A method for purifying acidic Fc fusion protein

Abstract

The present invention discloses a method for purifying an acidic Fc fusion protein, which relates to the technical field of acidic Fc fusion protein purification. Affinity chromatography, anion exchange chromatography and hydrophobic chromatography are used to wash and elute CHO cell culture supernatants containing an acidic Fc fusion protein; the elution is isocratic elution, and the pH difference between the equilibrium buffer and the elution buffer is not less than 1 pH unit. Compared with conventional buffer conditions, the purification method provided by the present invention can improve the purity of the target protein to about 99% under the premise of ensuring that process-related impurities meet the requirements.

Description

Technical Field

[0001] The present invention relates to the technical field of acidic Fc fusion protein purification, and more specifically to a method for purifying acidic Fc fusion protein. Background Art

[0002] Varicella-zoster virus (VZV) is a member of the alpha herpesvirus family of the Herpesviridae family. The virus is mainly composed of four parts: the center is a rod-shaped virus core, which is mainly double-stranded viral DNA; the outside of the core is a nucleocapsid, with a diameter of about 100nm, which is composed of 162 capsomeres to form an icosahedral symmetrical structure; the outside of the nucleocapsid is covered with an amorphous substance called the cortex, which contains proteins and enzymes; the outermost layer is the capsule, which is a typical lipid bilayer membrane structure with many protrusions, and the capsule is mainly composed of lipids, sugars and proteins.

[0003] At present, more than 30 VZV late gene products are known, including at least 8 glycoproteins: gE, gB, gH, gI, gC, gL, gK and gM. These glycoproteins are mainly present in the viral envelope and the cell membrane of infected cells, and are closely related to the pathogenicity and immunogenicity of the virus. Among them, gE (also known as gpI) has a molecular weight of 60~98kDa and is the most abundant and antigenic glycoprotein on the VZV envelope and infected cell membrane. It has a typical type I transmembrane protein structure, including a 544aa hydrophilic extracellular region, a 17aa hydrophobic transmembrane region and a 62aa charge-rich intracellular region, containing N-glycosylation and O-glycosylation, Cys-rich region, sialylation site and Ser / Thr tyrosine kinase II action site.

[0004] Compared with complete glycoprotein E or varicella-zoster virus particles, the immune response induced by the soluble extracellular domain of glycoprotein E is not sufficient and its immunogenicity is weak. Therefore, additional proteins can be introduced to form dimers to increase its immunogenicity. Fc fusion protein is a biopharmaceutical research direction that has developed rapidly in recent years. It uses the Fc segment of immunoglobulin as a molecular chaperone and fuses functional proteins with it through molecular biological methods, so that the functional proteins can form stable dimers and greatly prolong the half-life of functional proteins. Fc fusion protein maintains the activity of functional proteins, and at the same time has the characteristics of long half-life of immunoglobulin, cytotoxicity, simple production, and easy purification. It has become a star in the field of biopharmaceuticals.

[0005] The isoelectric point of common Fc fusion proteins is alkaline, similar to monoclonal antibodies, and there are chromatography solutions. However, the isoelectric point of VZV gE-Fc fusion protein is acidic and the properties of monomers, dimers, and polymers are similar. Conventional chromatography conditions make it difficult to obtain high-purity protein dimers, which is not conducive to subsequent applications.

[0006] Therefore, how to improve the purity of Fc fusion protein is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention is proposed.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] One aspect of the present invention is to provide a method for purifying an acidic Fc fusion protein, which comprises using affinity chromatography, anion exchange chromatography and hydrophobic chromatography to elute CHO cell culture supernatants containing the acidic Fc fusion protein; the elution is isocratic elution, and the pH difference between the equilibrium buffer and the elution buffer is not less than 1 pH unit.

[0010] In a preferred embodiment of the present invention, the pH value of the equilibrium buffer of the affinity chromatography is 7.0-7.5, and the pH value of the elution buffer is 3.5-4.0.

