CN112225818B - Method for removing/inactivating virus - Google Patents
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- CN112225818B CN112225818B CN202011112058.7A CN202011112058A CN112225818B CN 112225818 B CN112225818 B CN 112225818B CN 202011112058 A CN202011112058 A CN 202011112058A CN 112225818 B CN112225818 B CN 112225818B
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Abstract
The invention provides a method for removing/inactivating viruses, which comprises the following steps: s 1) providing a raw material liquid, wherein the raw material liquid comprises Fc fusion proteins generated by fermentation of CHO cell strains; and s 2) incubating the stock solution at a low pH for 0.5-8 hours, thereby producing a first purified solution; wherein the pH value of the low pH incubation is 3.0-4.0, and citric acid or salt thereof is used for regulating the pH value. The method of the invention not only can achieve the aim of removing/inactivating viruses in the Fc fusion protein, but also retains the activity of the target protein, namely the Fc fusion protein, so as to prepare the Fc fusion protein with high purity and high concentration.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for removing/inactivating viruses by proteins, and more particularly relates to the removal/inactivation of viruses in a process downstream of Fc fusion protein production.
Background
The use of mammalian cell expression systems allows the secreted proteins to be most similar to the native proteins in terms of immunogenicity and antigenicity, and protein processing, such as glycosylation, is also the most accurate, with CHO cells being widely used as expression vectors. Biotechnological products derived from CHO expression lines have the risk of viral contamination which can lead to very serious consequences in the clinic, which may originate from the original cell line itself, and also from foreign viruses accidentally introduced during production, which in order to ensure the safety of the biotechnological product requires the removal/inactivation of the virus during production to reduce the virus-like particle content in the unit dose to a certain standard.
There have been reports in the literature that the presence of endogenous retroviruses in CHO cells poses a certain risk for the safety of human administration. According to ICH Q5A biological product virus safety evaluation protocol, the process has 2 steps or more than 2 steps, and at least one step has virus removal/inactivation rate of more than 10 4 Can be considered an effective virus removal/inactivation process. Therefore, for CHO cell-derived pharmaceutical proteins, a certain method for removing or inactivating viruses is necessary in the production process, and laboratory inactivation verification test is also required for the inactivation process.
The current stage fusion protein downstream process generally includes fermentation broth filtration, crude purification and fine purification steps. The Fc fusion Protein generally removes related impurities and process impurities through Protein A affinity chromatography and adopts low pH value incubation for virus in the process, but in the prior art, the technical proposal of taking the process impurities and the low pH value inactivation process as main integral purification steps is not seen.
The virus removal under the existing process conditions is generally 10 4 About, for example, wang Shujing et al (verification of recombinant fusion protein column chromatography virus removal process, chinese biological impurities, volume 27 second stage in 2014, 241-244), removal of encephalomyocarditis virus (encephalomyocarditis virus, EMCV) and porcine parvovirus (porcine parvovirus, PPV) by dye affinity chromatography, anion exchange chromatography, gel chromatography 3 chromatography methods, and the results show that the three chromatography steps have a reduction value of 1.968, 1.984 and 4.391, respectively, for encephalomyocarditis virus (EMCV) removal, and three chromatography steps have a reduction value of Porcine Parvovirus (PPV) removalThe divide reduction values were 2.135, 3.936 and 2.048, respectively. The removal effect of gel chromatography on EMCV is best, 4.391log10.
Chinese patent application 201810010465.3 discloses a purification method in a low pH virus inactivation process
: use 1) low pH viral inactivation: adjusting the pH value 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) And (3) neutralization: neutralizing the low pH treated protein solution with an alkaline solution, and adjusting the pH to 5.5; 3) And (3) filtering: after standing for at least 0.5h, the solution was filtered through a 0.2 μm filter, and HCP and ResDNA were 1199ppm and 5.64pg/mg, respectively, after 1M phosphate treatment.
Chinese patent application 2015180038801. X discloses a method for purifying TNFR-Fc fusion proteins: and performing steps of affinity loading, affinity elution, HIC loading, HIC elution, AEX loading, AEX elution, MMC loading and MMCFF to obtain the HCP of 32ppm.
Chinese patent application 202010488460.9 discloses a method for reducing the content of acidic charge heterosomes of Fc fusion protein, wherein HCP residue is reduced from 43.1ppm to 11.0ppm, and the final quality target of the product is achieved.
Therefore, there is a need in the art to develop new methods for isolating and purifying proteins that are capable of not only inactivating/removing viruses with high efficiency, but also of effectively removing HCPs, HCDs, etc., while retaining protein activity.
Disclosure of Invention
The invention aims to provide an optimized method for separating and purifying protein, which adopts a process impurity combined with a low pH value inactivation process, can not only inactivate/remove viruses efficiently, but also remove HCP, HCD and the like effectively, and can retain the activity of the protein.
