Method for purifying F protein before fusion of recombinant human metapneumovirus

Abstract

The invention 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 liquor by using a deep filter to obtain fermentation clear liquor; capturing the recombinant hMPV F protein from the fermentation clarified liquid by using hydroxyapatite to obtain a purified liquid 1; carrying out virus inactivation on the purified solution 1 by adopting a low-pH method to obtain an inactivated solution; carrying out anion exchange chromatography on the inactivated solution to obtain a purified solution 2; carrying out composite weak anion chromatography on the purified solution 2 to obtain a purified solution 3; performing UF / DF on the purified liquid 3 to obtain an ultrafiltrate; and carrying out virus removal nanofiltration and sterile filtration on the ultrafiltrate to obtain a recombinant hMPV F stock solution. The method is simple and convenient, has good repeatability, is suitable for large-scale production, and can be effectively used for producing the recombinant human metapneumovirus pre-fusion F protein vaccine.

Description

Technical Field

[0001] The invention belongs to the technical field of protein purification, and specifically relates to a method for purifying recombinant human metapneumovirus pre-fusion F protein. Background Art

[0002] Human metapneumovirus (hMPV) is a common respiratory virus belonging to the genus Metapneumovirus of the family Pneumoviridae. It was first discovered in the Netherlands in 2001 and is now widely prevalent around the world. It is an important pathogen that causes acute lower respiratory tract infections in infants, immunocompromised people, and the elderly. About 6% of infants and young children hospitalized for acute lower respiratory tract infections are caused by hMPV.

[0003] Although there is no approved vaccine to prevent hMPV infection, the recombinant RSV F protein vaccines (ABRYSVO, AREXVY) approved by the FDA have pointed the way for the development of hMPV vaccines. Both belong to the Pneumoviridae family and have similar structural proteins and invasion mechanisms. Similar to RSV F protein, hMPV F protein is also the main target for the body to produce neutralizing antibodies against the virus. It exists on the surface of the virus in a metastable "prefusion" trimer state. Once it comes into contact with the host cell, 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 protease site, and does not release the P27 peptide after enzymatic cleavage. In order to stabilize the "pre-fusion" conformation of the hMPV F protein extracellular domain, the sequence was changed in three main aspects: (1) a trimerization domain was introduced at the C-terminus of the hMPV F protein, such as the 27-residue domain at the C-terminus of the T4 phage fibritin molecule; (2) the furin protease site was deleted and the F2-F1 linker was introduced; (3) amino acid substitutions were used to introduce interchain disulfide bonds and internal cavity filling to promote the hMPV F protein to maintain its pre-fusion state. Please refer to Guillaume B et al. (2021) "Interprotomer disulfide-stabilized variants of the humanmetapneumovirus fusion glycoprotein induce high titer-neutralizing responses", PNAS, DOI: 10.1073 / PNAS.2106196118. Therefore, hMPV subunit vaccines developed with highly conserved fusion (F) protein as candidate antigen and pre-fusion conformation as design concept are very promising hMPV vaccines. In order to maintain similarity with natural viral proteins (especially protein post-translational folding, glycosylation modification, etc.), human viral subunit vaccines are mainly expressed in mammalian / eukaryotic cell lines (such as CHO cells, 293 cells, Sf9 insect cells, etc.). Mammalian cell lines have a long fermentation cycle, and will secrete a large amount of host proteins, DNA and other impurities during their own growth or apoptosis. The expressed F protein also exists in non-trimeric forms such as monomers and polymers, and there are fragmentation and conformational changes.

[0004] Therefore, there is an urgent need to develop a method for purifying F protein that is uniform, highly pure, and maintains its pre-fusion conformation. Summary of the invention

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

[0006] The first object of the present invention is to provide a method for purifying recombinant human metapneumovirus pre-fusion F protein, comprising the following steps: P1: Filter the fermentation liquid using a deep filter to obtain fermentation clarified liquid; P2: using hydroxyapatite to capture the recombinant hMPV F protein from the fermentation clarification liquid to obtain purified liquid 1; P3: Inactivate the virus in purified solution 1 by low pH method to obtain an inactivated solution; P4: performing anion exchange chromatography on the inactivated solution to obtain purified solution 2; P5: performing composite weak anion chromatography on purified solution 2 to obtain purified solution 3; P6: Perform UF / DF on the purified solution 3 to obtain an ultrafiltrate; P7: The ultrafiltrate is subjected to virus removal nanofiltration and sterile filtration to obtain the recombinant hMPV F protein stock solution.

