Method for purifying recombinant PIV trimer protein

Abstract

The invention discloses a method for purifying recombinant PIV trimer protein, and relates to the technical field of protein purification. According to the method, a leaching step is added before target protein is eluted through cation composite chromatography for protein purification, a buffer solution containing 0.1-5 M of ammonium sulfate is adopted as a leaching solution, the removal rate of HCP is remarkably increased, the residual amount of the HCP for protein elution can be reduced to 83 ng / mg, and the yield of the HCP is improved. The risk that the residual quantity of HCP is too high can be remarkably reduced, and a new way is provided for purification and preparation of recombinant protein.

Description

Technical Field

[0001] The present invention relates to the technical field of protein purification, and in particular, to a method for purifying recombinant PIV trimer protein. Background Art

[0002] The downstream purification process of recombinant proteins expressed by CHO cells generally mainly includes culture medium clarification, capture, virus inactivation and filtration, polishing, and ultrafiltration concentration. Host cell protein (HCP) refers to a mixture of proteins derived from host cells. Many upstream parameters, including cell line selection, culture conditions, cell density, and cell viability, etc., will affect the types, quantities, and protein properties of host proteins. Since residual host proteins have the risk of interfering with the drug effect or triggering an immunogenic reaction, HCP is an impurity that needs to be removed during the downstream purification process.

[0003] In the downstream purification process of recombinant proteins, HCP is mainly removed step by step through various chromatography media with different ligand types, including ion chromatography media, hydrophobic chromatography media, and composite chromatography media with the combined action of ion and hydrophobicity. However, with the improvement of CHO cell expression level, the change of culture conditions and the increase of cell density will all lead to the increase of the types and contents of host proteins in the cell harvest fluid, and the difficulty of removing HCP increases.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for purifying recombinant PIV trimer protein.

[0006] The present invention is implemented as follows:

[0007] In a first aspect, an embodiment of the present invention provides a method for purifying recombinant PIV trimer protein, which includes: performing anion chromatography, virus inactivation, depth filtration, hydrophobic chromatography, and cationic composite chromatography on a cell culture solution expressing recombinant PIV trimer protein; wherein, the cationic composite chromatography includes elution and washing, and the eluent used for the washing includes: a washing buffer containing 0.1 - 5M ammonium sulfate.

[0008] In a second aspect, an embodiment of the present invention provides a method for preparing a stock solution of recombinant PIV trimer protein, which includes: mixing the recombinant PIV trimer protein purified by the purification method described in the foregoing embodiment with excipients to obtain a stock solution of recombinant PIV trimer protein.

[0009] The present invention has the following beneficial effects:

[0010] By adding a washing step before eluting the target protein through cationic complex chromatography for purifying the protein, the removal rate of HCP was significantly improved. The residual amount of HCP in the eluted protein could be reduced to 83 ng / mg, which could significantly reduce the risk of excessive residual HCP and provide a new approach for the purification and preparation of recombinant proteins. Description of the Drawings

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0012] Figure 1 It is the purification flow chart of recombinant PIV protein expressed by CHO cells;

[0013] Figure 2 It is the denaturing and reducing SDS-PAGE result of ultrafiltration concentration and replacement of clarified protein; among them, lanes 1-4 are in sequence: Marker; clarified protein; clarified protein after 4-fold ultrafiltration concentration; clarified protein after ultrafiltration concentration and replacement;

[0014] Figure 3 It is the SDS-PAGE result of the purified sample of recombinant PIV protein by anion chromatography; among them, lane 1 is: Marker; lane 2 is: sample loading; lane 4 is: anion chromatography eluted protein; lane 5 is: anion chromatography eluted impurity protein;

[0015] Figure 4 It is the SDS-PAGE result of the sample of recombinant PIV protein inactivated by low pH virus; among them, lane 1 is: Marker; lane 2 is: protein before inactivation; lane 3 is: protein after inactivation;

[0016] Figure 5 It is the SDS-PAGE result of the purified sample of recombinant PIV protein by hydrophobic chromatography; among them, lane 1 is: Marker; lane 2 is: sample loading; lane 3 is: hydrophobic chromatography eluted protein; lane 4 is: hydrophobic chromatography eluted impurity protein;

[0017] Figure 6 It is the SDS-PAGE result of the purified sample of recombinant PIV protein by cationic complex chromatography; among them, lane 1 is: sample loading; lane 2 is: cationic complex chromatography washed protein; lane 3 is: cationic complex chromatography eluted protein; lane 5 is: Marker. Detailed Embodiments

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0019] During the process of purifying recombinant proteins expressed by CHO cells, when performing cation-exchange chromatography, due to the difference in hydrophobic properties between the target protein and some host cell proteins (HCPs), after adding ammonium sulfate, the binding force of some HCPs to the cation-exchange medium is weaker. At this time, eluting with ammonium sulfate at a specific concentration defined in this application can effectively remove or reduce the content of HCPs.

