In one embodiment, the packing material is pre-rinsed with water (e.g., purified water, deionized water, such as water for injection) and NaOH aqueous solution in sequence.

[0051] In one embodiment, the solution is rinsed with water for 1-3 times, for example, 2 column volumes (CV). In one embodiment, the NaOH aqueous solution is a 0.05-0.5M, preferably 0.1-0.5M, NaOH aqueous solution, for example, a 0.1M NaOH aqueous solution; and the solution is rinsed with the NaOH aqueous solution for 1-5 column volumes, for example, 3 column volumes.

[0052] In one embodiment, the packing material is equilibrated to pH 7.0-8.5, such as pH 7.0-8.0, pH 7.5-8.5, or pH 7.5-8.0, before loading the sample.

[0053] In one embodiment, the equilibration is performed using an equilibration buffer, which can be any buffer capable of achieving the pH range described above.

[0054] For example, the equilibration buffer used for affinity chromatography can be a 10-50 mM phosphate buffer with pH 6.5-8.0 containing 100-200 mM NaCl.

[0055] In one embodiment, the sample loading amount for affinity chromatography does not exceed 70% of the dynamic loading capacity of the affinity packing material, preferably not exceeding 50% of the dynamic loading capacity of the affinity packing material.

[0056] In one embodiment, after loading, the sample is washed with the equilibration buffer described above until the UV detector returns to baseline. In another embodiment, the sample is washed with the equilibration buffer at a volume of 1-5 column.

[0057] In one embodiment, the UV detector is rinsed with a first rinsing buffer until it reaches a stable state; preferably, the first rinsing buffer is a 10-50 mM phosphate buffer with a pH of 6.5-8.0 (e.g., 7.5) containing 0.5-2 M NaCl (e.g., 1 M). In one embodiment, the UV detector is rinsed with the first rinsing buffer for 2-5 CV, for example, 3 CV.

[0058] In one embodiment, a second rinsing buffer is used to rinse until the conductivity stabilizes; preferably, the second rinsing buffer is a 10-50 mM phosphate buffer with a pH of 6.5-8.0 (e.g., 7.5). In one embodiment, the second rinsing buffer is used to rinse for 1-5 CV, for example, 3 CV.

[0059] In some implementations, the buffer used in the equilibration and rinsing process may also be any buffer capable of buffering within the above pH range, such as HEPES buffer, MOPS buffer, Tris buffer, citrate buffer, etc.

[0060] After rinsing until the conductivity stabilizes, elution is performed. In one embodiment, the eluent used in affinity chromatography is an amino acid buffer with a pH of 2.5-3.5; the amino acid buffer includes, but is not limited to, glycine buffer, arginine buffer, proline buffer, histidine buffer, etc., such as 20-200 mM glycine buffer (e.g., 50 mM, 100 mM, 150 mM glycine buffer) with a pH of 2.5-3.5.

[0061] In one embodiment, the column retention time of the sample in affinity chromatography is at least 6 minutes, for example, 6 to 60 minutes, preferably 20 to 30 minutes. Those skilled in the art can calculate the required flow rate for chromatography based on the column retention time of the sample.

[0062] In one implementation, after affinity chromatography, the virus is inactivated by adding an acidic buffer to the affinity chromatography collection solution and incubating.

[0063] In one embodiment, the incubation for inactivating the virus is carried out at pH 3.2-4.0, preferably 3.6-3.7.

[0064] In one embodiment, the acidic buffer used to inactivate the virus is selected from amino acid buffers, such as glycine buffer, arginine buffer, proline buffer, histidine buffer, etc.

[0065] In one embodiment, the acidic buffer solution used for virus inactivation has a pH of 3.5-4.5, preferably 3.6-4.5, 3.7-4.5, 3.6-4.0, or 3.7-4.0. For example, the acidic buffer solution used for virus inactivation may be a 20-200 mM glycine buffer or arginine buffer (e.g., 50 mM, 100 mM, 150 mM glycine buffer or arginine buffer) with a pH of 3.5-4.5.

[0066] In one embodiment, the acidic buffer incubation for virus inactivation is carried out at 18-26°C for 45 to 240 minutes, preferably 60 to 70 minutes. Optionally, after incubation in the acidic buffer, the pH of the sample is adjusted. The specific adjusting reagent and pH range are determined based on the subsequent chromatography. For example, the pH of the virus-inactivated sample can be adjusted to pH 6.5-8.5, preferably 6.8-8.0, for example, using an alkaline solution with a pH of 9.0-11.0. For example, it can be adjusted using a Tris alkaline solution (e.g., 1-3 mol / L) with a pH of 9.0-11.0.

[0067] After inactivating the virus, ion chromatography, hydrophobic chromatography, or a composite chromatography column can be used to perform both ion chromatography and hydrophobic chromatography simultaneously.

[0068] In one embodiment, the ion chromatography may be anion chromatography or cation chromatography.

[0069] In one embodiment, the anion chromatography is performed using anion exchange packing material, which includes anion exchangers containing diethylaminoethyl, quaternary aminoethyl, quaternary ammonium, triethylaminoethyl, aminoethyl, etc., preferably anion exchangers containing quaternary ammonium.

[0070] In one embodiment, the anion exchanger may be a strong anion exchanger or a weak anion exchanger, preferably a strong anion exchanger.

[0071] In one embodiment, the cation chromatography is performed using a cation exchange packing material, which includes a cation exchanger containing sulfonic acid groups, carboxymethyl groups, sulfopropyl groups, sulfomethyl groups, phosphate groups, carboxyl groups, etc., preferably a cation exchanger containing sulfopropyl groups.

[0072] In one embodiment, the cation exchanger may be a strong cation exchanger or a weak cation exchanger, preferably a strong cation exchanger.

[0073] In one embodiment, the hydrophobic chromatography is performed using a hydrophobic chromatography packing material, which includes a hydrophobic chromatography medium containing phenyl, octyl, butyl, butyl sulfide, butyl nitrogen, polyethylene glycol, and polyether, preferably a hydrophobic chromatography medium containing butyl or phenyl (e.g., phenyl glycidyl ether).

[0074] In one embodiment, the ion chromatography and hydrophobic chromatography can be performed individually or using a combination of hydrophobic and ion chromatography.

[0075] In a specific embodiment, ion chromatography may be performed first, followed by hydrophobic chromatography.

[0076] In one embodiment, the ion chromatography and hydrophobic chromatography are a composite chromatography of anion chromatography and hydrophobic chromatography. In one embodiment, the packing material for the composite chromatography comprises a chromatography medium containing quaternary ammonium groups and phenyl groups. In a preferred embodiment, the composite chromatography packing material is Diamond MIX-A packing material.

[0077] In a preferred embodiment, the composite chromatography includes equilibration, sample loading, and elution.

[0078] In one embodiment, in complex chromatography, the packing material is equilibrated with an equilibration buffer until the baseline is stable before loading; the equilibration buffer may be a 10-50 mM phosphate buffer with pH 8.5-9.5.

