A method for purifying recombinant human metapneumovirus pre-fusion f protein

Abstract

The application belongs to the technical field of protein purification, and particularly relates to a recombinant human metapneumovirus pre-fusion F protein purification method, which comprises the following steps: filtering fermentation broth by using a deep filter to obtain fermentation clarified liquid; capturing the recombinant hMPV F protein from the fermentation clarified liquid by using hydroxyapatite to obtain purified liquid 1; inactivating viruses in the purified liquid 1 by using a low-pH method to obtain inactivated liquid; performing anion exchange chromatography on the inactivated liquid to obtain purified liquid 2; performing composite weak anion chromatography on the purified liquid 2 to obtain purified liquid 3; performing UF / DF on the purified liquid 3 to obtain ultrafiltrate; and performing virus-removing nanofiltration and sterile filtration on the ultrafiltrate to obtain a recombinant hMPV F stock solution. The method is simple, reproducible, suitable for large-scale production, and can be effectively used for producing a recombinant human metapneumovirus pre-fusion F protein vaccine.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of protein purification, and particularly relates to a recombinant human metapneumovirus pre-fusion F protein purification method. BACKGROUND

[0002] Human metapneumovirus (hMPV) is a common respiratory virus belonging to the metapneumovirus genus of the family of pneumoviridae. It was first discovered in the Netherlands in 2001 and is now widely prevalent around the world. It is an important pathogenic microorganism causing acute lower respiratory tract infection in infants, immunodeficient persons and the elderly. About 6% of infants hospitalized due to acute lower respiratory tract infection are caused by hMPV.

[0003] Although there is no vaccine approved for preventing hMPV infection, recombinant RSV F protein vaccines (ABRYSVO, AREXVY) approved by FDA for marketing provide a direction for the development of hMPV vaccine, both of which belong to the family of pneumoviridae and have similar structural proteins and invasion mechanisms. Similar to RSV F protein, hMPV F protein is also the main target of neutralizing antibodies produced by the body against the virus, and exists in a metastable "prefusion" trimer state on the surface of the virus. Once in contact with host cells, it will undergo extensive conformational changes and fold into an energy-optimal and stable "postfusion" state, promoting the fusion of the virus and the cell. Unlike RSV F protein, hMPV F protein has only one furin cleavage site, and no P27 peptide segment is released after cleavage. In order to stabilize the "prefusion" conformation of the extracellular domain of hMPV F protein, the sequence is mainly changed in three aspects: (1) the C-terminal of hMPV F protein is introduced into a trimerization domain, such as the 27-residue domain at the C-terminus of the T4 bacteriophage fibritin molecule; (2) the furin cleavage site is deleted, and a F2-F1 linker is introduced; (3) interchain disulfide bonds and internal cavity filling are introduced by amino acid substitution to promote the maintenance of hMPV F protein in the prefusion state. Reference can be made to Guillaume B et al. (2021) "Interprotomer disulfide-stabilized variants of the human metapneumovirus fusion glycoprotein induce high titer-neutralizing responses", PNAS, DOI: 10.1073 / PNAS.2106196118. Therefore, the hMPV subunit vaccine developed with the highly conserved fusion (F) protein as the candidate antigen and the prefusion conformation as the design concept is a desirable hMPV vaccine. In order to maintain the similarity with the natural virus protein (especially the post-translational folding of the protein, glycosylation modification, etc.), human virus subunit vaccines are mainly expressed by mammalian / eukaryotic cell lines (such as CHO cells, 293 cells, Sf9 insect cells, etc.). The fermentation cycle of mammalian cell lines is long, and a large amount of host proteins, DNA and other impurities are secreted during the growth or apoptosis of the cell lines. The expressed F protein also exists in the form of monomer, multimer and other non-trimer forms, and there are situations of fragmentation and conformational changes.