[0020] The purification method of the present application is applicable to the purification process of recombinant proteins expressed by CHO cells, and there is no special limitation on the recombinant PIV trimer protein. The recombinant PIV trimer protein refers to the fusion protein F of Parainfluenza Virus (PIV), that is, the recombinant PIV trimer protein includes the PIV F protein trimer. PIV includes PIV3.

[0021] On the one hand, the embodiments of the present invention provide a method for purifying a recombinant PIV trimer protein, which includes: performing anion chromatography, virus inactivation, depth filtration, hydrophobic chromatography, and cation-exchange chromatography on the cell culture solution expressing the recombinant PIV trimer protein;

[0022] Among them, the cation-exchange chromatography includes elution and washing, and the eluent used for washing includes: a washing buffer containing 0.1 - 5 M ammonium sulfate.

[0023] In some embodiments, the working concentration of ammonium sulfate in the eluent can be any one or the range between any two of 0.1, 0.2, 0.4, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 M.

[0024] In some embodiments, the washing buffer includes any one of phosphate buffer, HEPES buffer, and Tris-HCl buffer.

[0025] In some embodiments, the concentration of the phosphate buffer is 1 - 50 mM, and this concentration can specifically be any one or the range between any two of 1, 2, 5, 10, 15, 18, 20, 22, 25, 30, 35, 40, 45, and 50 mM.

[0026] In some embodiments, the pH of the eluent is 7.0 to 8.0, and specifically can be any one of 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0 or the range between any two of them.

[0027] In some embodiments, the amount of the eluent is 4 to 8 column volumes or more. The column volume can specifically be any one of 4, 5, 6, 7, 8 CVs or the range between any two of them. When the elution volume ≥ 4 CV, the removal effect of HCP is obvious.

[0028] In some embodiments, the packing material for the cation exchange chromatography includes MMC packing material.

[0029] In some embodiments, the MMC packing material includes Agarosix MC90 MMC packing material.

[0030] In some embodiments, before elution, the cation exchange chromatography further includes: sample pretreatment, chromatography column pretreatment, equilibration, and sample loading.

[0031] In some embodiments, the sample pretreatment for the cation exchange chromatography includes: adjusting the conductivity of the sample after hydrophobic chromatography to 110 to 125 mS / cm, specifically can be any one of 110, 112, 114, 116, 118, 120, 122, 124, 125 mS / cm or the range between any two of them; adjusting the pH of the sample after hydrophobic chromatography to 7.0 to 8.0, specifically can be any one of 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0 or the range between any two of them.

[0032] In some embodiments, after sample loading, the cation exchange chromatography further includes performing re-equilibration.

[0033] In some embodiments, the equilibration buffer used for the equilibration and / or re-equilibration of the cation exchange chromatography includes: an equilibration buffer containing 0.1 to 2 M ammonium sulfate. The working concentration of ammonium sulfate in this equilibration buffer can be any one of 0.1, 0.2, 0.4, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, and 2 M or the range between any two of them.

[0034] In some embodiments, the equilibration buffer for the cation exchange chromatography includes 1 to 50 mM phosphate buffer. The concentration of this equilibration buffer can specifically be any one of 1, 2, 5, 10, 15, 18, 20, 22, 25, 30, 35, 40, 45, and 50 mM or the range between any two of them.

[0035] In some embodiments, the pH of the equilibration buffer for the cation exchange chromatography is 7.0 to 8.0, and specifically can be any one of 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0 or the range between any two of them.

[0036] In some embodiments, the eluent used for the elution of the cation exchange chromatography comprises: an elution buffer containing 0.1 to 2 M ammonium sulfate. The working concentration of ammonium sulfate in this elution buffer can be any one of 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8 and 2 M or the range between any two of them.