[0007] In the present invention, hydroxyapatite is selected to capture the target protein, which can remove most of the pigments, some nucleic acids and host proteins; then strong anion exchange chromatography is selected to remove most of the host proteins, most of the nucleic acids and endotoxins; and then composite weak anion chromatography is further selected to further purify and remove aggregates, a small amount of residual host proteins, nucleic acids and uninactivated viruses.

[0008] In some embodiments, the hydroxyapatite filler in the hydroxyapatite chromatography is type II hydroxyapatite (CHT type II) with a pore size of 40, 60, or 80 μm; In some embodiments, the P2 step specifically includes: The CHT chromatography column was balanced with phosphate buffer (PB buffer) until the baseline was stable; the conductivity of the fermentation clarified liquid was diluted with purified water to less than 8.0 mS / cm for loading; after the loading was completed, the chromatography column was balanced again with a phosphate buffer containing 0-70 mM NaCl until the baseline was stable; 70-500 mM NaCl phosphate buffer was used for elution, and the target protein eluate was collected to obtain the purified solution 1.

[0009] In some embodiments, the concentration of the phosphate buffer (PB buffer) is 10-30 mM, and the pH is 6.6-7.0.

[0010] In some embodiments, the P3 step specifically includes adjusting the pH of the purified solution 1 to ≤3.8 with ≤4M dilute hydrochloric acid, incubating at 18~26°C for 30~60min, and after inactivation, adjusting the pH to 6.5~7.0 with alkali solution or Tris-HCl to obtain the inactivated solution; the alkali solution is 0.1~0.5M NaOH; the concentration of Tris-HCl is 0.5~1.0M, and the pH is 8.0~9.0.

[0011] In some embodiments, the anion exchange filler in the anion exchange chromatography is a quaternary ammonium group [-CH 2 -N + (CH 3 ) 3 ] or diethylaminoethyl [-CH 2 -CH 2 -N + -(CH 2 -CH 3 ) 2 ] anion exchange filler with functional groups. In some embodiments, the anion exchange chromatography specifically comprises the following steps: The anion exchange chromatography column is balanced with a phosphate buffer (PB buffer) until the baseline is stable; the inactivation solution is adjusted to a conductivity of no more than 9.0 mS / cm for loading; after loading, the chromatography column is balanced again with a phosphate buffer until the baseline is stable; a phosphate buffer containing 0 to 300 mM NaCl is used for linear elution, and the target protein eluate is collected to obtain the purified solution 2. In some embodiments, the concentration of the phosphate buffer (PB buffer) is 10 to 30 mM, and the pH is 6.6 to 7.0.

[0012] In some embodiments, the composite weak anion chromatography specifically comprises the following steps: The chromatography column is equilibrated with phosphate buffer until the baseline is stable; the purified solution P2 (conductivity ≤ 20 ms / cm) is loaded onto the composite weak anion chromatography column with high salt; after loading, the chromatography column is equilibrated again with phosphate buffer containing 100-200 mM NaCl until the baseline is stable; elution is performed with phosphate buffer containing 0.5-1.0 M NaCl, and the target protein eluate is collected to obtain the purified solution 3.

[0013] In some embodiments, the filler in the composite chromatography is a multimodal strong anion filler with phenyl glycidyl ether and 2,3-epoxypropyltrimethylammonium chloride functional groups, such as Diamond Mix A Mustang, Smac MMA or Capto adhere.

[0014] In the present invention, high salt loading which cannot be achieved by conventional ion exchange can be achieved by using the benzene ring on the filler ligand. Therefore, the sample in the previous step does not need any treatment and can be directly loaded with high salt.

[0015] In some embodiments, the UF / DF specifically comprises the following steps: The purified solution 3 was ultrafiltrated using a 30-50 kDa ultrafiltration membrane package, with the replacement times being ≥ 6 to obtain an ultrafiltrate.