[0004] Therefore, there is an urgent need to develop a purification method for preparing F protein with uniformity, high purity and maintaining prefusion conformation. SUMMARY

[0005] In order to solve the above problems, the application provides a recombinant human metapneumovirus pre-fusion F (hereinafter referred to as hMPVF) protein purification method. The method is suitable for large-scale production and can obtain uniform, high-purity and conformationally stable recombinant human metapneumovirus pre-fusion F protein.

[0006] The first object of the application is to provide a recombinant human metapneumovirus pre-fusion F protein purification method, comprising the following steps:

[0007] P1: filtering the fermentation broth by using a depth filter to obtain a fermentation clarified broth;

[0008] P2: capturing the recombinant hMPV F protein from the fermentation clarified broth by using hydroxyapatite to obtain a purified liquid 1;

[0009] P3: inactivating the virus in the purified liquid 1 by using a low pH method to obtain an inactivated liquid;

[0010] P4: performing anion exchange chromatography on the inactivated liquid to obtain a purified liquid 2;

[0011] P5: performing composite weak anion chromatography on the purified liquid 2 to obtain a purified liquid 3;

[0012] P6: performing UF / DF on the purified liquid 3 to obtain an ultrafiltrate;

[0013] P7: performing virus removal nanofiltration and sterile filtration on the ultrafiltrate to obtain a recombinant hMPV F protein stock solution.

[0014] In the application, hydroxyapatite is selected to capture the target protein, which can remove most pigments, part of nucleic acids and host proteins; then strong anion exchange chromatography is selected to remove most host proteins, most nucleic acids and endotoxins; and then further composite weak anion chromatography is selected to further purify and remove aggregates, a small amount of residual host proteins, nucleic acids and un-inactivated viruses.

[0015] In some embodiments, the hydroxyapatite chromatography uses hydroxyapatite filler with a pore size of 40, 60 or 80 μm, type II hydroxyapatite (CHT type II);

[0016] In some embodiments, the P2 step specifically comprises:

[0017] equilibrating the CHT chromatography column to a baseline with a phosphate buffer (PB buffer); diluting the conductivity of the fermentation clarified broth with purified water to less than 8.0 mS / cm for loading; after loading, equilibrating the chromatography column to a baseline again with a phosphate buffer containing 0-70 mM NaCl; eluting with a phosphate buffer containing 70-500 mM NaCl, collecting the target protein eluate, and obtaining the purified liquid 1.

[0018] In some embodiments, the concentration of the phosphate buffer (PB buffer) is 10-30 mM, and the pH is 6.6-7.0.

[0019] In some embodiments, the P3 step specifically comprises adjusting the pH of the purified liquid 1 to ≤3.8 with ≤4 M dilute hydrochloric acid, incubating at 18-26°C for 30-60 min, adjusting the pH to 6.5-7.0 with an alkali solution or Tris-HCl after inactivation is complete, to obtain the inactivated liquid; the alkali solution is 0.1-0.5 M NaOH; the concentration of Tris-HCl is 0.5-1.0 M, and the pH is 8.0-9.0.

[0020] In some embodiments, the anion exchange filler in the anion exchange chromatography is an anion exchange filler with a quaternary ammonium group [-CH2-N + (CH3)3] or a diethylaminoethyl group [-CH2-CH2-N + (CH2-CH3)2] functional group. In some embodiments, the anion exchange chromatography specifically comprises the following steps:

[0021] equilibrating the anion exchange chromatography column with a phosphate buffer (PB buffer) to a baseline stable; adjusting the inactivated liquid to a conductivity of no more than 9.0 mS / cm for loading; after loading is complete, equilibrating the chromatography column again with a phosphate buffer to a baseline stable; using a phosphate buffer containing 0-300 mM NaCl for linear elution, collecting the target protein eluate, to obtain the purified liquid 2. In some embodiments, the concentration of the phosphate buffer (PB buffer) is 10-30 mM, and the pH is 6.6-7.0.