[0037] In some embodiments, the elution buffer for the cation exchange chromatography comprises 1 to 50 mM phosphate buffer.

[0038] In some embodiments, the packing material for the anion exchange chromatography comprises NanoGel-50Q.

[0039] In some embodiments, the anion exchange chromatography comprises: equilibration, sample loading and elution.

[0040] In some embodiments, the equilibration solution for the anion exchange chromatography comprises: 1 to 50 mM phosphate buffer with a pH of 7.0 to 8.0. The concentration of this phosphate buffer can specifically be any one of 1, 2, 5, 10, 15, 18, 20, 22, 25, 30, 35, 40, 45 and 50 mM or the range between any two of them; the pH can specifically be any one of 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0 or the range between any two of them.

[0041] In some embodiments, the eluent for the anion exchange chromatography comprises: an elution buffer containing 0.1 to 1 M NaCl with a pH of 7.0 to 8.0. In this elution buffer, NaCl can be any one of 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 M or the range between any two of them. The pH can specifically be any one of 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0 or the range between any two of them.

[0042] In some embodiments, the packing material for the hydrophobic interaction chromatography comprises: Monomix MC60-HIC Butyl packing material.

[0043] In some embodiments, the hydrophobic interaction chromatography comprises: equilibration, sample loading and elution.

[0044] In some embodiments, the equilibration buffer for hydrophobic chromatography comprises: an equilibration buffer containing 0.1 - 2 M ammonium sulfate, with a pH of 7.0 - 8.0. In this equilibration buffer, the concentration of ammonium sulfate can be any one of 0.1, 0.2, 0.4, 0.6, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, and 2 M or within the range between any two of them. The pH can specifically be any one of 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0 or within the range between any two of them.

[0045] In some embodiments, the elution buffer for hydrophobic chromatography comprises: an elution buffer containing 0.1 - 2 M ammonium sulfate, with a pH of 7.0 - 8.0. The concentration of ammonium sulfate in this elution buffer can be any one of 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, and 2 M or within the range between any two of them. The pH can specifically be any one of 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0 or within the range between any two of them.

[0046] In some embodiments, before performing the anion chromatography, the purification method further comprises subjecting the cell culture solution to clarification filtration.

[0047] In some embodiments, after performing the clarification filtration and before the anion chromatography, the purification method further comprises performing a first ultrafiltration concentration and replacement;

[0048] In some embodiments, after performing the cation complex chromatography, the purification method further comprises performing a second ultrafiltration concentration and replacement;

[0049] In some embodiments, the cut-off molecular weight of the ultrafiltration membrane used for the first ultrafiltration concentration and replacement and / or the second ultrafiltration concentration and replacement is 1 - 20 KDa, and specifically can be any one of 1, 5, 10, 15, 20 KDa or within the range between any two of them.

[0050] In some embodiments, the concentration in the first ultrafiltration concentration and replacement comprises: concentrating to 1 / 8 - 1 / 3 of the original volume, and specifically can be any one of 1 / 8, 1 / 7, 1 / 6, 1 / 5, 1 / 4, and 1 / 3 or within the range between any two of them.

[0051] In some embodiments, the concentration in the second ultrafiltration concentration and replacement comprises: concentrating to a recombinant PIV trimer protein containing 1 - 20 mg / mL. This concentration can specifically be any one of 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 mg / mL or within the range between any two of them.

[0052] In some embodiments, the virus inactivation includes low pH virus inactivation.

[0053] In some embodiments, the pH value of the low pH ranges from 3.4 to 3.7, and specifically can be any one of 3.4, 3.5, 3.6, and 3.7 or the range between any two of them.

[0054] In some embodiments, the inactivation time is 1.0 to 2.0 h, and specifically can be any one of 1, 1.2, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 h or the range between any two of them.

[0055] In some embodiments, the cells include CHO cells.

[0056] On the other hand, embodiments of the present invention provide a method for preparing a recombinant PIV trimeric protein stock solution, which includes: mixing the recombinant PIV trimeric protein purified by the purification method described in any of the foregoing embodiments with excipients to obtain a recombinant PIV trimeric protein stock solution.

[0057] In some embodiments, the excipients include: a protectant.

[0058] In some embodiments, the protectant includes: sucrose and Tween.

