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CN112225818A - Method for removing/inactivating viruses - Google Patents

Method for removing/inactivating viruses Download PDF

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CN112225818A
CN112225818A CN202011112058.7A CN202011112058A CN112225818A CN 112225818 A CN112225818 A CN 112225818A CN 202011112058 A CN202011112058 A CN 202011112058A CN 112225818 A CN112225818 A CN 112225818A
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fusion protein
virus
solution
chromatography
purified
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CN112225818B (en
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彭红卫
张磊
韩为跃
王冲
万云雷
俞亚波
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Shanghai Jingze Biological Technology Co ltd
Jiangsu Jingze Biomedical Co ltd
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Jiangsu Jingze Biomedical Co ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention provides a method for removing/inactivating viruses, which comprises the following steps: s1) providing a raw material liquid containing the Fc fusion protein produced by fermentation of the CHO cell line; and s2) incubating the feed solution at low pH for 0.5-8 hours to produce a first purified solution; wherein the low pH incubation has a pH of 3.0-4.0 and the pH is adjusted using citric acid or a salt thereof. The method of the invention not only can achieve the purpose of removing/inactivating viruses in the Fc fusion protein, but also retains the activity of the target protein, namely the Fc fusion protein, thereby preparing the high-purity high-concentration Fc fusion protein.

Description

Method for removing/inactivating viruses
Technical Field
The invention relates to the field of biotechnology, in particular to a method for removing/inactivating viruses by using protein, and more particularly to removal/inactivation of viruses in a downstream process for producing Fc fusion protein.
Background
The use of mammalian cell expression systems allows secreted proteins to be most similar to native proteins in terms of immunogenicity and antigenicity, and protein processing, such as glycosylation, is most accurate, and has found widespread use in CHO cells as expression vectors. The biotechnological products derived from the CHO expression line are at risk of viral contamination, which can lead to very serious clinical consequences, and the contamination may originate from the original cell line itself, or from foreign viruses accidentally introduced during the production process, and in order to ensure the safety of the biotechnological product, the virus needs to be removed/inactivated during the production process in order to reduce the content of virus-like particles per dose to a certain level.
It has been reported in the literature that the presence of endogenous retroviruses in CHO cells poses a certain risk to the safety of human administration. According to the ICH Q5A biological product virus safety evaluation regulation, the technology is required to have 2 steps or more than 2 steps, and at least one step has a virus removal/inactivation rate more than 104Can be considered as an effective virus removal/inactivation process. Therefore, for the pharmaceutical protein derived from CHO cells, the production process thereof must have a certain method for removing or inactivating viruses, and laboratory inactivation verification tests for the inactivation process are also required.
The downstream process of the fusion protein at present generally comprises the steps of filtering fermentation liquor, coarse purification and fine purification. The Fc fusion Protein is generally subjected to Protein A affinity chromatography and ion chromatography to remove related impurities and process impurities, and the virus is incubated at a low pH value in the process, but in the prior art, a technical scheme that the process impurities and a low pH value inactivation process are used as main integral purification steps is not available.
The virus removal rate under the existing process condition is generally 104On the other hand, for example, in the case of the wang shubi, etc. (verification of recombinant fusion protein column chromatography virus removal process, chinese biological product impurities, second stage 27 in 2014, 241-. Gel chromatography gave the best EMCV removal at 4.391log 10.
Chinese patent application 201810010465.3 discloses a purification method in a low pH virus inactivation process: use of 1) Low pH viral inactivation: adjusting the pH of the eluted protein solution after the protein A affinity chromatography purification to 3.6 by using an acidic solution, wherein the acidic solution is phosphoric acid; 2) neutralizing: neutralizing the protein solution after low pH treatment with an alkaline solution, and adjusting the pH to 5.5; 3) and (3) filtering: after standing for at least 0.5h, it was filtered through a 0.2 μ M filter and the HCP and ResDNA were 1199ppm and 5.64pg/mg, respectively, after 1M phosphate treatment.
Chinese patent application 201580038801.X discloses a method for purifying TNFR-Fc fusion proteins: and finally obtaining HCP of 32ppm by using the steps of affinity loading, affinity elution, HIC loading, HIC elution, AEX loading, AEX elution, MMC loading and MMCFF.
Chinese patent application 202010488460.9 discloses a method for reducing the content of acidic charge heteroplasmon in Fc fusion protein, and the HCP residue is reduced from 43.1ppm of sample loading to 11.0ppm, thus achieving the final quality target of the product.
Therefore, there is a need in the art to develop a novel method for separating and purifying proteins, which is capable of not only efficiently inactivating/removing viruses but also effectively removing HCPs, HCDs, etc., while maintaining protein activity.