[0022] In some embodiments, the composite weak anion chromatography specifically comprises the following steps:

[0023] equilibrating the chromatography column with a phosphate buffer to a baseline stable; loading the purified liquid P2 (conductivity ≤20 ms / cm) to the composite weak anion chromatography column at high salt; after loading is complete, equilibrating the chromatography column again with a phosphate buffer containing 100-200 mM NaCl to a baseline stable; using a phosphate buffer containing 0.5-1.0 M NaCl for elution, to collect the target protein eluate, to obtain the purified liquid 3.

[0024] In some embodiments, the filler in the composite chromatography is a multi-modal strong anion filler with a phenyl glycidyl ether and 2,3-epoxypropyl trimethyl ammonium chloride functional group, such as Diamond Mix A Mustang, Smac MMA, or Capto adhere.

[0025] In the present application, the benzene ring on the filler ligand can be loaded with high salt which cannot be achieved by conventional ion exchange, so the sample does not need any treatment and can be directly loaded with high salt.

[0026] In some embodiments, the UF / DF specifically comprises the following steps:

[0027] The purified liquid 3 is subjected to ultrafiltration and replacement with a 30-50 kDa ultrafiltration membrane, and the replacement ratio is ≥6, to obtain an ultrafiltrate.

[0028] In some embodiments, the virus removal filtration specifically comprises the following process: the ultrafiltrate is sequentially subjected to Virosart Max pre-filtration and Virosart HF virus removal filtration, and then subjected to sterile filtration to obtain the recombinant hMPV F protein stock solution.

[0029] In some embodiments, the recombinant hMPV F protein cell culture clarified liquid is collected after expressing the recombinant hMPV F protein in CHO, 293, Sf9, etc.

[0030] The second object of the present application is to provide a recombinant hMPV F protein stock solution prepared by the above-mentioned recombinant human metapneumovirus pre-fusion F protein purification method.

[0031] Compared with the prior art, the present application has the following advantages and positive effects:

[0032] The recombinant hMPV F protein expressed by CHO cells can be effectively separated and purified by the method of the present application. After purification, the SEC-HPLC comprehensive purity is greater than 98 %, the RP-HPLC purity is greater than 99 %, the HCP is less than 10 ppm, the HCD is less than 10 pg / dose, the SDS-PAGE electrophoresis band is clear and single, the method is simple and reproducible, and is suitable for large-scale production, and can be effectively used for producing recombinant human metapneumovirus pre-fusion F protein vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows.

[0034] Figure 1 : Electrophoresis detection map of complete purification process of hMPV F protein;

[0035] Figure 2A : High-pressure liquid chromatography (molecular sieve) detection map of recombinant hMPV F protein stock solution;

[0036] Figure 2B : High-pressure liquid chromatography (reverse phase C4) detection map of recombinant hMPV F protein stock solution;

[0037] Figure 3 SDS-PAGE electrophoresis detection map of recombinant hMPV F protein CHT type II chromatographic filler screening;

[0038] Figure 4 SDS-PAGE electrophoresis detection map of recombinant hMPV F protein Diamond Viru-S affinity chromatographic filler screening;

[0039] Figure 5A SDS-PAGE electrophoresis detection map of stability of recombinant hMPV F protein CHT eluent under different pH conditions;

[0040] Figure 5B SEC-HPLC detection map of stability of recombinant hMPV F protein CHT eluent under different pH conditions;

[0041] Figure 6 SDS-PAGE electrophoresis detection map of recombinant hMPV F protein UniGel 30Q strong anion chromatographic filler screening;

[0042] Figure 7 SDS-PAGE electrophoresis detection map of recombinant hMPV F protein Diamond MixA mustang composite weak anion chromatographic filler screening;

[0043] Figure 8 Isoelectric focusing electrophoresis map of recombinant hMPV F protein. DETAILED DESCRIPTION

[0044] The present application is further described below in conjunction with specific embodiments:

[0045] In order to better understand the present application, but not limited to the present application, the experimental methods in the following examples, such as no special instructions, are all conventional methods. The experimental materials used in the following examples, such as no special instructions, are all purchased from the conventional biochemical reagent store.