[0059] In some embodiments, in the stock solution, the mass volume percentage of the sugar is 1% to 20%, and specifically can be any one of 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20% or the range between any two of them; the mass volume percentage of Tween is 0.001% to 0.05%, and specifically can be any one of 0.001%, 0.005%, 0.010%, 0.015%, 0.020%, 0.025%, 0.03%, 0.035%, 0.040%, 0.045%, and 0.05% or the range between any two of them.

[0060] In the definition of mass volume percentage, 10% means 10 g of solute per 100 ml of solution.

[0061] In some embodiments, the pH of the stock solution is 7.0 to 8.0, and specifically can be any one of 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0 or the range between any two of them.

[0062] The features and properties of the present invention are further described in detail below in conjunction with embodiments.

[0063] Detection methods used in the examples:

[0064] SDS-PAGE detection

[0065] Electrophoresis parameter setting: (Loading Buffer is denaturing + reducing loading buffer (5X, manufacturer: Beyotime), sample preparation system is 40ul sample + 10ul loading buffer, gel is SurePage Bis Tris 4-20% (manufacturer: GenScript), electrophoresis buffer is Tris-MOPS-SDS Running Buffer (manufacturer: GenScript), electrophoresis parameters are 150V, 45min).

[0066] SEC-HPLC detection

[0067] Chromatographic column selection: Biocore SEC-300 7.8×300mm, 5μm (manufacturer: ChromCore); buffer: 20mM PB + 0.3M NaCl, pH7.5; other conditions: flow rate 0.5ml / min; detection time: 30min.

[0068] Antigen content detection

[0069] Capture antibody: PIA174; detection antibody: self-made HRP-labeled rabbit polyclonal antibody; double antibody sandwich ELISA detection method using PBST solution containing casein and sucrose as blocking solution and PBST solution containing BSA as dilution solution.

[0070] HCP residue detection

[0071] CYGNUS' CHINESE HAMSTER OVARY 3 rd Genreation HCP kit, ELISA detection method with the same factory diluent.

[0072] Example 1

[0073] A purification method of recombinant PIV trimer protein, which includes the following steps. The schematic diagram can be referred to Figure 1 .

[0074] (1) Obtain the culture medium of recombinant PIV protein CHO cells

[0075] CHO cells are resuscitated and passaged, and the culture medium containing more than 2g / L PIV protein (PIV3 F protein trimer, the F protein contains an extracellular region and a trimerization domain at the C-terminus) is obtained using the fed-batch culture process.

[0076] (2) Clarification and filtration

[0077] 1. Membrane package treatment: Take 1.1m 2The deep filter is vented, the deep filter is installed and connected, a pressure gauge is installed at the inlet end, the flow rate is controlled at 1.8 - 9.0 L / min. After venting, the vent port is closed, and the outlet port is opened. The pump is started and flushed with water. The water flow rate is gradually increased to 9.0 L / min within 1 min, and the volume of water flushing is 110 L. After completion, it is flushed with 11 L of the flushing solution (0.15 M NaCl + 20 mM PB, pH 7.5) at a flow rate of 9.0 L / min.

[0078] 2. Sample filtration: A 0.45 + 0.2 μm filter is connected in series at the rear end of the deep filter for filtration. The cell harvest fluid is taken, and the pump set flow rate is controlled at 0.9 - 3.6 L / min for filtration. The first 2 L of liquid at the front end is not collected, and the pressure is recorded. After the pressure increases, the flow rate can be appropriately reduced to control the pressure ≤ 1.5 bar. Then, the filter membrane is flushed with 10 L of the flushing solution (0.15 M NaCl + 20 mM PB, pH 7.5). After completion, it is combined with the filtered feed liquid to obtain clarified protein.

[0079] (3) First ultrafiltration concentration and replacement

[0080] 1. Membrane package treatment: Take 0.11 m 2 × 1 piece of 10 KD PES ultrafiltration membrane package for ultrafiltration. Adjust the pump speed so that the liquid flow rate is about 800 ml / min, and empty the ultrafiltration membrane package. First, flush the ultrafiltration membrane with water, and apply appropriate pressure to ensure that the volume of the liquid flowing out at the permeate end is 1 L. Then, take the cleaning solution to flush the ultrafiltration membrane, apply appropriate pressure to ensure that the volume of the liquid flowing out at the permeate end is 0.5 L, and pause for infiltration for 30 min. Flush the ultrafiltration membrane with water, apply appropriate pressure for flushing to ensure that the pH returns to neutral. Rinse the ultrafiltration membrane with ultrafiltration replacement solution 1.