[0079] In the use of multiplex chromatography, the sample loading capacity is preferably less than 20 mg / mL. In one embodiment, the multiplex chromatography can be eluted with an eluent, said eluent being a 20-80 mM phosphate buffer with pH 6.0-7.0 containing 0.2-0.6 M NaCl.

[0080] Alternatively, in some embodiments, the ion chromatography and hydrophobic chromatography are performed separately as cation chromatography and hydrophobic chromatography. In one embodiment, the packing material for the cation chromatography comprises a sulfopropyl cation exchanger; the packing material for the hydrophobic chromatography comprises a butyl hydrophobic chromatography medium. In a preferred embodiment, the cation chromatography is performed using SP-HP packing material; the hydrophobic chromatography is performed using Butyl HP packing material.

[0081] In a preferred embodiment, the cation chromatography includes equilibration, sample loading, optional equilibration, and elution; for example, the composite chromatography includes equilibration, sample loading, equilibration, and elution.

[0082] In one embodiment, the packing material for cation chromatography may be pre-washed by: rinsing the packing material for cation chromatography with 0.5-2M NaOH aqueous solution (e.g., 1M NaOH aqueous solution) for 2-5 CV (e.g., 3 CV), rinsing with water (e.g., purified water, deionized water, water for injection) for 3-8 CV (e.g., 5 CV), and then rinsing with a neutralization buffer until the conductivity is stable, for example, the neutralization buffer is a 100-200 mM Tris buffer with pH 7.5-9.5 (e.g., pH 8.0) containing 0.5-2M (e.g., 1M) NaCl.

[0083] In one embodiment, during cation chromatography, the sample is washed with an equilibration buffer before loading until the pH and conductivity baselines are stable; for example, the equilibration buffer in the cation chromatography may be a 10-100 mM Tris buffer (e.g., 50 mM Tris buffer) with pH 7.5-9.5 (e.g., pH 8.0). In one embodiment, the sample is washed with the equilibration buffer for 1-5 CV (e.g., 3 CV) during cation chromatography.

[0084] In one embodiment, the loading amount of the cation chromatography is less than 70% of the theoretical loading of the packing material.

[0085] In one embodiment, after loading the sample for cation chromatography, the sample is washed with the aforementioned equilibration buffer for cation chromatography until the baseline is stable, and then elution is performed.

[0086] The eluent for cation chromatography can be a 10-100 mM Tris buffer containing 0.1-1 M NaCl (e.g., 0.5 M NaCl) at pH 7.5-9.0, preferably using linear elution. For example, elution can be performed in cation chromatography using a linear elution method with 0% to 100% eluent.

[0087] In one embodiment, the column retention time of the sample in cation chromatography is >3 min, for example, from 3 min to 60 min. Those skilled in the art can calculate the required flow rate for chromatography based on the column retention time of the sample.

[0088] Preferably, hydrophobic chromatography can be performed after cation chromatography. In one embodiment, NaCl is added to the sample eluted by cation chromatography until a final concentration of 0.5-1.5 M NaCl (e.g., 1.0 M NaCl) is reached. The sample used for hydrophobic chromatography can be filtered to remove any possible particles, for example, by filtering through a 0.2 μm filter membrane.

[0089] In a preferred embodiment, the hydrophobic chromatography includes equilibration, sample loading, optional equilibration, and elution; for example, the hydrophobic chromatography includes equilibration, sample loading, equilibration, and elution.

[0090] In hydrophobic chromatography, the hydrophobic chromatography packing material is equilibrated with an equilibration buffer until the baseline is stable before loading the sample; for example, the equilibration buffer may be a buffer of pH 7.5-9.5 and 0.5-1.5M NaCl, such as 10-100mM Tris buffer.

[0091] In one embodiment, the sample loading volume for hydrophobic chromatography is less than 70% of the theoretical loading capacity of the packing material. Preferably, in one embodiment, after hydrophobic chromatography sample loading, the chromatography column can be washed with the aforementioned equilibration buffer for hydrophobic chromatography until the UV detector baseline stabilizes, and then elution is performed.

[0092] The eluent for hydrophobic chromatography can be 10-50 mM Tris buffer at pH 7.5-9.0, and linear elution is preferred.

[0093] For example, in hydrophobic chromatography, elution is performed using a linear elution method with 0% to 100% eluent.

[0094] In one embodiment, the column retention time of the sample in hydrophobic chromatography is >3 min, for example, from 3 min to 60 min. Those skilled in the art can calculate the required flow rate for chromatography based on the column retention time of the sample.

[0095] In some embodiments, the neutralization buffer, equilibration buffer, and eluent used in ion chromatography, hydrophobic chromatography, and complex chromatography may also be any buffer capable of buffering within the above pH range, such as glycine-sodium hydroxide buffer, phosphate buffer, Tris buffer, etc.

[0096] Optionally, hydroxyapatite chromatography may be performed before or after ion chromatography and / or hydrophobic chromatography to reduce aggregate content.

[0097] For hydroxyapatite chromatography, commercially available hydroxyapatite chromatography packing materials, such as CHTII type packing materials, can be used; hydroxyapatite chromatography may include equilibration, sample loading, optional equilibration, and elution. In one embodiment, the hydroxyapatite packing material is equilibrated by rinsing with an equilibration buffer before sample loading in hydroxyapatite chromatography until the baseline is stable; for example, the equilibration buffer is a 10-50 mM Tris buffer with a pH of 6.5-7.0.

[0098] In one embodiment, the loading amount of the hydroxyapatite chromatography is less than 20 mg / mL.

[0099] In one embodiment, after loading the hydroxyapatite sample into the hydroxyapatite chromatography solution, the sample is washed with the equilibration solution described above for hydroxyapatite chromatography until the baseline is stable, and then elution is performed.

[0100] In one embodiment, the eluent for hydroxyapatite chromatography is a 5-50 mM phosphate buffer with pH 6.0-7.0 and 200-500 mM NaCl.

[0101] Preferably, the hydroxyapatite chromatography packing is pre-washed with NaOH aqueous solution (e.g., 0.2-0.7M NaOH aqueous solution) and phosphate buffer (e.g., 0.1M-1M phosphate buffer) at pH 6.0-7.0 in sequence; for example, washing with NaOH aqueous solution for 0.5-2 hours, and then washing with 0.5M phosphate buffer for 1-5 CV (e.g., 3 CV).

[0102] In some embodiments, hydroxyapatite chromatography may be performed first, followed by composite chromatography; in this case, it is preferable to load the eluent from hydroxyapatite chromatography directly as a sample into the composite chromatography column.

[0103] In some embodiments, the equilibration buffer and elution buffer used in hydroxyapatite chromatography may also be any buffer capable of buffering within the above pH range, such as glycine-sodium hydroxide buffer, phosphate buffer, Tris buffer, etc.

[0104] Optionally, the sample may be filtered prior to all chromatographic operations in this application to remove any possible particles, for example, by filtering through a 0.2 μm filter membrane.

[0105] In some specific implementations, samples after ion chromatography, hydrophobic chromatography, or hydroxyapatite chromatography are subjected to devirulent filtration and / or sterile filtration, as well as concentration.