Disclosure of Invention
The invention aims to provide an optimized method for separating and purifying protein, which adopts process impurities combined with a low pH value inactivation process, can efficiently inactivate/remove viruses, can effectively remove HCP, HCD and the like, and can retain the activity of the protein.
In a first aspect of the present invention, there is provided a method for removing/inactivating Fc fusion protein viruses, comprising the steps of:
s1) providing a raw material liquid containing the Fc fusion protein produced by fermentation of the CHO cell line;
s2) incubating the stock solution at low pH for 0.5-8 hours to produce a first purified solution;
wherein the low pH incubation has a pH of 3.0-4.0 and the pH is adjusted using citric acid or a salt thereof.
In another preferred embodiment, the first purified solution contains an Fc fusion protein.
In another preferred embodiment, the citrate salt is selected from: sodium citrate, potassium citrate, calcium citrate and ammonium citrate.
In another preferred example, the raw material solution is a chromatography solution subjected to Protein a affinity chromatography.
In another preferred embodiment, the low pH incubation is performed at a pH of 3.1 to 3.8, preferably 3.2 to 3.6, more preferably 3.3. + -. 0.1, 3.4. + -. 0.1, 3.5. + -. 0.1, 3.6. + -. 0.1, 3.7. + -. 0.1, 3.8. + -. 0.1.
In another preferred embodiment, the low pH incubation time is 1-7 hours, preferably 2-4 hours, more preferably 2 hours, 3 hours, 4 hours.
In another preferred embodiment, the temperature of the low pH incubation is between-28 ℃ and 28 ℃, preferably between-5 ℃ and 25 ℃.
In another preferred example, the method further comprises the steps of:
s3) loading the first purified solution into a deep filter for deep filtration to obtain a deep filtrate;
s4) loading the deep filtrate into an anion chromatographic column for anion chromatography to obtain anion chromatographic solution;
s5) loading the anion chromatographic solution onto a cation chromatographic column for cation chromatography to obtain a cation chromatographic solution; and
optionally, s6) subjecting said anion chromatography liquid to nanofiltration, resulting in a second purified liquid.
In another preferred embodiment, the second purified solution comprises an Fc fusion protein.
In another preferred example, in step s4), the washing solution for anion chromatography is 10-60mmol/L citric acid, pH 5.0-6.0; preferably 25mmol/L citric acid.
In another preferred embodiment, in step s5), the eluent for the cation chromatography is 5-30mmol/L phosphoric acid, 0.1-5mol/L NaCl, pH 6.0-7.0; preferably 10mmol/L phosphoric acid, 0.5mol/L NaCl, pH 6.2. + -. 0.2.
In another preferred embodiment, in step s3), the depth filtration is performed using a filter having a filter flux of 10-250L/m2。
In another preferred embodiment, in step s3), the depth filtration is performed using a filter having a filter flux of 40-180L/m2Preferably 60-150L/m2More preferably 100-2。
In another preferred embodiment, in step s3), the depth filtration is performed using a filter having a pore size of 0.1-5 μm, preferably 0.15-4 μm, more preferably 0.15-3.5 μm.
In another preferred example, in step s4), anion chromatography is performed using a chromatography column Bestarose Diamond MIX-A.
In another preferred embodiment, in step s4), the anion chromatography is performed on a chromatography column equilibrated with an organic acid, preferably citric acid, Tris, more preferably citric acid, at a concentration of 5-50mmol/L, preferably 25 mmol/L.
In another preferred example, in step s5), the cation chromatography is performed using a chromatography column with a medium particle size of less than 60 μm.
In another preferred embodiment, the cation chromatography is a column equilibrated with an organic acid, preferably citric acid, Tris, more preferably citric acid.
In another preferred embodiment, the cation chromatographic column is Nuvia HR-S.
In another preferred example, in step s6), the nanofiltration membrane has a pore size of less than 20 nm.
In another preferred embodiment, the concentration of the citric acid is 0.5-3 mol/L.
In another preferred embodiment, the citric acid concentration is 1-2mol/L, preferably 1 mol/L.
In another preferred embodiment, the purity of the first purified liquid Fc fusion protein SEC-HPLC is greater than 99.5%.
In another preferred embodiment, the first purified liquid Fc fusion protein SEC-HPLC assay purity is greater than 99.7%.
In another preferred embodiment, the second purified liquid Fc fusion protein SEC-HPLC assay purity is greater than 99.7%.
In another preferred embodiment, the first purified liquid Fc fusion protein SEC-HPLC assay purity is greater than 99.8%.
In another preferred embodiment, the Fc fusion protein is a recombinant human vascular endothelial growth factor Fc fusion protein.