[0081] 2. Ultrafiltration concentration and replacement: The clarified protein is filtered through a 0.45 + 0.2 μm filter. Take the filtered feed liquid and adjust the transmembrane pressure TMP to 1 bar. After concentrating 4 times, it is continuously replaced with 4 times the sample volume with ultrafiltration replacement solution 1 (20 mM PB pH 7.5) to obtain the post - ultrafiltration protein.

[0082] The denaturing - reducing SDS - PAGE results of the clarified protein ultrafiltration concentration and replacement are shown in Figure 2 .

[0083] (4) Anion chromatography

[0084] 1. Use the post - ultrafiltration protein prepared in step (3) as the sample for loading. The packing material is NanoGel - 50Q. The operation steps are as follows:

[0085] 2. Flow rate setting: Set the system flow rate to 180 cm / h.

[0086] 3. Pretreatment: Rinse the chromatography column with 1M NaOH for 3 column volumes (CV), and then rinse the column with Solution B1 (20 mM PB + 1M NaCl, pH 7.5) for 4 CV.

[0087] 4. Equilibration: Equilibrate the chromatography column with Solution A1 (20 mM PB, pH 7.5) for 5 CV, adjust the UV detection wavelength to 280 nm, and zero the absorbance value.

[0088] 5. Loading: Load the protein after ultrafiltration prepared in step (3) as the sample, and maintain the loading amount at 15 mg / ml (Bradford method);

[0089] 6. Re - equilibration: After loading, rinse with Solution A1 for 5 CV and ensure that the UV absorption value drops to a stable state.

[0090] 7. Elution: Elute the chromatography column with Solution C1 (20 mM PB + 0.15M NaCl, pH 7.5) for 6 CV to obtain the anion - exchange chromatography - eluted protein. Detect with SDS - PAGE and SEC - HPLC.

[0091] The results of SDS - PAGE are shown in Figure 3 , and the results of SEC - HPLC are shown in the following table.

[0092] Table 1 SEC - HPLC integration results of anion - exchange chromatography - purified samples of recombinant PIV protein

[0093] Sample Name Purification of Target Peak Recovery of Target Peak Loading 32.38％ / Eluted Sample 74.10％ 92.67％

[0094] Test results: The purity of the target protein increased from about 32.38% to about 74.1%, and the yield of this step was about 92.67% (detected by SEC - HPLC).

[0095] (5) Low - pH virus inactivation

[0096] Take the anion - exchange chromatography - eluted protein prepared in step (4), adjust the pH of the sample to 3.5 with 1M citric acid, inactivate at room temperature for 1.5 h, and then use 2M Tris to adjust the pH of the sample back to 7.5 to obtain the inactivated protein, and conduct SDS - PAGE, SEC - HPLC and antigen content detection.

[0097] The results of SDS - PAGE are shown in Figure 4 , and the results of SEC - HPLC and antigen content are shown in Table 2.

[0098] Table 2 SEC - HPLC and antigen content detection results of low - pH virus - inactivated samples of recombinant PIV protein

[0099]

[0100] Test results: The yield of the target protein after inactivation at low pH is > 95%.

[0101] (6) Deep filtration of inactivated protein

[0102] 1. Membrane package treatment: Take 23 cm 2 Exhaust with purified water at a flow rate of 150 LMH, quickly adjust the flow rate to 600 LMH, and rinse at 100 L / m 2 . Adjust the flow rate to 150 LMH, and use buffer (20 mM PB, pH 7.5) to rinse the filter at 20 - 30 L / m 2 .

[0103] 2. Sample filtration: Take 220 ml of inactivated protein and filter it at a flow rate of 150 LMH to obtain the protein after deep filtration, and detect the antigen content and HCP residue.

[0104] The results are shown in the following table.

[0105] Table 3 Detection results of antigen content and HCP residue in the deep filtration sample of recombinant PIV protein inactivated protein

[0106]

[0107] Test results: The HCP removal rate after deep filtration of inactivated protein is > 90%, and the yield of recombinant PIV trimer protein is > 80%.