[0106] For example, a 20nm filter membrane can be used for virus removal; then an ultrafiltration membrane (e.g., a 30kDa ultrafiltration membrane) can be used for concentration.

[0107] In a specific implementation, the purification method according to this application includes:

[0108] 1) Affinity chromatography was performed on the fermentation broth;

[0109] 2) Incubate with acidic buffer solution to inactivate the virus;

[0110] 3) Perform hydroxyapatite chromatography on the incubated solution;

[0111] 4) The solution after hydroxyapatite chromatography was subjected to combined anion exchange chromatography and hydrophobic chromatography.

[0112] 5) Optionally, the solution after composite chromatography is filtered and concentrated.

[0113] In yet another specific embodiment, the purification method according to this application includes:

[0114] 1) Affinity chromatography was performed on the fermentation broth;

[0115] 2) Incubate with acidic buffer solution to inactivate the virus;

[0116] 3) Perform combined anion chromatography and hydrophobic chromatography on the incubated solution;

[0117] 4) Perform hydroxyapatite chromatography on the solution after composite chromatography;

[0118] 5) Optionally, the solution after hydroxyapatite chromatography is filtered and concentrated.

[0119] In yet another specific embodiment, the purification method according to this application includes:

[0120] 1) Affinity chromatography was performed on the fermentation broth;

[0121] 2) Incubate with acidic buffer solution to inactivate the virus;

[0122] 3) Perform cation exchange chromatography on the incubated solution; and

[0123] 4) Perform hydrophobic chromatography on the solution after cation chromatography.

[0124] 5) Optionally, the solution after hydrophobic chromatography is filtered and concentrated.

[0125] In existing purification methods, low-pH incubation with inorganic salt buffers is commonly used, which results in significant precipitation and protein loss during incubation. Furthermore, compared to the purification method of this invention, existing methods often employ size exclusion chromatography, which is less efficient and difficult to scale up to industrial levels for large-scale production. Additionally, existing purification methods require strict cleaning of size exclusion chromatography packing materials; any residual impurities can affect the purity of subsequent batches, leading to poor batch-to-batch stability and unstable purity data.

[0126] On the other hand, this application provides a method for preparing an EPO-Fc fusion protein, the method comprising:

[0127] 1) Fermentation of host cells expressing the EPO-Fc fusion protein;

[0128] 2) Perform affinity chromatography on the fermentation broth;

[0129] 3) Incubate with acidic buffer to inactivate the virus; and

[0130] 4) Perform ion chromatography and hydrophobic chromatography on the incubated solution to obtain the purified product;

[0131] Optionally, hydroxyapatite chromatography may be performed before or after ion chromatography and / or hydrophobic chromatography.

[0132] In this application, "EPO-Fc fusion protein" refers to a protein containing an Fc region and an erythropoietin polypeptide. In some embodiments, the Fc region is preferably derived from a human Fc region, such as from human IgG.

[0133] In a preferred embodiment of the invention, the EPO-Fc fusion protein forms a homodimer and therefore typically has one or more disulfide bonds.

[0134] The binding mode between the Fc region and the EPO peptide is not particularly limited as long as the two are functionally linked. As one embodiment, the EPO peptide is directly linked to the Fc region via a covalent bond. As another embodiment, the EPO peptide is indirectly linked to the Fc region. For example, in an EPO-Fc fusion protein, a linker may be included between the Fc region and the erythropoietin peptide.

[0135] In this invention, the "Fc region" contains the structural domain of the constant region of IgG derived from human IgG, which includes fragments and variants of the constant region.

[0136] The constant region of an immunoglobulin is defined as the same natural or synthetically produced polypeptide as the C-terminal region of the immunoglobulin, which may contain, alone or in any combination, a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain. The Fc region of the heavy chain consists of the CH2 and CH3 domains. The hinge region lies between the CH1 and CH2 domains.

[0137] In this invention, the Fc region of the IgG is preferably an Fc region variant in which amino acid changes have been introduced into the Fc region of wild-type IgG. More preferably, it is an Fc region variant in which the FcRn binding activity (hereinafter also referred to as FcRn binding activity) under acidic conditions is higher than the FcRn binding activity of the parent peptide before the change, and which contains at least one amino acid change.

[0138] Examples of changes to amino acids in the Fc region include substitution, insertion, and deletion of amino acids, with substitution being preferred. There is no particular limitation on the number of amino acids changed; it can be that only one amino acid is changed, or that two or more amino acids are changed. It is preferred that two or more amino acids are changed, more preferably about two to five.

[0139] In addition to binding to the EPO region, the polypeptide containing the Fc region of IgG used in this invention may also undergo modifications such as: alterations to enhance ADCC (antibody-dependent cytotoxicity) or CDC (complement-dependent cytotoxicity) activity; alterations to increase protease resistance; alterations to reduce effector function; alterations to reduce complement binding activity; alterations to increase antibody heterogeneity or stability; alterations to promote antigen disappearance; alterations to repeatedly bind to antigens of multiple molecules; alterations to reduce the pI of the constant region for the purpose of increasing blood retention; and alterations to maintain the binding capacity to other antigens. More specifically, refer to the Fc engineering techniques described in Current Pharmaceutical Biotechnology, 2016, 17, 1298-1314. These modifications may include, for example, any one of amino acid substitution, deletion, addition, insertion, modification, or a combination thereof, preferably amino acid substitution.

[0140] The Fc region variant used in this invention has FcRn binding activity, particularly showing high FcRn binding activity compared to the activity of the unmodified Fc region.

[0141] In this invention, the EPO polypeptide, in addition to containing wild-type or natural erythropoietin or recombinant erythropoietin from any animal species, preferably mammals, more preferably humans, and especially preferably humans, also contains erythropoietin-like molecules, which contain biologically active erythropoietin fragments or variants of erythropoietin.

[0142] The erythropoietin of the present invention is understood to specifically comprise an erythropoietin polypeptide having an amino acid sequence similar to that of wild-type erythropoietin. For example, one or more amino acid alterations may be included in the amino acid sequence of wild-type erythropoietin, provided that its biological or functional activity is maintained. Examples of such amino acid alterations include the addition, deletion, or substitution of amino acid residues.

[0143] Methods for introducing mutations into erythropoietin are well-known in this field. For example, mutations can be introduced using site-directed mutagenesis techniques. A wide range of site-specific mutagenesis techniques are also available.

[0144] The EPO-Fc fusion protein of the present invention may include a linker molecule, preferably a peptide linker, between the Fc moiety and the erythropoietin moiety. Fusion proteins with linkers may possess improved properties such as increased biological activity. Linkers typically contain 1-25 amino acids (e.g., 5-25 or 10-20 amino acids).

[0145] In a specific embodiment, the EPO-Fc fusion protein of the present invention has the amino acid sequence shown in SEQ ID NO: 1, or a sequence having 80% sequence identity with it.

[0146] In this application, there are no particular limitations on the host cells that can be used for the synthesis of the EPO-Fc fusion protein; for example, *E. coli* or various animal cells may be used. Host cells can be used, for example, as a production system for manufacturing or expressing the EPO-Fc fusion protein of the present invention. Production systems include in vitro and in vivo production systems. Examples of in vitro production systems include production systems using eukaryotic cells.