In another preferred embodiment, the Fc fusion protein is a recombinant human vascular endothelial growth factor receptor 1 and/or receptor 2Fc fusion protein.
In another preferred embodiment, the Fc fusion protein is a recombinant human vascular endothelial growth factor receptor 1 and/or receptor 2 linked to the Fc fragment gene of Ig 1, preferably VEGFR1 and VEGFR2 linked to the Fc fragment gene of immunoglobulin Ig 1, and a recombinant protein expressed in a eukaryotic expression system, preferably, a total of 2-3 extracellular regions of different receptors of VEGFR1 and VEGFR2 fused to the Fc fragment of immunoglobulin Ig 1.
In another preferred embodiment, the amino acid sequence of the Fc fusion protein comprises the amino acid sequence shown in SEQ ID No. 1. More preferably, the amino acid sequence of the Fc fusion protein is shown as SEQ ID No. 1.
In another preferred embodiment, the removed/inactivated virus is selected from the group consisting of: mouse parvovirus (MVM), reovirus type III (Reo3), pseudorabies virus (PRV), murine leukemia virus (X-MuLV), or a combination thereof.
In another preferred embodiment, the virus removal/inactivation ratio of the first or second purified liquid Fc fusion protein is more than 104Preferably greater than 105More preferably greater than 105.5。
In another preferred embodiment, the virus removal/inactivation ratio of the first or second purified solution is more than 104。
In another preferred embodiment, the removal/inactivation ratio of pseudorabies virus (PRV) in the first or second purified Fc fluid is more than 104Preferably greater than 105。
In another preferred embodiment, the first or second purified murine leukemia virus (X-MuLV) removal/inactivation ratio is greater than 104Preferably greater than 105。
In another preferred embodiment, the removal/inactivation rates of the pseudorabies virus (PRV) and the murine leukemia virus (X-MuLV) in the first or second purified solution are both greater than 104Preferably greater than 105。
In another preferred embodiment, the removal/inactivation ratio of the mouse parvovirus (MVM) in the first or second purified solution is more than 104Preferably greater than 105。
In another preferred embodiment, the removal/inactivation ratio of the reovirus type III (Reo3) of the first or second purified solution is more than 104Preferably greater than 105。
In another preferred example, the HCP removal rate of the Fc fusion protein from the second purified solution is 99% or more.
In another preferred embodiment, the removal rate of the Fc fusion protein HCD in the second purified solution is 99% or more.
In a second aspect of the invention, there is provided a pharmaceutical composition comprising i) an Fc fusion protein produced using the method of the first aspect, and ii) a pharmaceutically acceptable carrier.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Detailed Description
The present inventors have conducted extensive and intensive studies and, for the first time, have unexpectedly developed a method for separating and purifying proteins using low pH incubation, which not only can achieve the purpose of removing/inactivating viruses in Fc fusion proteins, but also retains the activity of Fc fusion proteins, which are target proteins, thereby producing high-purity and high-concentration proteins. On the basis of this, the present invention has been completed.
Term(s) for
Fc fusion protein: namely, the Fc fragment fusion protein of Ig G1, refers to a recombinant protein which is obtained by linking VEGFR1 and VEGFR2 genes with the Fc fragment gene of Ig G1 at the gene level and is expressed in a eukaryotic expression system. VEGFR1 and VEGFR2 genes are linked to the Fc fragment gene of immunoglobulin Ig G1, and are expressed as recombinant proteins in eukaryotic expression systems. A total of 2-3 extracellular domains of the different receptors for VEGFR1 and VEGFR2 were fused to the Fc fragment of immunoglobulin Ig G1.
Preferably, the amino acid sequence of the Fc fusion protein comprises a sequence shown in SEQ ID No. 1, and more preferably, the amino acid sequence of the Fc fusion protein is a sequence shown in SEQ ID No. 1:
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDG KRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSP SHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGS EMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK(SEQ ID No.:1)。
preferably, the Fc fusion protein of the present invention is expressed by the CHO-DG44 cell line, and the Fc fusion protein can be selected from fermentation supernatants or obtained by any primary purification step. Preferably, the concentration of the Fc fusion protein is 0.1-8g/L (mass concentration).
HCP: i.e., host protein residue. Preferably, it refers to a host protein derived from the CHO-DG44 cell line.
HCD (hydrogen chloride): i.e., host cell DNA. Preferably, it refers to a host DNA derived from the CHO-DG44 cell line.