[0108] (7) Hydrophobic chromatography

[0109] 1. Using the protein after deep filtration prepared in step (6) as the basic sample, and the packing material is Monomix MC60 - HIC Butyl, the operation steps are as follows:

[0110] 2. Sample pretreatment: Take the protein after deep filtration, add 3.6 M ammonium sulfate to adjust the final concentration of ammonium sulfate in the sample to 0.8 M, add 2 M NaOH to adjust the pH of the sample to 7.5, and finally filter it with a 0.45 + 0.2 μm capsule filter to obtain the sample for loading.

[0111] 3. Flow rate setting: Set the system flow rate to 180 cm / h.

[0112] 4. Pretreatment: Take 1 M NaOH to rinse the chromatography column for 2 CV, and then rinse the chromatography column with B2 solution (20 mM PB, pH 7.5) for 3 CV.

[0113] 5. Equilibration: Equilibrate the chromatography column with A2 solution (20 mM PB + 0.85 M ammonium sulfate, pH 7.5) for 3 CV, adjust the UV detection wavelength to 280 nm, and zero the absorbance value.

[0114] 6. Sample loading: The sample loading amount is maintained at 7 mg / ml (Bradford method).

[0115] 7. Rebalancing: After sample loading, rinse with Solution A2 for 5 CV and ensure that the UV absorption value drops to a stable state.

[0116] 8. Elution: Elute the chromatography column with Solution C2 (20 mM PB + 0.5 M ammonium sulfate, pH 7.5) for 5 CV to obtain the protein eluted by hydrophobic chromatography, and perform SDS-PAGE and SEC-HPLC detections.

[0117] The SDS-PAGE results of the sample purified by hydrophobic chromatography of recombinant PIV protein are shown in Figure 5 , and the SEC-HPLC results are shown in the following table.

[0118] Table 4 SEC-HPLC integration results of the sample purified by hydrophobic chromatography of recombinant PIV protein

[0119] Sample Name Purification of Target Peak Recovery of Target Peak Loading 90.82％ / Eluted Sample 98.17％ 80.93％

[0120] Test results: The purity of the target protein is increased from about 90.82% to about 98.17%, and the yield of this step is about 80.93% (results detected by SEC-HPLC).

[0121] (8) Cationic composite chromatography

[0122] 1. Using the protein eluted by hydrophobic chromatography prepared in step (7) as the basic sample and the filler as Agarosix MC90 MMC, the operation steps are as follows:

[0123] 2. Sample pretreatment: Take the protein eluted by hydrophobic chromatography, add 3.6 M ammonium sulfate to adjust the conductivity in the sample to 117 mS / cm, add 2 M NaOH to adjust the pH of the sample to 7.5, and finally filter through a 0.45 + 0.2 μm capsule filter to obtain the sample for loading.

[0124] 3. Flow rate setting: Set the system flow rate to 180 cm / h.

[0125] 4. Pretreatment: Rinse the chromatography column with 1 M NaOH for 2 CV, and then rinse the chromatography column with Solution B2 (20 mM PB, pH 7.5) for 3 CV.

[0126] 5. Balancing: Balance the chromatography column with Solution A2 (20 mM PB + 0.85 M ammonium sulfate, pH 7.5) for 3 CV, adjust the UV detection wavelength to 280 nm, and zero the absorbance value.

[0127] 6. Sample loading: The sample loading amount is maintained at 7 mg / ml (Bradford method).

[0128] 7. Rebalancing: After sample loading is completed, rinse with Solution A2 for 5 CV and ensure that the UV absorption value drops to a stable state.

[0129] 8. Elution washing: Elute the chromatography column with Solution D1 (20 mM PB + 0.72 M ammonium sulfate, pH 7.5) for 6 CV to elute the protein by cationic composite chromatography, and detect by SEC-HPLC.

[0130] 9. Elution: Elute the chromatography column with Solution C2 (20 mM PB + 0.5 M ammonium sulfate, pH 7.5) for 5 CV to elute the protein by cationic composite chromatography, and perform SDS-PAGE and SEC-HPLC detections.

[0131] The SDS-PAGE results of the purified sample of recombinant PIV protein by cationic composite chromatography are shown in Figure 6 , and the SEC-HPLC results are shown in Table 5.

[0132] Table 5 SEC-HPLC integration results of the purified sample of recombinant PIV protein by cationic composite chromatography

[0133] Sample Name Purification of Target Peak Recovery of Target Peak Loading 98.14％ / Eluted Sample 99.18％ 106.10％

[0134] Test results: The purity of the target protein increased from about 98.14% to about 99.18%, and the yield of this step was about 106.10% (detection result by SEC-HPLC). The residual HCP decreased from 2523 ng / mg to 83 ng / mg.