[0147] Eukaryotic cells that can be used as host cells include, for example, animal cells, plant cells, and fungal cells. Examples of animal cells include: mammalian cells such as CHO (J.Exp.Med.(1995)108:94.0), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, etc.; amphibian cells such as Xenopus oocytes (Valle et al., Nature(1981)291:338-340); and insect cells such as Sf9, Sf21, and Tn5. CHO-DG44, CHO-DX11B, COS7, HEK293, and BHK are preferred. CHO is particularly preferred when high-level expression is desired.

[0148] In some embodiments, the host cell contains a vector expressing the EPO-Fc fusion protein. Any suitable expression vector known in the art can be used to construct the expression vector with the polynucleotide encoding the aforementioned EPO-Fc fusion protein. Expression vectors suitable for eukaryotic cells, particularly mammalian cells, can be, for example, pcDNA series vectors (pcDNA3.1 vector, pcDNA3.2 vector, pcDNA3.3 vector, pcDNA3.4 vector (e.g., pcDNA3.4-TOPO TA vector)), pBK-CMV vector, pEGFP-N1 vector, etc., but are not limited to these. The vector can be introduced into the host cell using methods known to those skilled in the art, such as the calcium phosphate method, the DEAE dextran method, methods using cationic liposome DOTAP (manufactured by Boehringer Mannheim), electroporation, lipid transfection, microinjection, etc.

[0149] Depending on the host cell, appropriate fermentation methods can be used to produce EPO-Fc fusion protein through host cell fermentation.

[0150] In this application, CHO cells are preferably used as the host cells.

[0151] In some embodiments, serum-free culture media can be used to culture the host cells. In some embodiments, chemically defined culture media can be used to culture the host cells. For example, commercially available culture media for CHO cells include EX-CELL CD-CHO serum-free medium, Dynamis medium, CHOGrow 302 serum-free medium, etc.

[0152] The fermentation can be carried out by batch culture, fed-batch culture, or continuous culture. Those skilled in the art can add feed (including, for example, carbon sources, amino acids, vitamins, inorganic salts, etc.) to the above-mentioned culture medium during the fermentation process to meet different fermentation requirements.

[0153] In some embodiments, the fermentation can be carried out on a scale of 100 mL to 2000 L, for example, the production scale of a 30 L to 2000 L eukaryotic fermenter.

[0154] Those skilled in the art can adjust the specific parameters of the fermentation culture (e.g., fermentation temperature, pH value, fermentation time) according to actual production conditions. In an exemplary embodiment, the fermentation temperature is 33℃-37℃, preferably 35℃-37℃; the pH value is 6.4-7.4; and the fermentation time is 7-13 days, preferably 9-11 days.

[0155] The methods for pretreating the fermentation broth after fermentation and purifying the fusion protein are as defined in the foregoing.

[0156] The product obtained according to the purification and preparation processes described in this application can be used to prepare EPO-Fc stock solution for injection.

[0157] In some embodiments, the concentration of the EPO-Fc stock solution is 0.5-10 mg / mL. Preferably, the pH of the EPO-Fc stock solution is 5.5-7.5, and the buffer solution can be selected from: amino acid salt buffer, acetate buffer, citrate buffer, phosphate buffer, Tris buffer. In addition, the buffer EPO-Fc stock solution also contains: reagents for adjusting osmotic pressure, such as 10-200 mM NaCl (10-200 mM), arginine salt, etc.; surfactants, such as 0.001%-0.1% Tween 20, Tween 80, P188, etc. For cases containing high concentrations of EPO-Fc, suitable stabilizers may also be included, such as 2-15% sucrose, mannitol, trehalose, sorbitol, etc. The stock solution is stored at -20°C to -80°C.

[0158] In this application, the purification process can be used for large-scale commercial production, such as in 30L-2000L eukaryotic fermenters. For example, the stock solution produced by processing a sample from a 650L eukaryotic fermenter using the purification method of this invention can be used to produce approximately one million human products annually.

[0159] definition

[0160] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in cases of conflict, this specification, including the definitions, shall be binding. Therefore, in the context of this invention, the following definitions apply:

[0161] In this specification, "EPO-Fc fusion protein" refers to a protein comprising an EPO moiety and an Fc moiety. The "EPO moiety" as used herein includes full-length wild-type or naturally occurring erythropoietin from human or other sources, as well as erythropoietin-like molecules, including erythropoietin bioactive fragments, erythropoietin analogs, variants, mutants, and derivatives.

[0162] The term "Fc portion" as used herein includes domains derived from the constant region of immunoglobulins (preferably human immunoglobulins), including fragments, analogs, variants, mutants, or derivatives of the constant region. Suitable immunoglobulins include IgG, i.e., subclasses such as IgG1, IgG2, IgG3, and IgG4, as well as other classes.

[0163] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers containing amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are modified residues or non-naturally occurring residues, such as artificial chemical mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino polymers.

[0164] The term "isolated" is used herein to refer to molecules that are separated from their natural environment (i.e., in a non-natural form). Similarly, the term "purified" is used herein to refer to molecules that are substantially free of contaminants.

[0165] As used herein, a "buffer solution" refers to a buffer solution that resists pH changes through the action of its acid-base conjugate components. The buffer solution of the present invention has a pH in the range of about 3.0 to about 8.0; preferably about 4.0 to about 7.8. Examples of buffers that control the pH within this range include acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), phosphates (e.g., sodium phosphate), histidine, citrates, and other organic salt buffers.

[0166] The word “comprise” or “include” and its English variants such as comprises or comprising should be understood in an open, non-exclusive sense, meaning “including but not limited to”.

[0167] For purposes of description and disclosure, all patents, patent applications and other identified publications are expressly incorporated herein by reference. Any reference to these publications herein does not constitute an endorsement that such publication is part of the general knowledge in the art.

[0168] As used herein, the term "monomer" refers to an EPO-Fc fusion protein that is a homodimer formed by disulfide bonds. For example, due to varying levels of glycosylation modification, the molecular weight of EPO-Fc fusion protein monomers can range from 89 kDa to 120 kDa.

[0169] As used herein, the term "aggregate" refers to a polymer formed by the binding of at least two EPO-Fc monomers. Aggregates can be detected by SEC-HPLC or non-reducing SDS-PAGE. Aggregates may form during CHO expression or in unsuitable buffers. Consistency of aggregates in SEC-HPLC means consistency in both quantity and location compared to a comparative sample. Consistency in both location and quantity is considered harmless if quality release criteria are met.

[0170] As used herein, the term "degradation fragment" refers to a substance formed when a portion of the sequence of the intact EPO-Fc molecule is broken due to external forces. These external forces may occur during production, storage, and under inappropriate external conditions, including oxidation, hydrolysis, enzymatic hydrolysis, high temperature, and light exposure. These degradation fragments are typically smaller than the intact EPO-Fc molecule and may affect its biological activity and stability. For example, in this application, the "degradation fragment" may include a polypeptide fragment with a molecular weight smaller than that of the corresponding monomer. In a preferred embodiment of the invention, the EPO-Fc has a molecular weight between 89 kDa and 120 kDa due to glycosylation modification.