[0011] In a preferred embodiment of the present invention, the pH value of the equilibration buffer of the anion exchange chromatography is 7.0-7.5, and the pH value of the elution buffer is 5.5-6.0.

[0012] In a preferred embodiment of the present invention, the pH value of the equilibration buffer of the hydrophobic chromatography is 5.5-6.0, and the pH value of the elution buffer is 7.0-7.5.

[0013] In a preferred embodiment of the present invention, the pH value of the equilibration buffer and the elution buffer is adjusted with a pH buffer, and the pH buffer is selected from any one of phosphate, citrate, and phosphate-citrate buffer.

[0014] In a preferred embodiment of the present invention, the concentration of the phosphate is 10-50 mM, and the concentration of the citrate buffer is 0.1-0.2 M.

[0015] In a preferred embodiment of the present invention, the acidic Fc fusion protein consists of an Fc domain and a functional protein domain, the Fc domain includes Hinge-CH2-CH3, the functional protein domain selects the soluble extracellular domain of VZV glycoprotein E, and there is no linker connection between the Fc domain (as shown in SEQ ID NO.1) and the functional protein domain (as shown in SEQ ID NO.2).

[0016] In a preferred embodiment of the present invention, the Fc domain is selected from human IgG1.

[0017] It can be seen from the above technical solutions that, compared with the prior art, the present invention finds that, for proteins with similar properties, only changing the ionic strength of the buffer cannot achieve the desired recovery rate and purity. The target protein (dimer) has good stability under different pH conditions, and is not easy to form polymers, especially under low pH conditions.

[0018] In view of this, the present invention creatively adjusts the pH of the equilibrium buffer and elution buffer of the three chromatography techniques, that is, the pH values ​​of the two buffers are different and the difference is greater than 1 pH unit. The result surprisingly found that the purity of the target protein (dimer) after the first step of affinity chromatography can reach about 85%, all monomers can be removed after anion exchange chromatography, and almost all polymers can be removed by hydrophobic chromatography. Compared with conventional buffer conditions, the purification method provided by the present invention can improve the purity of the target protein to about 99% while ensuring that the process-related impurities meet the requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 The attached figure is the NR-SDS-PAGE image of affinity eluent-1, affinity eluent-2 and affinity eluent-3 in Example 2; the lanes from left to right are protein marker, affinity eluent-1, affinity eluent-2 and affinity eluent-3.

[0021] Figure 2 The accompanying drawing is a SEC-HPLC chromatogram of affinity eluent-2 in Example 2;

[0022] Figure 3 The accompanying drawing is a SEC-HPLC chromatogram of ion eluent-1 in Example 3;

[0023] Figure 4 The accompanying drawing is a SEC-HPLC chromatogram of ion eluent-2 in Example 3;

[0024] Figure 5 The accompanying drawing is a SEC-HPLC chromatogram of the hydrophobic eluent-1 in Example 4;

[0025] Figure 6The accompanying drawing is a SEC-HPLC chromatogram of the hydrophobic eluent-2 in Example 4. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The acidic Fc fusion protein consists of an Fc domain and a functional protein domain, the Fc domain includes Hinge-CH2-CH3, the functional protein domain selects the soluble extracellular domain of VZV glycoprotein E, there is no linker connection between the Fc domain and the functional protein domain, the amino acid sequence of the Fc domain is shown in SEQ ID NO.1, the amino acid sequence of the functional protein domain is shown in SEQ ID NO.2, and the Fc domain is selected from human IgG1.