SEC-HPLC Size Exclusion Chromatography (SEC) is a liquid chromatography method that takes advantage of the unique properties of porous gel stationary phases to produce a separation that is largely based on differences in molecular size. SEC-HPLC is used for measuring the purity, and a method is recommended by referring to molecular exclusion chromatography of <0514> in four parts of 'Chinese pharmacopoeia' 2015 edition and a high performance liquid chromatograph (Agilent, 1260).
Fluorescent quantitative PCR method: referring to the 'Chinese pharmacopoeia' 2015 edition of the four parts <3407> exogenous DNA residue determination method, the fluorescent quantitative PCR instrument is ABI (StepOne Plus). In the present invention, HCD was measured using a CHO cell residual DNA detection kit from Huzhou Shenke Biotechnology Ltd.
Enzyme linked immunosorbent assay (ELISA): HCPs were determined by reference to the method recommended by a microplate reader (molecular Devices, model SpectraMax i3 x). In the invention, the Kit used is CHO Host Cell Proteins ELISA Kit of Cygnus company.
The reduction of the removed/inactivated virus is the reduction of the virus used as the evaluation of the virus inactivation/removal effect according to the virus inactivation evaluation provision of the 2002 national drug administration of technical methods and validation guidelines for removing/inactivating viruses of blood products. If the virus reduction is 4logs, it means that the inactivation/removal of the virus by this step is reduced by 4.
Deep filtration: when the particle size is smaller than the diameter of the medium channel, a filter cake cannot be formed on the surface of the filter medium, and the particles enter the medium, approach the wall surface of the channel through inertia and diffusion action, and are deposited under the action of static electricity and surface force so as to be separated from the fluid. In the present invention, it is preferable to subject the sample (the nano-filtrate of the recombinant human antibody fusion protein) to depth filtration using a Millipore depth filtration membrane (Sadolis, model MX0 SP).
Virus inactivation assay method: refer to technical methods for removing/inactivating viruses from blood products and guidelines for verification (national drug Authority [2002] 160).
Anion exchange column
Suitable anion exchange columns for use in the present invention include Bestarose Diamond MIX-A.
In the present invention, the amount of the chromatography medium to be used is not particularly limited and may be generally determined depending on the amount of the Fc fusion protein contained in the raw material to be purified.
Cation exchange column
As used herein, the term "cation exchange column of the present invention" refers to a cation exchange resin that can efficiently separate Fc fusion proteins and other impurities specifically.
The cation exchange resin suitable for use in the present invention is not particularly limited, and in the present invention, cation chromatography uses a column having a medium particle size of less than 60um, preferably Nuvia HR-S.
Method for removing and/or inactivating viruses in Fc fusion protein (fermentation liquid)
The present invention provides a method for removing and/or inactivating viruses in an Fc fusion protein comprising any combination of any two, three or all steps including pH incubation by low pH incubation, depth filtration, anion chromatography, cation chromatography.
Specifically, the method comprises the following steps:
s1) providing a raw material liquid containing the Fc fusion protein produced by fermentation of the CHO cell line;
s2) incubating the stock solution at low pH for 0.5-8 hours to produce a first purified solution;
wherein the low pH incubation has a pH of 3.0-4.0, and the pH is adjusted with citric acid or its salt, preferably a pH of 3.1-3.8, preferably 3.2-3.6, more preferably 3.3 + -0.1, 3.4 + -0.1, 3.5 + -0.1, 3.6 + -0.1, 3.7 + -0.1, 3.8 + -0.1.
Preferably, the low pH incubation is for a period of 1-7 hours, preferably 2-4 hours, more preferably 2 hours, 3 hours, 4 hours.
Preferably, the low pH incubation is performed at-28 ℃ to 28 ℃, preferably-5 ℃ to 25 ℃.
Preferably, the method further comprises the step of:
s3) loading the first purified solution into a deep filter for deep filtration to obtain a deep filtrate;
s4) loading the deep filtrate into an anion chromatographic column for anion chromatography to obtain anion chromatographic solution;
s5) loading the anion chromatographic solution onto a cation chromatographic column for cation chromatography to obtain a cation chromatographic solution; and
optionally, s6) subjecting said anion chromatography liquid to nanofiltration, resulting in a second purified liquid.
Preferably, in step s4), the washing solution for anion chromatography is 10-60mmol/L citric acid, pH 5.00-6.0; preferably 25mmol/L citric acid.
Preferably, in step s5), the eluent for the cation chromatography is 10mmol/L phosphoric acid, 0.5mol/L LN aCl, pH 6.2. + -. 0.2.
Preferably, in step s3), depth filtration is performed using a filter flux of 10-250L/m2Preferably 40-180L/m2Preferably 60-150L/m2More preferably 100-2Wherein the pore size of the depth filtration filter is 0.1-5 μm, preferably 0.15-4 μm, more preferably 0.15-3.5 μm.