[0046] The DNA sequence of the recombinant hMPV F protein is integrated into CHO cells, and a GS (glutamine synthetase, GS) screening system is used to pressurize the screening of stable high-expression cell strains. A round of limited dilution + continuous photographing is used to select high-expression monoclonal cell strains. The monoclonal cell strains are recovered, and the seed cells are gradually expanded in a shake flask. The prepared seed cells are transferred to a 5L bioreactor, and a flow feeding culture method is used to produce the target protein. The fermentation broth is clarified and filtered using a depth filter, and the obtained is the fermentation clarified liquid. First, the CHT filler is used to capture the target protein from the fermentation clarified liquid in a binding / elution mode, and then the CHT eluate is subjected to low-pH virus inactivation treatment. The inactivated liquid is loaded onto the UniGel 30Q filler to further purify in a binding / elution mode. The UniGel 30Q eluate is finely purified through the Diamond MixA mustang filler in a binding / elution mode. The Diamond MixA mustang eluate is subjected to ultrafiltration replacement treatment, and then subjected to virus removal filtration and sterile filtration. The electrophoresis detection results of the complete purification process of the recombinant hMPV F protein are shown in Figure 1 (from left to right, lanes are M: protein Marker indicator; 1: fermentation clarified liquid; 2: CHT chromatography eluate; 3: low-pH inactivation liquid; 4: anion chromatography eluate; 5: composite chromatography eluate; 6: ultrafiltrate; 7: recombinant hMPV F protein stock solution), and the reverse-phase HPLC spectrum of the stock solution is shown in Figure 2A and the molecular sieve spectrum is shown in Figure 2B .

[0047] Example 1: Capture of recombinant hMPV F protein from fermentation clarified liquid:

[0048] Chromatography filler name: CHT type II;

[0049] Column equilibration: equilibrated with 20mM phosphate buffer (pH6.8) for 6-8 column volumes;

[0050] Loading: the clarified fermentation broth is adjusted to a conductivity of ≤8.0mS / cm with purified water before loading;

[0051] Re-equilibration: after loading is completed, re-equilibrate with 20mM phosphate buffer (pH6.8) containing 70mM NaCl for 6-8 column volumes;

[0052] Elution: linear elution with 20mM phosphate buffer (pH6.8) containing 70-500mM NaCl, and the elution peak is collected when UV280 reaches 200mAU, and the collection is stopped when UV280 drops to 200mAU;

[0053] The fermentation supernatant was purified by a CHT II chromatographic column, and the target protein was collected in a binding / elution mode. The purity of the antigen reached 85% in the first step of capture. The electrophoretogram of the chromatographic sample is shown in Figure 3 , wherein, from left to right, lanes are as follows: 1: fermentation supernatant sample (reduced); 2-3: capture flow-through (reduced); 4: elution collected peak (reduced); M: protein marker; 5: fermentation supernatant sample (non-reduced); 6-7: capture flow-through (non-reduced); 8: elution collected peak (non-reduced). The CHT II filler has a significant effect on capturing the recombinant hMPV F protein. The target protein is in a trimeric state in a non-reduced state, which is consistent with the theoretical design. It is suitable for the capture and purification of the recombinant hMPV F protein.