[0016] In some embodiments, the specific process of the virus removal filtration is as follows: the ultrafiltrate is sequentially subjected to Virosart Max pre-filtration and Virosart HF virus removal filtration, and then sterile filtration to obtain the recombinant hMPV F protein stock solution.

[0017] In some embodiments, the recombinant hMPV F protein cell culture supernatant is the cell culture supernatant collected after the recombinant hMPV F protein is expressed in CHO, 293, Sf9 and other cells.

[0018] The second object of the present invention is to provide a recombinant hMPV F protein stock solution prepared according to the recombinant human metapneumovirus pre-fusion F protein purification method.

[0019] Compared with the prior art, the advantages and positive effects of the present invention are: By adopting the method of the present invention, the recombinant hMPV F protein expressed by CHO cells can be effectively separated and purified. 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 bands are clear and single, the method is simple, the repeatability is good, it is suitable for large-scale production, and can be effectively used to produce recombinant human metapneumovirus pre-fusion F protein vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0021] Figure 1 :The electrophoresis detection profile of the complete purification process of hMPV F protein; Figure 2A : High pressure liquid phase (molecular sieve) detection spectrum of recombinant hMPV F protein stock solution; Figure 2B : High pressure liquid chromatography (reverse phase C4) detection spectrum of recombinant hMPV F protein stock solution; Figure 3 : Screening of recombinant hMPV F protein CHT Ⅱ type chromatography filler by SDS-PAGE electrophoresis detection; Figure 4 :Screening of recombinant hMPV F protein Diamond Viru-S affinity chromatography medium and SDS-PAGE electrophoresis detection pattern; Figure 5A :The stability SDS-PAGE electrophoresis detection profile of recombinant hMPV F protein CHT eluate under different pH conditions; Figure 5B : SEC-HPLC detection profile of the stability of recombinant hMPV F protein CHT eluate under different pH conditions; Figure 6 :Screening of recombinant hMPV F protein UniGel 30Q strong anion chromatography filler by SDS-PAGE electrophoresis detection; Figure 7 :Screening of recombinant hMPV F protein Diamond MixA mustang composite weak anion chromatography filler SDS-PAGE electrophoresis detection pattern; Figure 8 : Isoelectric focusing electrophoresis of recombinant hMPV F protein. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific embodiments: To facilitate a better understanding of the present invention, but not to limit the present invention, the experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0023] The DNA sequence of the recombinant hMPV F protein was integrated into CHO cells, and the GS (glutamine synthetase, GS) screening system was used to screen stable high-expressing cell lines under pressure, and a round of limiting dilution + continuous photography was used to select high-expressing monoclonal cell lines. The monoclonal cell lines were revived, and the seed cells were gradually expanded and cultured by shaking flasks. The prepared seed cells were transferred to a 5L bioreactor, and the target protein was produced by fed-batch culture. The fermentation broth was clarified and filtered using a deep filter, and the resulting fermentation clarification was the fermentation clarification. First, hydroxyapatite (CHT) filler was used to capture the target protein from the fermentation clarification in a bind / elute mode, and then the CHT eluate was treated with low pH virus inactivation. The inactivated solution was loaded onto UniGel 30Q filler for further purification in a bind / elute mode. The UniGel 30Q eluate was finely purified by Diamond MixA mustang filler in a bind / elute mode. The Diamond MixA mustang eluate was subjected to ultrafiltration replacement treatment, followed by virus removal filtration and sterile filtration. The results of the complete purification process of recombinant hMPV F protein are shown in the electrophoresis test. Figure 1 (From left to right, lanes are: M: protein marker indicator; 1: fermentation clarification 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). The HPLC reverse phase spectrum of the stock solution is shown in Figure 2A and molecular sieve spectra see Figure 2B .