Preferably, in step s4), anion chromatography is performed using a column Bestarose Diamond MIX-A equilibrated with an organic acid, preferably citric acid, Tris, more preferably citric acid, at a concentration of 25 mmol/L.
Preferably, in step S5), cationic chromatography is performed using a chromatography column with a medium particle size of less than 60 μm, preferably Nuvia HR-S; the cation chromatography column equilibrated with an organic acid is preferably citric acid, Tris, and more preferably citric acid.
Preferably, in step s6), the nanofiltration membrane has a pore size of less than 20 nm.
Preferably, the concentration of the citric acid is 0.5-3 mol/L; preferably 1 to 2mol/L, more preferably 1 to 1.5 mol/L.
Preferably, the purity of the first purified liquid Fc-fusion protein SEC-HPLC is greater than 99.5%, preferably greater than 99.7%.
Preferably, the second purified liquid Fc-fusion protein SEC-HPLC assay purity is greater than 99.7%, preferably greater than 99.8%.
Preferably, the Fc fusion protein is a recombinant human vascular endothelial growth factor Fc fusion protein, more preferably the Fc fusion protein is a recombinant human vascular endothelial growth factor receptor 1 and/or receptor 2Fc fusion protein linked to an Fc fragment gene of Ig G1, preferably VEGFR1 and VEGFR2 genes linked to an Fc fragment gene of immunoglobulin Ig G1, and is a recombinant protein expressed in a eukaryotic expression system. A total of 2-3 extracellular domains of the different receptors for VEGFR1 and VEGFR2 were fused to the Fc fragment of immunoglobulin Ig G1.
Preferably, the amino acid sequence of the Fc fusion protein is shown in SEQ ID No. 1.
Preferably, the removed/inactivated virus is selected from the group consisting of: mouse parvovirus (MVM), reovirus type III (Reo3), pseudorabies virus (PRV), murine leukemia virus (X-MuLV), or a combination thereof.
Preferably, the virus removal/inactivation rate of the Fc fusion protein of the first or second purification solution is more than 104Preferably greater than 105。
Preferably, the removal/inactivation rate of the first or second purified liquid Fc fusion protein pseudorabies virus (PRV) is more than 104Preferably greater than 105。
Preferably, the first or second purified liquid Fc fusion protein murine leukemia virus (X-MuLV) removal/inactivation rate is greater than 104Preferably greater than 105。
Preferably, the removal/inactivation rates of the first or second purified liquid Fc fusion protein for pseudorabies virus (PRV) and murine leukemia virus (X-MuLV) are both more than 104Preferably greater than 105。
Preferably, saidThe removal/inactivation rate of the Fc fusion protein of the first or the second purification solution to the mouse parvovirus (MVM) is more than 104Preferably greater than 105。
Preferably, the removal/inactivation rate of the first or second purified liquid Fc fusion protein reovirus type III (Reo3) is more than 104Preferably greater than 105。
Preferably, the HCP removal rate of the Fc fusion protein from the first or second purified solution is 99% or more.
Preferably, the removal rate of the Fc fusion protein HCD in the first or second purified solution is 99% or more.
Pharmaceutical composition
Preferably, the pharmaceutical composition comprises i) an Fc fusion protein prepared using the method described above, and ii) a pharmaceutically acceptable carrier.
Preferably, the Fc fusion protein SEC-HPLC assay purity is greater than 99.5%, preferably greater than 99.7%, more preferably 99.8%.
Preferably, the Fc fusion protein is a recombinant human vascular endothelial growth factor Fc fusion protein, more preferably the Fc fusion protein is a recombinant human vascular endothelial growth factor receptor 1 and/or receptor 2Fc fusion protein, more preferably the VEGFR1 and VEGFR2 genes are linked to an Fc fragment gene of immunoglobulin Ig 1 and are expressed in a eukaryotic expression system. More preferably, a total of 2-3 extracellular domains of the different receptors for VEGFR1 and VEGFR2 are fused to the Fc fragment of immunoglobulin Ig G1. Preferably, the amino acid sequence of the Fc fusion protein is shown in SEQ ID No. 1.
The main advantages of the invention include:
(1) the purification method has good virus removal/inactivation effect, and can prepare high-purity Fc fusion protein with the purity of more than 99.8 percent.
(2) In the purified product, mouse parvovirus (MVM), reovirus type III (Reo3) and host HCP and HCD are effectively removed/inactivated, and the requirements of medicaments can be met.