[0054] Example 1: Capturing the recombinant hMPV F protein from fermentation supernatant:

[0055] Chromatographic filler name: Diamond Viru-S;

[0056] Column equilibration: equilibrated with 20 mM phosphate buffer (pH 6.8) for 6-8 column volumes;

[0057] Loading: adjust the conductivity of the clarified fermentation broth to ≤5 mS / cm with purified water before loading;

[0058] Re-equilibration: after loading, re-equilibrate with 20 mM phosphate buffer (pH 6.8) for 6-8 column volumes;

[0059] Elution: linear elution with 20 mM phosphate buffer (pH 6.8) containing 0-500 mM NaCl. Start collecting the elution peak when UV280 reaches 200 mAU, and stop collecting the peak when UV280 drops to 200 mAU;

[0060] The fermentation supernatant was purified by a Diamond Viru-S chromatographic column, and most of the target protein was present in the flow-through, with a small amount bound to the chromatographic column. The electrophoretogram of the chromatographic sample is shown in Figure 4 , wherein, from left to right, lanes are as follows: M: protein marker; 1: fermentation supernatant sample; 2-5: capture flow-through; 6-7: different elution peak collected samples. Diamond Viru-S chromatography is not suitable for the capture and purification of the recombinant hMPV F protein.

[0061] Example 1: Capturing the recombinant hMPV F protein from fermentation supernatant:

[0062] Chromatographic filler name: Q Bestarose BB;

[0063] Column equilibration: equilibrated with 20 mM phosphate buffer (pH 6.8) for 6-8 column volumes;

[0064] Loading: The clarified fermentation broth was adjusted to conductivity ≤5.0 mS / cm with purified water before loading;

[0065] Re-equilibration: After the loading was completed, the column was re-equilibrated with 20 mM phosphate buffer (pH 6.8) for 6-8 column volumes;

[0066] Elution: Linear elution was performed with 20 mM phosphate buffer (pH 6.8) containing 0-500 mM NaCl. The elution peak was collected when UV280 reached 200 mAU, and the collection was stopped when UV280 dropped to 200 mAU;

[0067] The clarified fermentation broth was purified by Q Bestarose BB chromatography column, and the target protein was collected in a binding / elution mode. The antigen purity was about 75%, and the Q Bestarose BB eluate was yellow and contained a large amount of pigment, which interfered with the determination of antigen concentration by ultraviolet spectrophotometer. Therefore, Q Bestarose BB chromatography was not suitable for the capture purification of recombinant hMPV F protein.

[0068] Example 2, Virus inactivation process of recombinant hMPV F protein:

[0069] The eluate in Example 1 was subjected to virus inactivation by low-pH virus inactivation method. The eluate in Example 1 was gradually adjusted to pH 3.5 with acid, and after 60 min at room temperature, the pH was adjusted to 7.0 with base. During the adjustment of low pH, samples were continuously taken to detect whether the structure of the target antigen changed. The sample electrophoresis and SEC-HPLC spectrum are shown in Figure 5A (from left to right lane, 1-7: pH 6.5, pH 6.0, pH 5.5, pH 5.0, pH 4.5, pH 4.0, pH 3.5; 8: Protein Marker) and Figure 5B During the adjustment of low pH, the structure of the target protein remained unchanged, indicating that the low-pH virus inactivation method was suitable for virus inactivation of recombinant hMPV F protein.

[0070] Example 3, Moderate purification of recombinant hMPV F protein:

[0071] Chromatography packing name: UniGel 30Q;

[0072] Column equilibration: Equilibrated with 20 mM phosphate buffer (pH 6.8) for 6-8 column volumes;

[0073] Loading: The inactivated liquid was adjusted to conductivity ≤9.0 mS / cm with 20 mM phosphate buffer (pH 6.8) before loading;

[0074] Re-equilibration: After loading, equilibrate the column with 20 mM Phosphate buffer (pH 6.8) for 6-8 column volumes;

[0075] Elution: Linear elution with 20 mM Phosphate buffer (pH 6.8) containing 0-300 mM NaCl, start to collect the elution peak when UV280 reaches 200 mAU, stop when UV280 drops to 200 mAU;

[0076] The inactivation solution was purified by UniGel 30Q chromatography column, and the target protein was collected in a binding / elution mode. The protein purity was about 95%. The UniGel 30Q elution sample electrophoresis and RP-HPLC spectrum are shown in FIG. 2. Figure 6 , wherein, from left to right lane, M: protein Marker; 1: CHT eluent sample; 2-4: elution peak fraction collection sample; the results show that the purity of the recombinant hMPV F protein is significantly improved, and UniGel 30Q chromatography is suitable for the medium purification of the recombinant hMPV F protein.