[0135] (9) Second ultrafiltration concentration and replacement

[0136] 1. Membrane package treatment: Take 0.11 m 2 ×1 piece of 10 KD PES ultrafiltration membrane package for ultrafiltration. Adjust the pump speed so that the liquid flow rate is about 800 ml / min, and empty the ultrafiltration membrane package; first rinse the ultrafiltration membrane with water, apply appropriate pressure to ensure that the liquid outflow volume at the permeate end is not less than 1 L; then take the cleaning solution to rinse the ultrafiltration membrane, apply appropriate pressure to ensure that the liquid outflow volume at the permeate end is not less than 0.5 L, and pause for infiltration for not less than 30 min; rinse the ultrafiltration membrane with water, apply appropriate pressure for rinsing to ensure that the pH returns to neutral; rinse the ultrafiltration membrane with ultrafiltration replacement solution 1.

[0137] 2. Ultrafiltration concentration and replacement: Filter the eluted sample of cationic composite chromatography with a 0.45 + 0.2 μm filter. Take the filtered feed liquid and adjust the transmembrane pressure TMP to 1 bar, concentrate to the theoretical concentration of the sample of 5 mg / ml (Bradford method), and continuously replace it with ultrafiltration replacement solution 2 (5 mM PB pH 7.5) for 8 times the sample volume to obtain ultrafiltration protein 2, and perform SEC-HPLC detection.

[0138] (10) Stock solution preparation

[0139] Take ultrafiltration protein 2, add 5 mM PB + 50% sucrose, pH 7.5 to the sample until the final concentration of sucrose in the sample is 10% (w / v), add 10% (w / v) Tween 80 to the sample until the final concentration of Tween 80 in the sample is 0.025% (w / v), and filter the sample through a 0.45 + 0.2 μm filter for sterilization to obtain the stock solution.

[0140] Example 2

[0141] Based on the purification method provided in Example 1, set up negative controls and non-elution controls to verify the effect of adding an elution step before elution in cationic composite chromatography, as shown in Table 6.

[0142] Table 6 Detection results of HCP in the eluted protein and the eluted protein of recombinant PIV protein in cationic composite chromatography

[0143] Whether there is a washing step Sample Name HCP Residue ng / mg HCP Concentration ng / ml / Loading 9628 6672 Yes Washing Protein 2340 536 Yes Eluting Protein 160 192 No Eluting Protein 321 492

[0144] It can be seen that adding an elution step before eluting the target protein in cationic composite chromatography significantly improves the removal rate of HCP. After adding the elution step, the residual HCP in the eluted protein decreased from 321 ng / mg to 160 ng / mg.

[0145] Example 3

[0146] Based on the purification method provided in Example 1, take the dosage (elution volume) of the eluent in cationic composite chromatography as a single factor variable, set up multiple groups of experiments to verify the effect of the elution volume in cationic composite chromatography, as shown in Table 7.

[0147] Table 7 Detection results of HCP in recombinant PIV protein in cationic composite chromatography with different elution volumes

[0148] Sample Name HCP Residue ng / mg HCP Concentration ng / ml Loading 3525 5245.7 Washing 2CV Transient Protein 10892 403 Washing 4CV Transient Protein 2280 408.2 Washing 6CV Transient Protein 868 201.45 Eluting Protein 83 158.58

[0149] It can be seen that as the elution volume increases, the residual HCP decreases significantly. After the elution volume ≥ 4 CV, the HCP removal effect is obvious. When the elution volume is 6 CV, the residual amount of HCP in the eluted protein decreases to 83 ng / mg.

[0150] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A purification method for recombinant PIV trimeric protein, characterized in that, It includes: Performing anion chromatography, virus inactivation, depth filtration, hydrophobic chromatography, and cationic composite chromatography on the cell culture solution expressing the recombinant PIV trimer protein; Among them, the cationic composite chromatography includes elution and elution, and the eluent used for the elution includes: an elution buffer containing 0.1 to 5 M ammonium sulfate.

2. The purification method according to claim 1, wherein The eluent includes: an elution buffer containing 0.1 to 2 M ammonium sulfate; Optionally, the elution buffer includes any one of phosphate buffer, HEPES buffer, and Tris-HCl buffer; Optionally, the concentration of the phosphate buffer is 1 to 50 mM; Optionally, the pH of the eluent is 7.0 to 8.0; Optionally, the dosage of the eluent is 4 to 8 column volumes or more.