(3) The method has simple and convenient operation of the technical process, does not need special reagents, does not contain the technical steps or operations which are not easy to amplify, such as gel chromatography, dialysis and the like, and is beneficial to large-scale production.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Examples
Experimental Material
The Fc fusion protein sample is from Shanghai Jingze biotechnology limited, and is expressed by CHO-DG44 cell strain, wherein the amino acid sequence of the obtained Fc fusion protein is shown as SEQ ID No. 1.
Determination of HCP: the Cygnus CHO Host Cell Proteins ELISA Kit.
And (3) determining HCD: CHO cell residual DNA detection kit of Huzhou Shenke biotechnology limited company.
1. Low pH incubation
Selection of a buffer: directly adjusting the pH value of a sample to 5.5 by using 1mol/L Tris, and placing the sample at the temperature of 2-8 ℃ to be detected; and (3) taking the same batch of samples, adjusting the pH of the Fc fusion protein to 3.0 by using an acidic titration solution (the concentration is 1mol/L), keeping the same batch of samples at room temperature (18-26 ℃) for 3 hours, and then adjusting the pH back to 5.5 by using an alkaline titration solution. The monomer purity was measured and the results are shown in Table 1
TABLE 1 Effect of different acids or acid buffers on the purity of Fc fusion protein monomers
Figure BDA0002728906980000101
The results show that: the purity of the obtained Fc fusion protein monomer is lower than that of citric acid and citrate buffer solution under the condition of obtaining the same pH (3.0) by using 1mol/L hydrochloric acid, phosphoric acid and buffer solution titration solution with the same concentration.
Adjusting the pH value of the Fc fusion protein to 3.0 by using citric acid and citrate buffer solution, keeping the purity of the Fc fusion protein more than 99.1% for 3 hours at room temperature (18-26 ℃), stabilizing the Fc fusion protein, and detecting the biological activity within the range of 80-120%. Thus, the product was initially considered stable at pH3.3-3.7 for 3 hours in an acidic titration solution of 1mol/L citric acid and citrate (pH 3.0).
Incubation time and pH selection: and (3) directly adjusting the pH value of the control sample to 5.5 by using citric acid and citrate buffer solution, and placing the control sample at the temperature of 2-8 ℃ for detection. Adjusting the pH value to 3.2, 3.4, 3.6, 3.8 and 4.0 by using citric acid or citrate buffer solution, and adjusting the pH value back to 5.5 after incubating for 2h, 4h, 6h and 8h at room temperature (18-26 ℃). The effect of low pH incubation on the product is shown in Table 2 below
Biological activity: reference is made to the "chinese pharmacopoeia" 2015 edition of three <3531> nimotuzumab biological activity assays, based on the amount of Fc fusion protein required to inhibit half of VEGF.
TABLE 2
Figure BDA0002728906980000111
The above results show that: and (3) incubating under five pH conditions (pH3.2, 3.4, 3.6, 3.8 and 4.0), wherein the purity of the sample is lower than 99.7% when the sample is incubated for 8 hours, the purity of the sample is basically not obviously changed when the sample is incubated for 1 hour, 2 hours, 4 hours and 6 hours, and the purity is higher than 99.5%. The purity of the culture medium is 99.5-99.8% after five kinds of pH conditions (pH3.2, 3.4, 3.6, 3.8 and 4.0) are incubated, and the purity tends to be reduced along with the prolonging of the incubation time. The biological activity detection result has a decreasing trend along with the prolonging of the incubation time, but all the results are in the range of 75-115%.
Biological Activity (Cell based bioassay) and purity (SEC-HPLC (%))
At 25 ℃, 4g/L of Fc fusion Protein solution purified by Protein A affinity chromatography, 5L, 1mol/L of citric acid, pH3.4 adjusted, incubated for 2h, and measured for biological activity (Cell based bioassay) and purity (SEC-HPLC (%))
The incubation results are shown in table 4:
TABLE 4
Figure BDA0002728906980000121
After incubation, the stability of the samples was determined by adjusting the pH to 5.5 with citric acid, and the results are shown in Table 5:
TABLE 5
Temperature of Time of day Purity SEC-HPLC (%)
At room temperature 0 99.8
At room temperature 28D 99.7
2～8℃ 56D 99.8
The results show that: incubating and inactivating the virus at the pH value of 3.6 for 2h, and after the pH is adjusted back to 5.5, the sample is stable when being stored at the room temperature (18-26 ℃) for 14 days, and the monomer purity (SEC-HPLC) is 99.8%; the monomer purity (SEC-HPLC) after being stored for 28 days at the temperature of 2-8 ℃ is consistent with the measurement result of the first day of storage and is 99.8 percent. This shows that this step has no significant effect on the purity and activity of the product, ensuring the safety and effectiveness of the product.