[0077] Comparative Example 3, medium purification of recombinant hMPV F protein:

[0078] Chromatography filler name: Diamond SP mustang;

[0079] Column equilibration: equilibrate the column with 20 mM Phosphate buffer (pH 6.8) for 6-8 column volumes;

[0080] Loading: adjust the conductivity of the inactivation solution to ≤5.0 mS / cm with 20 mM Phosphate buffer (pH 6.8) before loading;

[0081] Re-equilibration: After loading, equilibrate the column with 20 mM Phosphate buffer (pH 6.8) for 6-8 column volumes;

[0082] Elution: Linear elution with 20 mM Phosphate buffer (pH 6.8) containing 0-300 mM NaCl, start to collect the elution peak when UV280 reaches 200 mAU, stop when UV280 drops to 200 mAU;

[0083] The inactivation solution was purified by Diamond SP mustang chromatography column, and the target protein mainly existed in the flow-through liquid. The antigen purity was about 85%, indicating that Diamond SP mustang chromatography is suitable for the medium purification of the recombinant hMPV F protein.

[0084] Example 4, fine purification of recombinant hMPV F protein:

[0085] Chromatography filler name: Diamond MixA mustang;

[0086] Column equilibration: equilibrate the column with 20mM Phosphate Buffer (pH6.8) for 6-8 column volumes;

[0087] Loading: load the sample after adjusting conductivity of the UniGel 30Q eluate to ≤20.0 mS / cm with 20mM Phosphate Buffer (pH6.8);

[0088] Re-equilibration: after loading, re-equilibrate the column with 20mM Phosphate Buffer (pH6.8) containing 100-200mM NaCl for 6-8 column volumes;

[0089] Elution: elute with 20mM Phosphate Buffer (pH6.8) containing 0.5-1.0 M NaCl linearly, start to collect the elution peak when UV280 reaches 150mAU, stop when UV280 drops to 200mAU;

[0090] The UniGel 30Q eluate was purified by Diamond MixA mustang column, and the target protein was collected in binding / elution mode, with a purity of about 99%. Diamond MixA mustang is a multi-mode that can interact with target antigens through electrostatic interaction, hydrogen bonding and hydrophobic interaction, and has obvious advantages in removing impurities such as aggregates, fragments and HCP in the sample. It is also an important virus removal step. The electrophoretogram of the eluate of the chromatographic filler is shown in Figure 7 From left to right, the lanes are M: protein Marker; 1: UniGel 30Q eluate sample; 2-4: elution peak fraction collected sample. The results show that the purity of the recombinant hMPV F protein is significantly improved, and Diamond MixA mustang chromatography is suitable for fine purification of recombinant hMPV F protein.

[0091] Comparative Example 4, fine purification of recombinant hMPV F protein:

[0092] Chromatographic filler name: Phenyl (HS);

[0093] Column equilibration: equilibrate the column with 20mM Phosphate Buffer (pH6.8) containing 2M NaCl for 6-8 column volumes;

[0094] Loading: load the sample after diluting the Diamond Q mustang eluate with an equal volume of 20mM Phosphate Buffer (pH6.8) containing 4M NaCl;

[0095] Re-equilibration: after loading, re-equilibrate the column with 20mM Phosphate Buffer (pH6.8) containing 2M NaCl for 6-8 column volumes;

[0096] Elution: Linear elution with 20 mM phosphate buffer (pH 6.8), start to collect elution peak when UV280 reaches 150 mAU, stop when UV280 drops to 200 mAU;

[0097] The eluate was purified by Phenyl (HS) column, the target protein mainly existed in the flow-through, and the purity of the antigen was about 95%, which indicated that Phenyl (HS) could not improve the purity of recombinant hMPV F protein and was not suitable for fine purification of recombinant hMPV F protein.