[0171] As used in this article, room temperature (RT) generally refers to the temperature of an indoor environment under standard atmospheric pressure. For example, room temperature can be 18-26°C.

[0172] As used in this article, the term "deep filtration" is a filtration technology based on the principle of membrane separation. It uses a porous membrane as the filter medium, and by applying pressure, a mixture is forced through the porous membrane, thereby achieving the separation of particles of different sizes. During deep filtration, cells and large particles are trapped on or inside the membrane surface, while the solution and fusion proteins flow out through the membrane pores, thus achieving separation.

[0173] As used herein, the term "affinity chromatography" refers to a liquid chromatography method that relies on the reversible, site-specific binding of proteins and their ligands, such as hormones and their receptors, enzymes and their substrates, antibodies and antigens, etc. This specific interaction can be utilized by immobilizing one phase of the interacting substances, called the affinity ligand, onto a column as the stationary phase for affinity chromatography. Through the selective adsorption of the affinity ligand, the target protein elutes from the complex fraction. The complex fraction containing the target protein is loaded onto a chromatography column with a suitable buffer system and pH, allowing the target protein to bind to the affinity ligand while other fractions flow through directly. The target protein can then be eluted from the column, the elution method depending on the nature of the interaction between the target protein and the ligand. Affinity chromatography allows for the selection of various types of binding agents (ligands) and solid-phase supports (matrix). By optimizing different properties such as specificity, selectivity, reproducibility, cross-linking chemistry, and cost-effectiveness, affinity chromatography can be used for large-scale antibody purification to obtain the desired yield and purity.

[0174] As used herein, the term "conductivity" refers to the ability of an aqueous solution to allow an electric current to flow between two electrodes. In a solution, current flows via ion transport. Therefore, increasing the amount of ions present in an aqueous solution results in a higher conductivity. Conductivity is the ability of ions in a solution to carry electric current; therefore, the conductivity of a solution can be altered by changing the ion concentration.

[0175] As used herein, the term "anion exchange chromatography" refers to chromatography using a column packed with anion exchange resin. Anion exchange resin is a synthetic resin added to other aqueous solutions to exchange specific anions in the solution with its own anions. Anion exchange columns can adsorb anion-containing proteins above their isoelectric point. Antibodies have a high isoelectric point; therefore, when using a neutral pH buffer, antibodies do not adhere to the anion exchange resin and overflow. However, impurities containing host cell proteins have a low isoelectric point and can be adsorbed onto the anion exchange resin and removed. Thus, the purification step can be performed using the principles described above.

[0176] As used herein, the term "cation exchange chromatography" refers to chromatography using a column packed with a cation exchange resin. Impurities, such as host cell proteins and isomeric antibodies, can be removed by performing cation exchange chromatography. A cation exchange resin is a synthetic resin that functions to exchange cations in an aqueous solution with its own cations. In the case of antibodies, it has a high isoelectric point and therefore carries cations in pH buffers below its isoelectric point. Therefore, a cation exchange resin capable of adsorbing antibodies carrying these cations is used.

[0177] As used herein, the term "hydrophobic chromatography" refers to a chromatographic technique that separates proteins based on differences in their surface hydrophobicity. It utilizes the interaction between proteins and hydrophobic groups at high salt concentrations to achieve separation. At high salt concentrations, the hydrophobic groups of the protein are exposed and interact with hydrophobic ligands on the stationary phase, thereby achieving protein separation and purification. The strength of this interaction depends on the protein's hydrophobicity and the salt concentration. High salt concentrations enhance the interaction between the protein and the hydrophobic ligands, while low salt concentrations weaken this interaction. Therefore, protein binding and elution can be controlled by adjusting the salt concentration. Typically, decreasing the salt concentration or increasing the polarity of the buffer solution is used to elute proteins.

[0178] As used herein, the isoelectric point (pI) is the average effective charge on the surface of a protein molecule, that is, the pH at which the potential of the electric double layer of a protein molecule becomes 0, meaning that the protein's functional groups dissociate, making the number of cationic and anionic groups equal, thus resulting in an effective charge of 0. Therefore, the fusion proteins used for purification in this invention are not limited to this, and fusion proteins with isoelectric points of 6 to 11, optionally 7 to 10, can be used.

[0179] Example

[0180] For clarity, the invention is further illustrated by examples, but these examples are not intended to limit the scope of this application. All reagents used in this application are commercially available and can be used without further purification.

[0181] The CHO cells used in the following examples to produce the rhEPO-Fc fusion protein with the amino acid sequence shown in SEQ ID NO: 1 were prepared according to the method described in the example of US7250493B2. In short, a vector containing nucleic acid encoding EPO-Fc was transfected into CHO host cells, stable expression cell lines were obtained through screening, and a cell bank was established and stored in liquid nitrogen or a gaseous liquid nitrogen tank. Before performing the following examples, the cells were removed from liquid nitrogen, revived in a 37°C water bath, and expanded.

[0182] In the following examples, the purity of proteins (including target proteins, degradation fragments, and aggregates) was determined using SEC-HPLC. Specific parameters were as follows: size-exclusion column (TOSOH, 7.8 × 300 mm, 5 μm); mobile phase (20 mM PB (pH 7.5) + 100 mM NaCl); HPLC instrument equipped with VWD / DAD (Waters Corporation).

[0183] The chromatographic conditions are as follows:

[0184] Chromatographic column: TOSOH, 7.8×300mm, 5μm;

[0185] Detection wavelength: 280nm; Detector type: VWD;

[0186] Flow rate: 0.7 mL / min; Column temperature: 25℃;

[0187] Injection volume: 20 μL; Collection (elution) time: 24 min.

[0188] Example 1: Isolation and purification of rhEPO-Fc

[0189] CHO cell lines expressing the rhEPO-Fc fusion protein, after resuscitation and passage, were fermented in a 3L fermenter for 8-13 days. After fermentation, deep filtration was used to remove cells and other substances, and the fermentation broth was retained. Protein content was determined after deep filtration using a small Protein A chromatography filter under conditions similar to those used in subsequent affinity chromatography. All samples after affinity chromatography were then analyzed using a UV detector at 280 nm.

[0190] The affinity chromatography packing material used is Mabselect SURE (Cytiva, catalog number 17543805), the hydroxyapatite chromatography packing material used is CHT II type packing material (Bio-Rad, catalog number 157-8500), and the hydrophobic and anionic composite chromatography packing material used is DiamondMix-A (hydrophobic and anionic composite packing material; Borglon, catalog number A10105).