[0024] Example 1, Capturing recombinant hMPV F protein from fermentation clarification liquid:

[0025] Chromatographic filler name: CHT type Ⅱ; Column equilibration: equilibrate with 20 mM phosphate buffer (pH 6.8) for 6 to 8 column volumes; Loading: Adjust the conductivity of the clarified fermentation broth to ≤8.0mS / cm with purified water before loading; Re-equilibration: After loading, re-equilibrate with 20 mM phosphate buffer (pH 6.8) containing 70 mM NaCl for 6 to 8 column volumes; Elution: linear elution with 20 mM phosphate buffer (pH 6.8) containing 70-500 mM NaCl. Start collecting the elution peak when UV280 reaches 200 mAU and stop collecting the peak when UV280 drops to 200 mAU. The fermentation clarification liquid was purified by CHT II column, and the target protein was collected in binding / elution mode. The purity of the captured antigen in the first step reached 85%. The electrophoresis pattern of the chromatographic sample is shown in Figure 3 Among them, the lanes from left to right are, 1: fermentation clarified liquid sample (reduced); 2~3: capture flow-through (reduced); 4: elution collection peak (reduced); M: protein marker; 5: fermentation clarified liquid sample (non-reduced); 6~7: capture flow-through (non-reduced); 8: elution collection peak (non-reduced). CHT Ⅱ type filler has obvious effect in capturing recombinant hMPV F protein. The target protein is in a trimer state in the non-reduced state, which is consistent with the theoretical design and is suitable for the capture and purification of recombinant hMPV F protein.

[0026] Comparative Example 1, capturing recombinant hMPV F protein from fermentation clarification liquid:

[0027] Chromatography medium name: Diamond Viru-S; Column equilibration: equilibrate with 20 mM phosphate buffer (pH 6.8) for 6 to 8 column volumes; Loading: Adjust the conductivity of the clarified fermentation broth to ≤5mS / cm with purified water before loading; Re-equilibration: After loading, re-equilibrate with 20 mM phosphate buffer (pH 6.8) for 6 to 8 column volumes; 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. The fermentation clarification liquid was purified by Diamond Viru-S chromatography column. Most of the target protein was present in the flow-through liquid, and a small part was bound to the chromatography column. The electrophoresis pattern of the chromatography sample is shown in Figure 4 , where the lanes from left to right are: M: protein marker; 1: fermentation clarification sample; 2-5: capture flow-through; 6-7: samples collected from different elution peaks. Diamond Viru-S chromatography is not suitable for the capture and purification of the recombinant hMPV F protein.

[0028] Comparative Example 2, capturing recombinant hMPV F protein from fermentation clarification liquid:

[0029] Chromatographic medium name: Q Bestarose BB; Column equilibration: equilibrate with 20 mM phosphate buffer (pH 6.8) for 6 to 8 column volumes; Loading: Adjust the conductivity of the clarified fermentation broth to ≤5.0 mS / cm with purified water before loading; Re-equilibration: After loading, re-equilibrate with 20 mM phosphate buffer (pH 6.8) for 6 to 8 column volumes; 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. The fermentation clarified liquid was purified by Q Bestarose BB chromatography column, and the target protein was collected in binding / elution mode. The antigen purity was about 75%. The Q Bestarose BB eluate was yellow and contained a large amount of pigment, which interfered with the determination of antigen concentration by UV spectrophotometer. Therefore, Q Bestarose BB chromatography was not suitable for the capture and purification of recombinant hMPV F protein.

[0030] Example 2, Recombinant hMPV F protein virus inactivation process:

[0031] The eluate in Example 1 was inactivated by low pH virus inactivation. The eluate in Example 1 was gradually adjusted to pH 3.5 with acid, and then placed at room temperature for 60 min. The pH was then adjusted to pH 7.0 with alkali. During the low pH adjustment process, continuous sampling was performed to detect whether the target antigen structure changed. The sample electrophoresis and SEC-HPLC patterns are shown in Figure 5A(Lane 1 to lane 7 from left to right: pH 6.5, pH 6.0, pH 5.5, pH 5.0, pH 4.5, pH 4.0, pH 3.5; Lane 8: Protein Marker) and Figure 5B ,During the process of adjusting the low pH, the structure of the target protein remained unchanged, indicating that the low pH virus inactivation method is suitable for the inactivation of recombinant hMPV F protein virus.