3. The purification method according to claim 1, wherein, The packing material for the cationic composite chromatography includes MMC packing material; Optionally, the MMC packing material includes Agarosix MC90 MMC packing material; Optionally, before performing the elution, the cationic composite chromatography further includes: sample pretreatment, chromatography column pretreatment, equilibration, and sample loading; Optionally, the sample pretreatment for the cationic composite chromatography includes: adjusting the conductivity of the sample after hydrophobic chromatography to 110 to 125 mS / cm and the pH to 7.0 to 8.0; Optionally, after sample loading, the cationic composite chromatography further includes performing re-equilibration; Optionally, the equilibration solution used for the equilibration and / or re-equilibration of the cationic composite chromatography includes: an equilibration buffer containing 0.1 to 2 M ammonium sulfate; Optionally, the equilibration buffer for the cationic composite chromatography includes 1 to 50 mM phosphate buffer; Optionally, the pH of the equilibration buffer for the cationic composite chromatography is 7.0 to 8.0; Optionally, the eluent used for the elution of the cationic composite chromatography includes: an elution buffer containing 0.1 to 2 M ammonium sulfate; Optionally, the elution buffer for the cationic composite chromatography includes 1 to 50 mM phosphate buffer.

4. The purification method according to any one of claims 1 to 3, characterized in that, The packing material for the anion chromatography includes NanoGel-50Q; Optionally, the anion chromatography includes: equilibration, sample loading, and elution; Optionally, the equilibration solution for the anion chromatography includes: a phosphate buffer of 1 to 50 mM with a pH of 7.0 to 8.0; Optionally, the eluent for the anion chromatography includes: an elution buffer containing 0.1 to 1 M NaCl with a pH of 7.0 to 8.

0.

5. The purification method according to any one of claims 1 to 3, characterized in that, The packing material for the hydrophobic chromatography includes: Monomix MC60-HIC Butyl packing material; Optionally, the hydrophobic chromatography includes: equilibration, sample loading, and elution; Optionally, the equilibration solution for the hydrophobic chromatography includes: an equilibration buffer containing 0.1 to 2 M ammonium sulfate with a pH of 7.0 to 8.0; Optionally, the eluent for the hydrophobic chromatography includes: an elution buffer containing 0.1 to 2 M ammonium sulfate with a pH of 7.0 to 8.

0.

6. The purification method according to any one of claims 1 to 3, characterized in that, Before performing the anion chromatography, the purification method further includes performing clarification filtration on the cell culture solution; Optionally, after performing the clarification filtration and before the anion chromatography, the purification method further includes performing the first ultrafiltration concentration replacement; Optionally, after performing the cation exchange chromatography, the purification method further includes performing a second ultrafiltration concentration and replacement; Optionally, the molecular weight cut-off of the ultrafiltration membrane used in the first ultrafiltration concentration and replacement and / or the second ultrafiltration concentration and replacement is 1-20 KDa; Optionally, the concentration in the first ultrafiltration concentration and replacement includes: concentrating to 1 / 8 to 1 / 3 of the original volume; Optionally, the concentration in the second ultrafiltration concentration and replacement includes: concentrating to contain 1-20 mg / mL of the recombinant PIV trimer protein.

7. The purification method according to any one of claims 1 to 3, characterized in that, The virus inactivation includes low pH virus inactivation; Optionally, the pH value of the low pH is selected from 3.4 to 3.7; Optionally, the inactivation time is 1.0 to 2.0 h.

8. The purification method according to any one of claims 1 to 3, characterized in that, The cells include CHO cells.

9. A method for preparing a recombinant PIV trimeric protein stock solution, characterized in that, It includes: Mixing the recombinant PIV trimer protein purified by the purification method according to any one of claims 1 to 8 with excipients to obtain the stock solution of the recombinant PIV trimer protein.

10. The preparation method according to claim 9, characterized in that, The excipients include: protectants; Optionally, the protectants include: sucrose and Tween; Optionally, in the stock solution, the mass volume percentage of the sugar is 1% to 20%, and the mass volume percentage of the Tween is 0.001% to 0.05%; Optionally, the pH of the stock solution is 7.0 to 8.0.

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