[0191] The specific steps are as follows:

[0192] Affinity chromatography: The column diameter is 26 mm and the column height is 20 cm. The affinity packing material is rinsed with 2CV of water for injection, followed by 3CV of 0.1 M NaOH aqueous solution. Then, the 3CV is rinsed with equilibration buffer (20 mM phosphate buffer, 150 mM NaCl, pH 7.5) at a low flow rate until the pH decreases to 7.5-8.5. The sample loading volume should not exceed 70% of the dynamic loading capacity of the affinity packing material. The column retention time for the sample (pH 6.0-8.0, phosphate buffer containing 50-200 mM NaCl) should be at least 6 min. After sample loading, the 3CV is rinsed with equilibration buffer until the UV detector detection value at 280 nm returns to baseline. The 3CV is then rinsed with a first elution buffer (20 mM phosphate buffer, 1 M NaCl, pH 7.5) until the UV reaches a stable state. Finally, the 3CV is rinsed with a second elution buffer (20 mM phosphate buffer, pH 7.5) until the conductivity stabilizes. Elution was performed using elution buffer (100 mM glycine buffer, pH 3.2). Sample collection began when the UV value exceeded 100 mAU and ceased when it dropped to 100 mAU. The sample concentration was determined using a UV detector at 280 nm.

[0193] Acidic buffer incubation: Adjust the pH of the affinity chromatography collection buffer to 3.7 with 100 mM glycine buffer (pH 3.7) and incubate for 120 min at room temperature (18–26 °C). Adjust the pH of the virus-inactivated sample to 7.0 with 2 mol / L Tris alkaline solution (pH 9.0–10.5).

[0194] Hydroxyapatite chromatography: Wash with 0.5M NaOH at a low flow rate for 1 h, wash with 0.5M phosphate buffer (pH 6.5) for 3 CVs, and wash with equilibration buffer (20 mM Tris buffer, pH 6.5) until the baseline is stable. The sample loading should be less than 20 mg / mL. After loading, wash with equilibration buffer until the baseline is stable. Elute with elution buffer (10 mM phosphate buffer, 300 mM NaCl, pH 6.5) until the UV detector value at 280 nm is below 150 mAU.

[0195] Hydrophobic and anionic complex chromatography: Equilibrate the Mix-A column with equilibration buffer (10 mM phosphate buffer, pH 9.0) until the baseline is stable, load the sample at a concentration of less than 30 mg / mL, and elute with elution buffer (50 mM phosphate buffer, 0.5 M NaCl, pH 6.5) until the UV detector value is below 150 mAU.

[0196] Virus removal filtration and concentration: Virus removal filtration was performed using a 20nm filter membrane, followed by concentration to 5mg / mL using a 30kDa ultrafiltration membrane. The buffer solution was then replaced with the original buffer solution (10mM phosphate buffer, 90mM NaCl, pH 6.5).

[0197] After filtration through a 0.2μm filter membrane, it is stored at -80℃.

[0198] Purity was determined using SEC-HPLC, and the purity was >95%. Additionally, endotoxin levels were <2.5 EU / mL, 10 μm insoluble particles <6000 / mL, 25 μm insoluble particles <600 / mL, total glycosidic bond content >15%, sialic acid content >7.5%, and activity assays met quality standards.

[0199] In this embodiment, purification was performed in the same manner except that the order of CHT and Mix-A chromatography was interchanged, and similar results could be obtained.

[0200] The purification process described above (the specific purification steps are consistent with the above examples, except that the column diameter is increased to 600 mm while the column height remains unchanged, specifically including affinity chromatography, low pH incubation, CHT chromatography, and hydrophobic and anion complex chromatography, virus removal nanofiltration, ultrafiltration, and sterile filtration) was scaled up using a 650 L fermenter. CHO cell lines expressing the rhEPO-Fc fusion protein were cultured at 36±1 °C for 9 days. The purification method according to the present invention, for the separation and purification of samples at commercial production scale, yielded similar purification results as on small-scale samples. Purity was determined using SEC-HPLC, and the purity was >95%. Furthermore, endotoxin was <2.5 EU / mL, 10 μm insoluble particles <6000 / mL, 25 μm insoluble particles <600 / mL, total glycosidic bond content >15%, sialic acid content >7.5%, and activity assays met quality standards.

[0201] In the purification scheme described above according to this application, the overall protein yield is based on...

[0202] Calculations show that the yields in the small-scale and large-scale purification processes of this embodiment are similar, both achieving an overall protein yield of 30-50%.

[0203] In addition, a long-term stability study of the purified products from small-scale samples and commercial-scale samples was conducted at 2-8°C for 12 months. Specific parameters and results are shown in Table 1 below.

[0204] Table 1

[0205]

[0206] Table 1 shows the composition of each component at different scales, as well as the results of the 12-month accelerated stability study. The results indicate that the sample purity exhibits a similar trend in the accelerated stability study, validating the successful scale-up of the process to commercial production.

[0207] Example 2. Preparation of rhEPO-Fc

[0208] The revived and passaged CHO cell lines expressing the rhEPO-Fc fusion protein were fermented in a 3L fermenter for 8-13 days. After fermentation, deep filtration was used to remove cells and other substances, and the clear fermentation broth was retained. The protein content was quantified using the same method as in Example 1.

[0209] The affinity chromatography packing materials used are NMab Pro (Suzhou Nanomicro Technology), cation SP-HP (Cytiva, catalog number 17115101), and hydrophobic packing material Butyl HP (Cytiva, catalog number 17543204).

[0210] Affinity chromatography: The chromatography column is 26 mm in diameter and 20 cm in height. The affinity packing material is rinsed with 2 CV of water for injection, followed by 3 CV of 0.1 M NaOH aqueous solution. Then, the 3 CV is rinsed with equilibration buffer (20 mM phosphate buffer, 150 mM NaCl, pH 7.5) at a low flow rate until the pH drops to 7.0-8.0. The sample loading volume should not exceed 70% of the dynamic loading capacity of the affinity packing material, and the sample retention time on the column should be at least 6 min. After loading, the 3 CV is rinsed with equilibration buffer until the UV detector's detection value at 280 nm returns to baseline. The 3 CV is then rinsed with a first elution buffer (20 mM phosphate buffer, 1 M NaCl, pH 7.5) until the UV reaches a stable state. Finally, the 3 CV is rinsed with a second elution buffer (20 mM phosphate buffer, pH 7.5) until the conductivity stabilizes. Elution was performed using elution buffer (50 mM arginine buffer, pH 3.2). Sample collection began when the UV value exceeded 100 mAU and ceased when it dropped to 100 mAU. Protein content in the sample was determined using a UV detector at 280 nm.

[0211] Acidic buffer incubation: Adjust the pH of the affinity chromatography collection buffer to 3.7 with 50 mM arginine buffer (pH 4) and incubate for 120 min at room temperature (18–26 °C). Adjust the pH of the virus-inactivated sample to 8.0 with 2 mol / L Tris alkaline solution (pH 9.0–10.5).

[0212] For cation chromatography, wash 3 CVs with 1M NaOH, 5 CVs with purified water, and then wash with neutralization buffer (150 mM Tris buffer, 1 M NaCl, pH 8.0) until conductivity stabilizes. Wash 3 CVs with equilibration buffer (50 mM Tris buffer, pH 8.0) until the pH and conductivity baselines are stable. Load the sample at less than 70% of the theoretical packing capacity, ensuring a sample retention time >3 min on the column. After loading, wash with equilibration buffer until the UV280 baseline is stable. Elute linearly with elution buffer (50 mM Tris buffer, 0.5 M NaCl, pH 8.0), eluting from 0% to 100% eluent over 60 min until the main peak is below 100 mAU at UV280. Quantify proteins at 280 nm using a UV detector.