[0032] Example 3, intermediate purification of recombinant hMPV F protein:

[0033] Chromatographic filler name: UniGel 30Q; Column equilibration: equilibrate with 20 mM phosphate buffer (pH 6.8) for 6 to 8 column volumes; Loading: Adjust the conductivity of the inactivated solution to ≤9.0 mS / cm with 20 mM phosphate buffer (pH 6.8) before loading; Re-equilibration: After loading, re-equilibrate with 20 mM phosphate buffer (pH 6.8) for 6 to 8 column volumes; Elution: linear elution with 20 mM phosphate buffer (pH 6.8) containing 0-300 mM NaCl. Start collecting the elution peak when UV280 reaches 200 mAU and stop collecting the peak when UV280 drops to 200 mAU. The inactivated solution was purified by UniGel 30Q column, and the target protein was collected in binding / elution mode. The protein purity was about 95%. The electrophoresis and RP-HPLC spectra of UniGel 30Q eluted samples are shown in Figure 6 , where the lanes from left to right are, M: protein marker; 1: CHT eluate sample; 2-4: elution peak segmented collection samples; the results showed that the purity of the recombinant hMPV F protein was significantly improved, and UniGel 30Q chromatography was suitable for the moderate purification of the recombinant hMPV F protein.

[0034] Comparative Example 3, moderate purification of recombinant hMPV F protein:

[0035] Chromatography medium name: Diamond SP mustang; Column equilibration: equilibrate with 20 mM phosphate buffer (pH 6.8) for 6 to 8 column volumes; Loading: Adjust the conductivity of the inactivated solution to ≤5.0 mS / cm with 20 mM phosphate buffer (pH 6.8) before loading; Re-equilibration: After loading, re-equilibrate with 20 mM phosphate buffer (pH 6.8) for 6 to 8 column volumes; Elution: linear elution with 20 mM phosphate buffer (pH 6.8) containing 0-300 mM NaCl. Start collecting the elution peak when UV280 reaches 200 mAU and stop collecting the peak when UV280 drops to 200 mAU. The inactivated solution was purified by Diamond SP Mustang chromatography column. The target protein was mainly present in the flow-through and the antigen purity was about 85%, indicating that Diamond SP Mustang chromatography was suitable for the moderate purification of recombinant hMPV F protein.

[0036] Example 4, fine purification of recombinant hMPV F protein:

[0037] Chromatography filler name: Diamond MixA mustang; Column equilibration: equilibrate with 20 mM phosphate buffer (pH 6.8) for 6 to 8 column volumes; Sample loading: The UniGel 30Q eluate was adjusted to a conductivity of ≤20.0 mS / cm with 20 mM phosphate buffer (pH 6.8) before loading; Re-equilibration: After loading, re-equilibrate with 20 mM phosphate buffer (pH 6.8) containing 100-200 mM NaCl for 6-8 column volumes; Elution: linear elution with 20 mM phosphate buffer (pH 6.8) containing 0.5-1.0 M NaCl. Start collecting the elution peak when UV280 reaches 150 mAU and stop collecting the peak when UV280 drops to 200 mAU. The UniGel 30Q eluate was purified by Diamond MixA mustang chromatography column, and the target protein was collected in binding / elution mode with a protein purity of about 99%. Diamond MixA mustang is a multi-mode column that can interact with the target antigen through electrostatic interaction, hydrogen bonding and hydrophobic interaction. It has obvious advantages in removing impurities such as aggregates, fragments, HCP, etc. in the sample, and is also an important step in virus removal. See the electrophoresis pattern of the eluted sample of the chromatography filler for details. Figure 7 , from left to right, lanes are: M: protein marker; 1: UniGel 30Q eluate sample; 2~4: elution peak segmented collection samples. The results showed that the purity of recombinant hMPV F protein was significantly improved, and Diamond MixA mustang chromatography was suitable for the fine purification of recombinant hMPV F protein.