[0028] Example 1 Expression of acidic Fc fusion protein

[0029] The monoclonal cells (Chinese hamster ovary cells (CHO-K1) containing the acidic Fc fusion protein gene) were revived and inoculated into a 250ml shake flask containing 80ml CHO medium (QuaCell® CHO CD02 medium). The vented flask cap was screwed tightly and cultured on a shaker at 36~37℃, 5% CO2, and 125r / min. After 3~4 days of culture, the cells were transferred to a 1.6L shake flask containing 400ml CHO medium for Fed-Batch culture. After 3~4 days of culture, the cell count reached 5~8*10 per ml. 6 After viable cells were detected, daily feeding began with the addition of 3% QuaCell® CHO Feed02A medium and 0.3% QuaCell® CHO Feed02B medium. Daily sampling was performed to detect cell density, cell viability, agglomeration rate, particle size, glucose content, lactic acid content and osmotic pressure. If the glucose content was lower than 10mmole / L, 50% glucose solution was added to the shake flask until the glucose content reached 20mmole / L. The cells were harvested after 14 days of culture or when the cell viability was lower than 85%. Cells and cell debris were removed by centrifugation at 3000rpm for 20min, and the cell culture supernatant was collected.

[0030] Example 2 Affinity chromatography of acidic Fc fusion protein

[0031] The nature of Protein A affinity chromatography filler is high pH combined with low pH elution, and the elution pH is optimized for acidic Fc fusion proteins because the elution pH affects the purity and yield of the target protein.

[0032] Chromatography system: Hanbang Bio-100

[0033] Chromatography medium: Boglund AT Protein A Diamond

[0034] Equilibration buffer: 20 mM PB, 150 mM NaCl, pH 7.4

[0035] Elution buffer: 0.1M citrate, pH 3.2, 3.5, 4.0

[0036] Neutralization buffer: 2M Tris

[0037] First, equilibrate the Protein A affinity chromatography column with 5 column volumes of equilibration buffer until A 280 After returning to the baseline level, the CHO cell culture supernatant after centrifugation was loaded onto the affinity chromatography column, and then the equilibration buffer was used to wash 10 column volumes to A 280 After returning to the baseline level, the protein was eluted with elution buffers of pH 4.0, pH 3.5, and pH 3.2, and affinity eluents-1, 2, and 3 were collected. The Protein A affinity chromatography column was then washed with 0.1 M NaOH solution for 5 column volumes, replaced with ultrapure water, and finally stored in 20% ethanol. A neutralization buffer was added to affinity eluent-1 to adjust the pH to 6.5-7.0, and then the flocculent precipitate was removed by 0.45 μm filtration.

[0038] NR-SDS-PAGE spectrum is shown in Figure 1 The protein concentrations of affinity eluent-1, affinity eluent-2, and affinity eluent-3 were measured using an ultra-micro spectrophotometer. The results are shown in Table 1.

[0039] Table 1 Content of affinity eluate obtained at different elution pH

[0040]

[0041] The affinity eluate-2 was detected by Hitachi D-2000 Elite HPLC (Tosoh G3000PWXL chromatographic column, 7.8 mm × 30 cm, Tosoh (Shanghai) Biotechnology Co., Ltd.)). The chromatogram is shown in Figure 2 The peak area ratios of polymers, dimers and monomers were calculated respectively, and the results are shown in Table 2.

[0042] Table 2 SEC-HPLC purity results of affinity eluate-2

[0043]

[0044] It can be seen from Tables 1 and 2 that most of the protein was eluted when the pH of the elution buffer was 3.5, accounting for 91.8%. Figure 1 In the protein marker lane, the molecular weights of the protein bands from bottom to top are 45kDa, 72kDa, 100kDa, 130kDa, 160kDa, 200kDa, 250kDa, and 300kDa. Figure 1 It can be found that the size of the proteins eluted at pH 3.2 is mainly distributed between 160kDa and 250kDa and above 300kDa. The size of the target protein (dimer) is about 200kDa. Since it is a glycoprotein, the protein bands in the NR-SDS-PAGE spectrum are diffuse. According to the depth of the protein bands, it can be seen that the proteins above 300kDa (polymers) account for about 25%, which is higher than the proteins eluted at pH 4.0 and pH 3.5. The purity of the target protein at pH 4.0 is very high, but the recovery rate is low, indicating that adjusting the pH of the affinity eluent to between 3.5 and 4.0 can remove some polymers while ensuring the recovery rate, so that the purity of the target protein (dimer) reaches about 85%.