[0213] After hydrophobic and cation exchange chromatography, NaCl was added to the sample to a final concentration of 1M NaCl. The sample was then filtered through a 0.2μm filter membrane to remove insoluble particles. The Butyl HP column was equilibrated with equilibration buffer (50mM Tris buffer, 1M NaCl, pH 8.0) until the baseline stabilized. The sample loading was less than 70% of the theoretical loading capacity of the packing material. The column was washed with equilibration buffer until the UV baseline stabilized. Linear elution was then performed with elution buffer (20mM Tris buffer, pH 8.0), taking 60 min to elute from 0% to 100%. Collection was stopped when the main peak was below 150 mAU at UV280.

[0214] Virus removal filtration and concentration: Virus removal filtration was performed using a 20 nm nanofiltration membrane, followed by concentration to 5 mg / mL using a 30 kDa ultrafiltration membrane. The buffer solution was then replaced with the original buffer solution (10 mM citrate buffer, 90 mM NaCl, pH 7.0).

[0215] After filtration using a 0.2–0.45 μm filter membrane, store at -80 degrees Celsius.

[0216] Purity was determined using SEC-HPLC. The prepared material yielded a purity >95%, endotoxin <2.5 EU / mL, 10 μm insoluble particles <6000 / mL, and 25 μm insoluble particles <600 / mL. Total glycosidic bond content >15%, sialic acid content >7.5%, and activity assays met quality standards.

[0217] According to Example 2, the overall protein yield of different batches was between 20% and 40%.

[0218] Comparative Example 1. Preparation of rhEPO-Fc

[0219] The revived and passaged CHO cell lines expressing the rhEPO-Fc fusion protein were fermented in a 3L fermenter for 8-13 days. After fermentation, deep filtration was used to remove cells and other substances, and the clear fermentation broth was retained. The protein content was quantified using the same method as in Example 1.

[0220] The affinity chromatography packing materials used are Mabselect SURE (Cytiva, catalog number 17543805), anion Q-FF (Cytiva, catalog number 17051004), and size exclusion chromatography S300 (Cytiva, catalog number 17059905).

[0221] Affinity chromatography: The column diameter is 26 mm and the column height is 20 cm. The affinity packing material is rinsed with 2CV of water for injection, followed by 3CV of 0.1 M NaOH aqueous solution. Then, the 3CV is rinsed with equilibration buffer (20 mM phosphate buffer, 150 mM NaCl, pH 7.5) at a low flow rate until the pH decreases to 7.5-8.5. The sample loading volume should not exceed 70% of the dynamic loading capacity of the affinity packing material, and the sample retention time on the column should be at least 6 min. After loading, the 3CV is rinsed with equilibration buffer until the UV detector's detection value at 280 nm returns to baseline. The 3CV is then rinsed with eluent (20 mM phosphate buffer, 1 M NaCl, pH 7.5) until the UV reaches a plateau, and then with eluent (20 mM phosphate buffer, pH 7.5) until the conductivity stabilizes. Elution is performed using eluent (50 mM citrate, pH 3.2). Receiving begins when the UV value exceeds 100 mAU and stops when it drops below 100 mAU. The sample content was determined by detecting the sample at 280 nm using an ultraviolet detector.

[0222] Incubation in acidic buffer: Adjust the pH of the affinity chromatography collection buffer to 3.7 using 150 mM citrate buffer (pH 4.5). During pH adjustment with inorganic salts, an increase in target protein aggregates may occur, even leading to protein precipitation and loss. Incubate at room temperature (18–26 °C) for 120 min. Adjust the pH of the virus-inactivated sample to 7.0 using 2 mol / L Tris alkaline solution (pH 9.0–10.5). Remove insoluble particles using a 0.2 μm filter membrane. Quantify the protein using a UV detector at 280 nm.

[0223] Anion exchange chromatography was performed using a 26 mm diameter column and a 15 cm height. The column was washed with 1 M NaOH for 3 CV washes, followed by 1 M NaCl washes for 3 CV washes. The column was then equilibrated with anion exchange buffer (20 mM Tris, pH 7.0) for 5 CV washes to ensure the sample pH was consistent with the buffer. The sample loading volume was greater than the theoretical loading capacity of the packing material but less than 100 g sample / L packing material. The retention time on the column was >3 min, and the flow-through sample was collected.

[0224] Sample concentration: Concentrate to 15 mg / mL using a 50 kDa ultrafiltration membrane. Replace the buffer with size exclusion chromatography equilibration buffer: 20 mM histidine buffer, 150 mM NaCl, pH 7.0. Filter through a 0.2 μm filter membrane to remove insoluble particles.

[0225] Size exclusion chromatography: The column diameter is 26 mm and the column height is 40 cm. Wash with 0.5 M NaOH for 5 CVs, wash with 1 M NaCl for 3 CVs, and wash with equilibration buffer (20 mM histidine buffer, 150 mM NaCl, pH 7.0) for 5 CVs. Confirm that pH, conductivity, and UV280 baseline are stable. Load the sample at 5% CV for 60 minutes. Start collecting the main peak when UV280 is greater than 100 mAU; stop collecting when it is below 100 mAU.

[0226] Virus removal filtration and concentration: Virus removal filtration was performed using a 20nm nanofiltration membrane, followed by concentration to 5mg / mL using a 30kDa ultrafiltration membrane.

[0227] After filtration through a 0.2-0.45μm filter membrane, store at -80℃.

[0228] Purity was determined using SEC-HPLC. The prepared product had a purity >90%, endotoxin <2.5 EU / mL, 10 μm insoluble particles <6000 / mL, 25 μm insoluble particles <600 / mL, total glycosidic bond content >15%, sialic acid content >7.5%, and activity assays met quality standards.

[0229] The overall protein yield of Comparative Example 1 was 10-30%.

[0230] rhEPO-Fc amino acid sequence (SEQ ID NO: 1)

[0231]

Claims

1. A method for purifying an EPO-Fc fusion protein, the method comprising: 1) Affinity chromatography was performed on the fermentation broth; 2) Incubate with acidic buffer solution to inactivate the virus; as well as 3) Perform ion chromatography and hydrophobic chromatography on the incubated solution to obtain the purified product; Optionally, hydroxyapatite chromatography may be performed before or after ion chromatography and / or hydrophobic chromatography.

2. The purification method as described in claim 1, wherein, The affinity chromatography employs one or more of the following: The affinity chromatography is Protein A chromatography; preferably, the Protein A chromatography is performed using Mabselect SURE packing or NMab Pro packing. The sample used for affinity chromatography is in a buffer solution with pH 6.0-8.0 containing 50-200 mM NaCl; The affinity chromatography includes equilibration, sample loading, optional equilibration, optional elution, and elution; preferably, it includes equilibration, sample loading, equilibration, first elution, second elution, and elution. The packing material was equilibrated to pH 7.0-8.5 before loading the sample; The sample loading volume for the affinity chromatography shall not exceed 70% of the dynamic loading capacity of the affinity packing material; or The elution buffer used in affinity chromatography is an amino acid buffer with a pH of 2.5-3.5; the amino acid buffer is preferably selected from any one of glycine buffer, arginine buffer, proline buffer, and histidine buffer, and is preferably a 20-200 mM glycine buffer with a pH of 2.5-3.