[0038] Comparative Example 4, fine purification of recombinant hMPV F protein:

[0039] Chromatographic filler name: Phenyl (HS); Column equilibration: equilibrate with 20 mM phosphate buffer (pH 6.8) containing 2 M NaCl for 6 to 8 column volumes; Sample loading: Diamond Q mustang eluate was diluted with an equal volume of 20 mM phosphate buffer (pH 6.8) containing 4 M NaCl and then loaded; Re-equilibration: After loading, re-equilibrate with 20 mM phosphate buffer (pH 6.8) containing 2 M NaCl for 6 to 8 column volumes; Elution: linear elution was performed with 20 mM phosphate buffer (pH 6.8). The elution peak was collected when UV280 reached 150 mAU and stopped when UV280 dropped to 200 mAU. The Diamond Q mustang eluate was purified by Phenyl (HS) chromatography column. The target protein was mainly present in the flow-through and the antigen purity was about 95%. This 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.

[0040] Example 5, ultrafiltration replacement of recombinant hMPV F protein:

[0041] The ultrafiltration membrane package was a 50 kD ultrafiltration membrane package (Sartorius); Membrane cleaning: clean the membrane with injection water and 0.5M NaOH respectively, then circulate with 0.5M NaOH for no less than 30 minutes, and finally clean 10 membrane volumes with injection water; Membrane package rinsing: rinse 10 membrane volumes with 20 mM phosphate buffer (pH 6.8); The purified solution 3 was replaced (replacement times ≥ 6 times) using a 50kD ultrafiltration membrane pack (capacity ≤ 100 L / m², 1L / m2 / min, TMP ≤ 1bar) to a stock buffer solution containing 50mM to 200mM NaCl of disodium hydrogen phosphate / sodium dihydrogen phosphate, 50mM NaCl and 0.10% PS80.

[0042] Example 6, virus removal filtration of recombinant hMPV F protein:

[0043] Pre-filter membrane: Virosart Max (Sartorius); Virus removal filter membrane: Virosart HF (Sartorius); Membrane cleaning: rinse 5 membrane volumes with sterile water; Rinse the filter membrane: Rinse 4 volumes of the membrane with stock buffer (pH 6.8); The ultrafiltrate was pre-filtered and filtered for virus removal (loading capacity ≤ 150 L / m²) in a constant pressure mode (2 bar). The filtered sample was the virus-removal filtrate.

[0044] Final stock solution: The virus-free filtrate is sterile filtered through a 0.2 μm filter membrane to obtain the recombinant hMPV F protein stock solution.

[0045] Example 7, Isoelectric focusing electrophoresis of recombinant hMPV F protein:

[0046] Isoelectric focusing (IEF) is an important method for determining the isoelectric point (pI) of proteins. During electrophoresis, when the net charge of a protein is zero, it will stay at its current position and no longer move forward. The pI of a protein can be determined by staining and comparing it with a standard. Protein pI plays an important role in guiding protein purification, stability studies, and formulation screening.

[0047] Gel: Novex™ pH 3-10 IEF Protein Gel, 1.0 mm (Invitrogen); Sample loading buffer: IEF Sample Buffer pH 3–10 (2×); IEF Anode Buffer: IEF Anode Buffer (50×); Cathode Buffer: IEF Cathode Buffer pH 3–10 (10×); IEF Marker 3-10 Standard The operation was carried out according to the instructions provided by the manufacturer: 100V constant voltage for 1h; 200V constant voltage for 1h; 500V constant voltage for 30min; end the electrophoresis staining, decolorization to check the results, take pictures and save. The results are shown in the figure. The pI of recombinant hMPV F is mainly between 6.9 and 7.8, which is quite different from the isoelectric point calculated by computer (6.2). The possible reason is that the computer calculates the isoelectric point of protein mainly based on the primary amino acid sequence, while the recombinantly expressed hMPV F has post-translational modifications (such as glycosylation) and spatial structure, so isoelectric focusing electrophoresis can better show the true protein isoelectric point.

[0048] The yields of key steps in the purification process of the purification method of the present invention, the purity of the process samples, and the residual amounts of HCP and HCD are shown in the table below.