The results of verifying the virus removal/inactivation effects and the removal/inactivation effects (residual indicator virus titer (LgTCID50/0.1ml)) of murine leukemia virus (X-Mulv) and pseudorabies virus (PRV) in the samples according to the "blood product removal/inactivation Virus technique and verification guide principles" (national drug Authority [2002] 160) are shown in Table 3
TABLE 3-1
Virus Viral reduction
X-Mulv ≥4.78
PRV ≥4.83
The results show that the low pH incubation method of the invention can effectively remove mouse leukemia virus (X-Mulv) and pseudorabies virus (PRV).
2. Deep filtration
Low pH incubations were incubated with a depth filter NP7PDE21(PALL) (0.2-3.5um) equilibrated with 25mmol/L citrate buffer at a flux of 120L/m2The HCP (ELISA) was 192ppm and the HCD (fluorescence quantitative PCR) was 8 pg/mg.
TABLE 3-2
Item HCP(ELISA) HCD (fluorescent quantitative PCR method)
Before Low pH incubation 418ng/mg 91pg/mg
After low pH incubation and deep filtration 192ng/mg 8pg/mg
Removal rate 54％ 91％
The removal rates of HCP and HCD reach 54 percent and 91 percent respectively.
3. Anion chromatography
The filtrate from the depth filtration of the previous step was applied to a Bestarose Diamond MIX-A column (packing from Bogelong Shanghai Biotechnology Co., Ltd.) equilibrated at pH 5.3-5.7 in "25 mmol of citric acid buffer" for chromatography. The peak containing the recombinant human antibody fusion protein of this example was detected in the collected flow-through by ELISA.
The virus removal/inactivation effect, the removal/inactivation effect of mouse parvovirus (MVM) and reovirus type III (Reo3) of the sample (residual indicator virus titer (LgTCID50/0.1m l)) was verified with reference to "blood product removal/inactivation virus technique and verification guide (national drug administration [2002] 160)".
The virus reduction was calculated by the Karber method using the 96-well cell pathology method. The assay was repeated twice for each batch of samples. The Karber method formula is: LgTCID50 ═ L-d (S-0.5), where L is the logarithm of the highest dilution; d is the difference between the dilution logarithms; s is the sum of the positive pore ratios. The virus reduction is the difference between the virus titer (LgTCID50/0.1mL) measured in the zero control sample and the sample after the inactivation process.
The results are given in Table 6 below
TABLE 6
Figure BDA0002728906980000131
The experimental result shows that the reduction of the mouse leukemia virus (X-Mulv) virus is 4.48log, the reduction of the mouse parvovirus (MVM) virus is 4.45log, the method has good removal effect on HCP (ELISA) and HCD (fluorescence quantitative PCR), and the results are respectively 90ng/mg and 0.1pg/mg after the determination.
4. Cation chromatography
The flow-through obtained in the above step was applied to a Nuvia HR-S column (Bio-Rad), gradient elution was carried out using 0.01M/L phosphate buffer (pH 6.0 to 6.4) and 0.01M/L phosphate buffer containing 0.5M/L NaCl (pH 6.0 to 6.4), and the eluates were collected.
5. Nanofiltration
And (3) selecting an SV4 filter membrane of Pall company to carry out virus removal and filtration on the collected cation eluent, carrying out nanofiltration on a filtered sample, namely a recombinant human antibody fusion protein nanofiltration solution, detecting the purified fusion protein by SEC-HPLC (Agilent), and enabling the peak position to be consistent with a predicted theoretical value.
The purity of the product was determined by SEC-HPLC (Agilent), HC P by enzyme-linked immunosorbent assay (ELISA), and purity by SEC-HPLC (Agilent) at 2-8 ℃ for 7 days and 28 days.
The virus removal/inactivation effect, the removal/inactivation effect of mouse parvovirus (MVM) and reovirus type III (Reo3) of the sample (residual indicator virus titer (LgTCID50/0.1m l)) was verified with reference to "blood product removal/inactivation virus technique and verification guide (national drug administration [2002] 160)".
The virus reduction was calculated by the Karber method using the 96-well cell pathology method. The assay was repeated twice for each batch of samples. The Karber method formula is: LgTCID50 ═ L-d (S-0.5), where L is the logarithm of the highest dilution; d is the difference between the dilution logarithms; s is the sum of the positive pore ratios. The virus reduction is the difference between the virus titer (LgTCID50/0.1mL) measured in the zero control sample and the sample after the inactivation process.