[0045] Example 3 Anion Exchange Chromatography of Acidic Fc Fusion Protein

[0046] Chromatography system: Hanbang Bio-100

[0047] Chromatography column: Cytiva Q HP

[0048] Equilibration buffer A: 10 mM PB, pH 7.0

[0049] Elution buffer B: 10 mM PB, 0.5 M NaCl, pH 5.7

[0050] Regeneration buffer: 2M NaCl

[0051] First, equilibrate the Cytiva Q HP anion chromatography column with equilibration buffer A for 5 column volumes to A. 280 After returning to the baseline level, the affinity eluate (affinity eluate-2 from Example 2) was loaded onto the anion chromatography column, and then 5 column volumes of equilibration buffer A were used to wash the column to A. 280Return to the baseline level. The equilibration buffer A was replaced with the elution buffer B within 10 column volumes. There was only one elution peak in the whole process, which was collected in separate tubes, a total of 7 tubes. Each tube of eluate was detected by Hitachi D-2000 Elite HPLC (Tosoh G3000PWXL chromatographic column, 7.8mm×30cm, Tosoh (Shanghai) Biotechnology Co., Ltd.), and the results are shown in Table 3.

[0052] Table 3 SEC-HPLC purity results of linear elution ion eluent

[0053]

[0054] According to Table 3, for monomer, dimer and multimer protein solutions with similar properties, the purity of the target protein (dimer) obtained by collecting according to the elution peak during linear elution is not significantly different from that of the affinity elution solution; the purity is also not ideal when collecting according to the tube during linear elution.

[0055] According to the conductivity of each tube of eluent, the elution mode was adjusted to isocratic elution. 45% B buffer (equilibrium buffer A and elution buffer B were mixed in a volume ratio of 11:9) was used to wash the impurities, and 60% B buffer (equilibrium buffer A and elution buffer B were mixed in a volume ratio of 2:3) was used to elute the target protein and collect it, i.e., ion eluent-1.

[0056] In addition, the pH of the equilibration buffer A and the elution buffer B were adjusted to 7.0, and the elution method was isocratic elution. 45% B buffer was also used to wash the impurities, and 60% B buffer was used to elute the target protein and collect it, namely, ion eluent-2.

[0057] The above two ion eluents were detected by Hitachi D-2000 Elite HPLC (Tosoh G3000PWXL chromatographic column, 7.8 mm × 30 cm, Tosoh (Shanghai) Biotechnology Co., Ltd.)). The chromatograms are shown in Figure 3-Figure 4 , the results are shown in Table 4.

[0058] Table 4 SEC-HPLC purity results of isocratic elution ion eluent

[0059]

[0060] According to Table 4, the pH of the equilibration buffer A and the elution buffer B are different and differ by more than 1 pH unit. The use of isocratic elution can significantly improve the purity of the target protein (dimer).

[0061] Example 4 Hydrophobic chromatography of acidic Fc fusion protein

[0062] Chromatography system: Hanbang Bio-100

[0063] Chromatography column: MaXtar Phenyl HR

[0064] Equilibration buffer A: 50 mM PB, 0.5 M ammonium sulfate, pH 6.0

[0065] Elution buffer B: 50 mM PB, pH 7.4

[0066] Sample treatment buffer: 50 mM PB, 1.0 M ammonium sulfate, pH 6.0

[0067] First, equilibrate the MaXtar Phenyl HR hydrophobic chromatography column with equilibration buffer A for 5 column volumes to A. 280 Back to the baseline level, the ion eluent (ion eluent-1 from Example 3) was pre-treated with sample treatment buffer at a ratio of 1:1 and then loaded onto the hydrophobic chromatography column, followed by further washing with equilibrium buffer A for 5 column volumes to A 280 After returning to the baseline level, the equilibration buffer A was replaced with the elution buffer B within 20 column volumes. There was only one elution peak in the whole process, which was collected in separate tubes, for a total of 8 tubes. Each tube of eluate was detected by Hitachi D-2000 Elite HPLC (Tosoh G3000PWXL column, 7.8 mm × 30 cm, Tosoh (Shanghai) Biotechnology Co., Ltd.), and the results are shown in Table 5.