[0098] Example 5, ultrafiltration displacement of recombinant hMPV F protein:

[0099] The ultrafiltration membrane package was 50 kD ultrafiltration membrane package (Sartorius);

[0100] Membrane package cleaning: the membrane package was cleaned with water and 0.5 M NaOH respectively, then was hydrolyzed with 0.5 M NaOH for not less than 30 minutes, and finally was cleaned with 10 membrane volumes of water;

[0101] Membrane package wetting: 10 membrane volumes of 20 mM phosphate buffer (pH 6.8) were used for wetting;

[0102] The purified liquid 3 was subjected to displacement (displacement multiple ≥ 6 times) with 50 kD ultrafiltration membrane package (loading capacity ≤ 100 L / m2, 1 L / m2 / min, TMP ≤ 1 bar) to the original buffer solution containing 50 mM-200 mM NaCl, 50 mM NaCl and 0.10% PS80.

[0103] Example 6, virus removal filtration of recombinant hMPV F protein:

[0104] Pre-filter membrane: Virosart Max (Sartorius);

[0105] Virus removal filtration membrane: Virosart HF (Sartorius);

[0106] Membrane cleaning: 5 membrane volumes of sterile water were used for washing;

[0107] Membrane wetting: 4 membrane volumes of the original buffer solution (pH 6.8) were used for buffer wetting;

[0108] The ultrafiltration liquid was subjected to pre-filtration and virus removal filtration (loading capacity ≤ 150 L / m2) in turn by using constant pressure mode (2 bar), and the filtered sample was the virus removal filtration liquid.

[0109] Final original solution: the virus removal filtration liquid was sterilized by 0.2 μm filter membrane to obtain the original solution of recombinant hMPV F protein.

[0110] Example 7, Isoelectric focusing electrophoresis of recombinant hMPV F protein

[0111] Isoelectric focusing electrophoresis (IEF) is an important means to determine the isoelectric point (pI) of a protein. The protein will stop moving forward when the net charge of the protein is zero during the electrophoresis process, and the pI of the protein can be determined by comparison with standard products after staining. The pI of a protein plays an important role in guiding protein purification, stability research, and formulation prescription screening.

[0112] Gel: Novex™ pH 3-10 IEF Protein Gel, 1.0 mm (Invitrogen);

[0113] Sample loading buffer: IEF Sample Buffer pH 3-10 (2x);

[0114] IEF Anode Buffer: IEF Anode Buffer (50x);

[0115] Cathode Buffer: IEF Cathode Buffer pH 3-10 (10x);

[0116] IEF Marker 3-10 standard product

[0117] According to the instructions provided by the manufacturer: 100V constant voltage for 1h; 200V constant voltage for 1h; 500V constant voltage for 30min; end electrophoresis staining, decolorization, view results, take pictures and save. The results are shown in the figure. The pI of the recombinant hMPV F is mainly in the range of 6.9-7.8, which is quite different from the calculated isoelectric point (6.2). The possible reason is that the computer calculates the isoelectric point of the protein mainly based on the primary amino acid sequence, while the recombinantly expressed hMPV F has post-translational modification (such as glycosylation) and spatial structure, so the isoelectric focusing electrophoresis can better represent the true isoelectric point of the protein.

[0118] The yield of the key step in the purification process, the purity of the process sample, and the HCP and HCD residual amount of the purification method of the application are shown in the following table.

[0119] Name Yield % RP-HPLC (%) SEC-HPLC (%) HCP ppm HCD (pg / dose) Stock solution 40.15 99.5 98.7 6.25 6.68

[0120] The above embodiments are merely exemplary and are not intended to limit the present application. Based on the above description, those skilled in the art can make various changes and modifications to the present application without departing from the scope of the present application. Therefore, the scope of the present application should be defined by the appended claims rather than by the foregoing description.