5.

3. The purification method as described in claim 1 or 2, wherein, Following affinity chromatography, the virus is inactivated with acidic buffer using one or more of the following methods: Incubation for virus inactivation is carried out at pH 3.3-3.9, preferably 3.6-3.7; The acidic buffer used for virus inactivation is selected from one or more of the following: amino acid buffers, such as glycine buffer, arginine buffer, proline buffer, histidine buffer; preferably, the pH of the acidic buffer used for virus inactivation is 3.5-4.5, more preferably 3.6-4.5, 3.7-4.5, 3.6-4.0, or 3.7-4.0; preferably, the acidic buffer used for virus inactivation is a 20-200 mM glycine buffer or arginine buffer with a pH of 3.5-4.5; or The acidic buffer solution used to inactivate the virus is incubated at 18-26°C for 45 min to 240 min, preferably 60 min to 70 min. Optionally, after incubation in an acidic buffer solution, the pH of the sample is adjusted to a pH of 6.5-8.5, preferably 6.8-8.0, preferably using an alkaline solution with a pH of 9.0-11.0, and more preferably using a Tris alkaline solution with a pH of 9.0-11.

0.

4. The purification method according to any one of claims 1-3, wherein, After virus inactivation, ion chromatography, hydrophobic chromatography, or simultaneous ion chromatography and hydrophobic chromatography using a composite chromatography column are performed, employing one or more of the following methods: The ion chromatography is either anion chromatography or cation chromatography; The anion chromatography is performed using anion exchange packing material, which preferably includes anion exchangers containing diethylaminoethyl, quaternary aminoethyl, quaternary ammonium, triethylaminoethyl, and aminoethyl, and more preferably anion exchangers containing quaternary ammonium. The anion exchanger is a strong anion exchanger or a weak anion exchanger, preferably a strong anion exchanger; The cation chromatography is performed using a cation exchange packing material, which preferably includes a cation exchanger containing sulfonic acid group, carboxymethyl group, sulfopropyl group, sulfomethyl group, phosphate group, and carboxyl group, and more preferably a cation exchanger containing sulfopropyl group; The cation exchanger is a strong cation exchanger or a weak cation exchanger; or The hydrophobic chromatography is performed using hydrophobic chromatography packing material, which preferably includes a hydrophobic chromatography medium containing phenyl, octyl, butyl, butyl sulfide, butyl nitrogen, polyethylene glycol, and polyether.

5. The purification method as described in claim 4, wherein, The ion chromatography and hydrophobic chromatography are a combination of anion chromatography and hydrophobic chromatography; Preferably, the packing material for the composite chromatography includes a chromatography medium containing quaternary ammonium groups and phenyl groups; Preferably, the composite chromatography includes equilibration, sample loading, and elution; Preferably, the packing material is equilibrated with equilibration buffer until the baseline is stable before loading the sample; Preferably, the composite chromatography is eluted with an eluent, wherein the eluent is a 20-80 mM phosphate buffer with pH 6.0-7.0 containing 0.2-0.6 M NaCl.

6. The purification method according to claim 4, wherein, The ion chromatography and hydrophobic chromatography are separate cation chromatography and hydrophobic chromatography processes. Preferably, the packing material for the cation chromatography comprises a cation exchanger containing sulfopropyl groups, and the packing material for the hydrophobic chromatography comprises a hydrophobic chromatography medium containing butyl groups. Preferably, the cation chromatography includes equilibration, sample loading, optional equilibration, and elution; more preferably, the composite chromatography includes equilibration, sample loading, equilibration, and elution. Preferably, in cation chromatography, the sample is washed with equilibration buffer before loading until the pH and conductivity baselines are stable. Preferably, the equilibration buffer in the cation chromatography can be a 10-100 mM Tris buffer with a pH of 7.5-9.5; Preferably, the eluent for cation chromatography is a 10-100 mM Tris buffer containing 0.1-1 M NaCl at pH 7.5-9.0, and linear elution is preferably used; preferably, elution is performed in cation chromatography using a linear elution method with 0% to 100% eluent. Alternatively, the hydrophobic chromatography may include equilibration, sample loading, optional equilibration, and elution; preferably, the hydrophobic chromatography may include equilibration, sample loading, equilibration, and elution. Preferably, in hydrophobic chromatography, the hydrophobic chromatography packing material is equilibrated with a equilibration buffer until the baseline is stable before loading the sample; preferably, the equilibration buffer is a buffer with pH 7.5-9.5 and 0.5-1.5M NaCl, more preferably 10-100mM Tris buffer; Preferably, after loading the hydrophobic chromatography sample, the chromatography column is washed with the above-mentioned equilibration buffer for hydrophobic chromatography until the UV detector baseline is stable, and then elution is performed; Preferably, the eluent for hydrophobic chromatography is a 10-50 mM Tris buffer with a pH of 7.5-9.0, and linear elution is preferably used; preferably, elution is performed in hydrophobic chromatography using a linear elution method with 0% to 100% eluent. Optionally, hydrophobic chromatography is performed after cation chromatography; preferably, NaCl is added to the sample after cation chromatography elution until the final concentration reaches 0.5-1.5M NaCl; preferably, the above-mentioned sample used for hydrophobic chromatography is filtered to remove possible particles, preferably through a 0.2μm filter membrane.

7. The purification method according to any one of claims 1-6, wherein, The hydroxyapatite chromatography includes equilibration, loading, optional equilibration, and elution. Preferably, the hydroxyapatite packing is rinsed with equilibration buffer before loading into hydroxyapatite chromatography until the baseline is stable; preferably, the equilibration buffer used for hydroxyapatite chromatography is a 10-50 mM Tris buffer with pH 6.5-7.

0. Preferably, the eluent for hydroxyapatite chromatography is a 5-50 mM phosphate buffer with pH 6.0-7.0 and 200-500 mM NaCl; Optionally, hydroxyapatite chromatography is performed first, followed by composite chromatography; preferably, the eluent from hydroxyapatite chromatography is directly loaded as a sample into the composite chromatography column.

8. The purification method according to any one of claims 1-7, wherein, Samples after ion chromatography, hydrophobic chromatography, or hydroxyapatite chromatography are subjected to virus-free filtration and / or sterile filtration, as well as concentration. Preferably, a 20nm filter membrane is used for virus removal filtration; then an ultrafiltration membrane is used for concentration.

9. A method for preparing an EPO-Fc fusion protein, the method comprising: 1) Fermentation of host cells expressing the EPO-Fc fusion protein; 2) Perform affinity chromatography on the fermentation broth; 3) Incubate with acidic buffer to inactivate the virus; and 4) Perform ion chromatography and hydrophobic chromatography on the incubated solution to obtain the purified product; Optionally, hydroxyapatite chromatography may be performed before or after ion chromatography and / or hydrophobic chromatography.

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