The results are given in Table 6 below
TABLE 6
Figure BDA0002728906980000141
Experimental results show that the reduction of mouse parvovirus (MVM) virus by the method of the invention reaches 5.01log, and the reduction of reovirus type III (Reo3) reaches 5.12 log. In addition, the method of the invention has good removal effect on HCP (ELISA) and HCD (fluorescence quantitative PCR method), and the results are respectively 2ng/mg and 0.07pg/mg after determination. Compared with HCP (ELISA) and HCD (fluorescent quantitative PCR method), the removal rate of the protein before low-pH incubation reaches 99.52 percent and 99.92 percent respectively. Further, the Fc fusion protein obtained was stored at 2-8 ℃ for 7D and 28D with unchanged purity.
In conclusion, compared with the prior art, the method has excellent virus removal/inactivation effect, and simultaneously, the method also has the advantages of removing HCP and HCD and having excellent effect.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> JINGZE biomedical corporation of Jiangsu
SHANGHAI JINGZE BIOLOGICAL TECHNOLOGY Co.,Ltd.
<120> method for removing/inactivating virus
<130> P2020-1617
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 432
<212> PRT
<213> Artificial sequence ()
<400> 1
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Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val
20 25 30
Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
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Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
50 55 60
Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu
65 70 75 80
Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg
85 90 95
Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile
100 105 110
Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr
115 120 125
Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys
130 135 140
His Gln His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly
145 150 155 160
Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr
165 170 175
Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met
180 185 190
Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr
195 200 205
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
210 215 220
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
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Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
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Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
260 265 270
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
275 280 285
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
290 295 300
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
305 310 315 320
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
325 330 335
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
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Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
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Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
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Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
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Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
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Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425 430

Claims (12)

1. A method for removing/inactivating viruses, comprising the steps of:
s1) providing a raw material liquid containing the Fc fusion protein produced by fermentation of the CHO cell line; and
s2) incubating the stock solution at low pH for 0.5-8 hours to produce a first purified solution;
wherein the low pH incubation has a pH of 3.0-4.0 and the pH is adjusted using citric acid or a salt thereof.
2. The method of claim 1, wherein the method further comprises the steps of:
s3) loading the first purified solution into a deep filter for deep filtration to obtain a deep filtrate;
s4) loading the deep filtrate into an anion chromatographic column for anion chromatography to obtain anion chromatographic solution;
s5) loading the anion chromatographic solution onto a cation chromatographic column for cation chromatography to obtain a cation chromatographic solution; and
optionally, s6) subjecting said anion chromatography liquid to nanofiltration, resulting in a second purified liquid.
3. The method of claim 2, wherein in step s3), depth filtration uses a filter flux of 10-250L/m2。
4. The method according to claim 2, wherein in step s5), the cation chromatography is performed using a chromatography column with a medium particle size of less than 60 μm.
5. The method of claim 1 or 2, wherein the citric acid concentration is 0.5-3 mol/L.
6. The method of claim 1 or 2, wherein the first or second purified liquid Fc fusion protein SEC-HPLC assay is greater than 99.5% pure.
7. The method of claim 1 or 2, wherein the Fc fusion protein is a recombinant human vascular endothelial growth factor Fc fusion protein.
8. The method of claim 1 or 2, wherein the removed/inactivated virus is selected from the group consisting of: mouse parvovirus (MVM), reovirus type III (Reo3), pseudorabies virus (PRV), murine leukemia virus (X-MuLV), or a combination thereof.
9. The method of claim 1 or 2, wherein the virus removal/inactivation ratio of the first or second purified solution is greater than 104。
10. A pharmaceutical composition comprising i) an Fc fusion protein produced using the method of claim 1 or 2, and ii) a pharmaceutically acceptable carrier.
11. The method of claim 1 or 2, wherein the Fc fusion protein comprises the amino acid sequence set forth in SEQ ID No. 1.
12. The method of claim 1 or 2, wherein the low pH incubation is at a pH of 3.1-3.8.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105175548A (en) * 2015-08-13 2015-12-23 齐鲁制药有限公司 Purification method of recombinant human vascular endothelial growth factor receptor-antibody fusion protein
CN105026433B (en) * 2014-01-24 2018-10-19 上海恒瑞医药有限公司 VEGF and PDGFR β bispecific fusion proteins and application thereof
CN110272491A (en) * 2018-03-13 2019-09-24 江苏恒瑞医药股份有限公司 A kind of purifying process of anti-PD-1 antibody

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105026433B (en) * 2014-01-24 2018-10-19 上海恒瑞医药有限公司 VEGF and PDGFR β bispecific fusion proteins and application thereof
CN105175548A (en) * 2015-08-13 2015-12-23 齐鲁制药有限公司 Purification method of recombinant human vascular endothelial growth factor receptor-antibody fusion protein
CN110272491A (en) * 2018-03-13 2019-09-24 江苏恒瑞医药股份有限公司 A kind of purifying process of anti-PD-1 antibody

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