[0121] While particular embodiments of the application have been illustrated and described, it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the application. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this application.

Claims

1. A method for purifying recombinant human metapneumovirus pre-fusion F protein, characterized by, The method comprises: P1: filtering the fermentation broth by using a deep filter to obtain a fermentation clarified broth; P2: capturing the recombinant hMPV F protein from the fermentation clarified broth by using hydroxyapatite chromatography to obtain a purified liquid 1; P3: inactivating viruses in the purified liquid 1 by using a low-pH method to obtain an inactivated liquid; P4: performing anion exchange chromatography on the inactivated liquid to obtain a purified liquid 2; P5: performing composite weak anion chromatography on the purified liquid 2 to obtain a purified liquid 3; P6: performing UF / DF on the purified liquid 3 to obtain an ultrafiltration liquid; P7: performing virus-removing nanofiltration and sterile filtration on the ultrafiltration liquid to obtain a recombinant hMPV F protein stock solution. In the hydroxyapatite chromatography, the hydroxyapatite filler has a pore size of 40, 60 or 80 μm and is type II hydroxyapatite; the step P2 specifically comprises equilibrating the hydroxyapatite chromatography column with a phosphate buffer to a baseline stability; diluting the conductivity of the fermentation clarified broth with purified water to less than 8.0 mS / cm for sample loading; after the sample loading is completed, equilibrating the chromatography column again with a phosphate buffer containing 0-70 mM NaCl to a baseline stability; eluting with a phosphate buffer containing 70-500 mM NaCl, and collecting the eluate of the target protein to obtain the purified liquid 1. The step P3 specifically comprises adjusting the pH of the purified liquid 1 to ≤3.8 with ≤4M dilute hydrochloric acid, incubating at 18-26°C for 30-60 min, adjusting the pH to 6.5-7.0 with an alkali or Tris-HCl after the inactivation is completed, and obtaining the inactivated liquid; the alkali is 0.1-0.5M NaOH; the Tris-HCl has a concentration of 0.5-1.0M and a pH of 8.0-9.

0. The anion exchange filler in the anion exchange chromatography is an anion exchange filler with quaternary ammonium group [-CH2-N + (CH3)3] functional group; The anion exchange chromatography specifically comprises the following steps: equilibrating the anion exchange chromatography column with a phosphate buffer to a baseline stability; adjusting the inactivated liquid to a conductivity of not higher than 9.0 mS / cm for sample loading; after the sample loading is completed, equilibrating the chromatography column again with a phosphate buffer to a baseline stability; linearly eluting with a phosphate buffer containing 0-300 mM NaCl, and collecting the eluate of the target protein to obtain the purified liquid 2. The composite weak anion chromatography specifically comprises the following steps: equilibrating the chromatography column with a phosphate buffer to a baseline stability; loading the purified liquid P2 into the composite weak anion chromatography column; after the sample loading is completed, equilibrating the chromatography column again with a phosphate buffer containing 100-200 mM NaCl to a baseline stability; eluting with a phosphate buffer containing 0.5-1.0M NaCl, and collecting the eluate of the target protein to obtain the purified liquid 3.

2. The method of claim 1, wherein the recombinant human metapneumovirus pre-fusion F protein is purified by affinity chromatography using a monoclonal antibody that binds to the pre-fusion F protein. The UF / DF specifically comprises: performing ultrafiltration liquid exchange on the purified liquid 3 by using a 30-50 kDa ultrafiltration membrane pack, and the exchange ratio is ≥6 to obtain an ultrafiltration liquid.

3. The method for purifying recombinant human metapneumovirus pre-fusion F protein according to claim 1, characterized in that, The specific process of the virus-removing nanofiltration and sterile filtration is as follows: sequentially performing Virosart Max prefiltration and Virosart HF virus-removing filtration on the ultrafiltration liquid; and obtaining the recombinant hMPV F protein stock solution after the sterile filtration.

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