[0068] Table 5 SEC-HPLC purity results of the hydrophobic eluent of linear elution

[0069]

[0070] According to Table 5, for monomer, dimer and multimer protein solutions with similar properties, when linear elution is performed and collected according to the elution peak, the purity of the obtained target protein (dimer) is not much improved compared with the ion elution solution; when linear elution is performed and collected according to the tube, the purity is also not ideal.

[0071] According to the conductivity of each tube of hydrophobic eluent, the elution mode was adjusted to isocratic elution. 77% B buffer (equilibrium buffer A and elution buffer B were mixed at a volume ratio of 23:77) was used to wash the impurities, and 100% B buffer (i.e., elution buffer B) was used to elute the target protein and collect it, i.e., hydrophobic eluent-1.

[0072] In addition, the pH of the equilibration buffer A and the elution buffer B were adjusted to 7.4, and the elution method was isocratic elution. 77% B buffer was also used to wash the impurities, and 100% B buffer was used to elute the target protein and collect it, namely, hydrophobic eluent-2.

[0073] The above two hydrophobic eluents were detected by Hitachi D-2000 Elite HPLC (Tosoh G3000PWXL chromatographic column, 7.8 mm×30 cm, Tosoh (Shanghai) Biotechnology Co., Ltd.), the chromatograms are shown in Figures 5~6, and the results are shown in Table 6.

[0074] Table 6 SEC-HPLC purity results of isocratic elution hydrophobic eluent

[0075]

[0076] According to Table 6, the pH of the equilibration buffer A and the elution buffer B are different and differ by more than 1 pH unit. The use of isocratic elution can significantly improve the purity of the target protein (dimer).

[0077] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0078] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for purifying an acidic Fc fusion protein, characterized in that: Affinity chromatography, anion exchange chromatography and hydrophobic chromatography are used successively to wash and elute the CHO cell culture supernatant containing the acidic Fc fusion protein; the elution is isocratic elution, and the pH difference between the equilibrium buffer and the elution buffer is not less than 1 pH unit; in the affinity chromatography, the pH value of the equilibrium buffer is 7.0-7.5, and the pH value of the elution buffer is 3.5-4.0; in the anion exchange chromatography, the pH value of the equilibrium buffer is 7.0-7.5, and the pH value of the elution buffer is 5.5-6.0; in the hydrophobic chromatography, the pH value of the equilibrium buffer is 5.5-6.0, and the pH value of the elution buffer is 7.0-7.5; the acidic Fc fusion protein consists of an Fc domain and a functional protein domain, the Fc domain includes Hinge-CH2-CH3, the functional protein domain selects the soluble extracellular domain of VZV glycoprotein E, and there is no linker connection between the Fc domain and the functional protein domain; the amino acid sequence of the Fc domain is as shown in SEQ ID NO.1; the amino acid sequence of the functional protein domain is shown in SEQ ID NO.

2.

2. The purification method according to claim 1, wherein the pH value of the equilibration buffer and the elution buffer is adjusted with a pH buffer, and the pH buffer is selected from any one of phosphate, citrate, and phosphate-citrate buffer.

3. The purification method according to claim 2, wherein the concentration of the phosphate is 10-50 mM, and the concentration of the citrate buffer is 0.1-0.2 M.

4. The purification method according to claim 1, characterized in that The Fc domain is selected from human IgG1.

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