[0049] name Yield % RP-HPLC (%) SEC-HPLC (%) HCP ppm HCD (pg / dose) Original solution 40.15 99.5 98.7 6.25 6.68 The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

[0050] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will also be appreciated by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A method for purifying recombinant human metapneumovirus pre-fusion F protein, characterized in that: The method comprises: P1: Filter the fermentation liquid using a deep filter to obtain fermentation clarified liquid; P2: using hydroxyapatite to capture the recombinant hMPV F protein from the fermentation clarification liquid to obtain purified liquid 1; P3: Inactivate the virus in purified solution 1 by low pH method to obtain an inactivated solution; P4: performing anion exchange chromatography on the inactivated solution to obtain purified solution 2; P5: performing composite weak anion chromatography on purified solution 2 to obtain purified solution 3; P6: Perform UF / DF on the purified solution 3 to obtain an ultrafiltrate; P7: The ultrafiltrate is subjected to virus removal nanofiltration and sterile filtration to obtain the recombinant hMPV F protein stock solution.

2. The method for purifying recombinant human metapneumovirus pre-fusion F protein according to claim 1, characterized in that: The hydroxyapatite filler in the hydroxyapatite chromatography is type II hydroxyapatite with a pore size of 40, 60 or 80 μm.

3. The method for purifying recombinant human metapneumovirus pre-fusion F protein according to claim 1, characterized in that: The P2 step specifically includes balancing the hydroxyapatite chromatography column with a phosphate buffer until the baseline is stable; diluting the conductivity of the fermentation clarified liquid with purified water to less than 8.0 mS / cm for loading; after the loading is completed, balancing the chromatography column again with a phosphate buffer of 0-70mM NaCl until the baseline is stable; eluting with a phosphate buffer of 70-500mM NaCl, collecting the target protein eluate, and obtaining the purified solution 1.

4. The method for purifying recombinant human metapneumovirus pre-fusion F protein according to claim 1, characterized in that: The P3 step specifically includes adjusting the pH of the purified solution 1 to ≤3.8 with ≤4M dilute hydrochloric acid, incubating at 18-26°C for 30-60min, and adjusting the pH to 6.5-7.0 with alkaline solution or Tris-HCl after inactivation to obtain the inactivated solution; the alkaline solution is 0.1-0.5M NaOH; the concentration of Tris-HCl is 0.5-1.0M, and the pH is 8.0-9.

0.

5. The method for purifying recombinant human metapneumovirus pre-fusion F protein according to claim 1, characterized in that: The anion exchange filler in the anion exchange chromatography is a quaternary ammonium group [-CH2-N + (CH3)3] functional group anion exchange filler.

6. The method for purifying recombinant human metapneumovirus pre-fusion F protein according to claim 5, characterized in that: The anion exchange chromatography specifically includes the following steps: balancing the anion exchange chromatography column with a phosphate buffer until the baseline is stable; adjusting the conductivity of the inactivation solution to no more than 9.0 mS / cm for loading; after the loading is completed, balancing the chromatography column again with a phosphate buffer until the baseline is stable; using a phosphate buffer containing 0 to 300 mM NaCl for linear elution, collecting the target protein eluate, and obtaining the purified solution 2.

7. The method for purifying recombinant human metapneumovirus pre-fusion F protein according to claim 1, characterized in that: The composite weak anion chromatography specifically includes the following steps: balancing the chromatography column with a phosphate buffer until the baseline is stable; loading the purified solution P2 onto the composite weak anion chromatography column; after loading, balancing the chromatography column again with a phosphate buffer containing 100-200 mM NaCl until the baseline is stable; eluting with a phosphate buffer containing 0.5-1.0 M NaCl, collecting the target protein eluate, and obtaining the purified solution 3.

8. The method for purifying recombinant human metapneumovirus pre-fusion F protein according to claim 1, characterized in that: The UF / DF specifically includes: using a 30-50 kDa ultrafiltration membrane package to ultrafilter the purified solution 3, the liquid replacement multiple is ≥6, and an ultrafiltrate is obtained.

9. The method for purifying recombinant human metapneumovirus pre-fusion F protein according to claim 1, characterized in that: The specific process of the virus removal filtration is as follows: the ultrafiltrate is sequentially subjected to Virosart Max pre-filtration and Virosart HF virus removal filtration; and then sterile filtration is performed to obtain the recombinant hMPV F protein stock solution.

10. The recombinant hMPV F protein stock solution prepared by the recombinant human metapneumovirus prefusion F protein purification method according to any one of claims 1 to 9.

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