In a first aspect of the invention, there is provided a method for removing/inactivating an Fc fusion protein virus comprising the steps of:
s 1) providing a raw material liquid, wherein the raw material liquid comprises Fc fusion proteins generated by fermentation of CHO cell strains;
s 2) incubating the raw material liquid for 0.5-8 hours at low pH, thereby preparing a first purified liquid;
wherein the pH value of the low pH incubation is 3.0-4.0, and citric acid or salt thereof is used for regulating the pH value.
In another preferred embodiment, the first purified solution comprises 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 embodiment, the raw material liquid is a chromatographic liquid subjected to Protein A affinity chromatography.
In another preferred embodiment, the low pH incubation uses 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.
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 low pH incubation temperature is from-28 ℃ to 28 ℃, preferably from-5 ℃ to 25 ℃.
In another preferred embodiment, the method further comprises the steps of:
s 3) loading the first purified solution on a deep filter for deep filtration to obtain a deep filtrate;
s 4) loading the deep filtrate on an anion chromatographic column for anion chromatography to obtain anion chromatographic liquid;
s 5) loading the anion chromatographic liquid on a cation chromatographic column for cation chromatography to obtain cation chromatographic liquid; and
optionally, s 6) nanofiltration of said cationic layer-separated solution to obtain a second purified solution.
In another preferred embodiment, the second purified solution comprises an Fc fusion protein.
In another preferred embodiment, in step s 4), the washing liquid of the anion chromatography is 10-60mmol/L citric acid, pH 5.0-6.0; preferably 25mmol/L citric acid.
In another preferred embodiment, in step s 5), the eluate of the cationic chromatography is 5-30mmol/L phosphoric acid, 0.1-5mol/LNaCl, pH 6.0-7.0; preferably 10mmol/L phosphoric acid, 0.5mol/LNaCl, pH 6.2.+ -. 0.2.
In a further preferred embodiment, in step s 3), depth filtration is carried out with a filter flux of from 10 to 250L/m 2 。
In a further preferred embodiment, in step s 3), depth filtration is carried out with a filter flux of 40-180L/m 2 Preferably 60-150L/m 2 More preferably 100-150L/m 2 。
In another preferred embodiment, in step s 3), the depth filtration is performed using a filter having a pore size of 0.1 to 5. Mu.m, preferably 0.15 to 4. Mu.m, more preferably 0.15 to 3.5. Mu.m.
In another preferred embodiment, in step s 4), anion chromatography is performed using a chromatography column Bestarose Diamond MIX-A.
In another preferred embodiment, in step s 4), the anion chromatography is carried out using an organic acid-equilibrated column, preferably citric acid, tris, more preferably citric acid, at a concentration of 5-50mmol/L, preferably 25mmol/L.
In another preferred example, in step s 5), cation chromatography is performed using a column having a medium particle diameter of less than 60 μm.
In another preferred embodiment, the cation chromatography is performed using a chromatography column equilibrated with an organic acid, preferably citric acid, tris, more preferably citric acid.
In another preferred embodiment, the cationic chromatographic column is Nuvia HR-S.
In another preferred embodiment, in step s 6), the nanofiltration membrane pore size is less than 20nm.
In another preferred embodiment, the citric acid concentration is 0.5-3mol/L.
In another preferred embodiment, the citric acid concentration is 1-2mol/L, preferably 1mol/L.
In another preferred embodiment, the first purified Fc-fusion protein has a SEC-HPLC assay purity of greater than 99.5%.
In another preferred embodiment, the first purified Fc-fusion protein has a SEC-HPLC assay purity of greater than 99.7%.
In another preferred embodiment, the second purified Fc-fusion protein has a SEC-HPLC assay purity of greater than 99.7%.
In another preferred embodiment, the first purified Fc-fusion protein has a SEC-HPLC assay purity of 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 an Fc fragment gene of Ig G1, preferably a recombinant protein in which VEGFR1 and VEGFR2 genes are linked to an Fc fragment gene of immunoglobulin Ig G1 and expressed in a eukaryotic expression system, preferably a total of 2-3 extracellular regions of different receptors of VEGFR1 and VEGFR2 are fused to an Fc fragment of immunoglobulin Ig G1.
In another preferred embodiment, the amino acid sequence of the Fc fusion protein comprises the amino acid sequence as set forth in SEQ ID No. 1. More preferably, the amino acid sequence of the Fc fusion protein is the sequence shown in SEQ ID No. 1.
In another preferred embodiment, the removed/inactivated virus is selected from the group consisting of: mouse picovirus (MVM), reovirus type III (Reo 3), pseudorabies virus (PRV), murine leukemia virus (X-MuLV), or combinations thereof.
In another preferred embodiment, the first or second purified Fc fusion protein virus removal/inactivation rate is greater than 4log, preferably greater than 5log, more preferably greater than 5.5log.
In another preferred embodiment, the first or second purification fluid has a virus removal/inactivation rate of greater than 4log.
In another preferred embodiment, the first or second purified Fc solution pseudorabies virus (PRV) removal/inactivation rate is greater than 4log, preferably greater than 5log.
In another preferred embodiment, the first or second purified liquid murine leukemia virus (X-MuLV) removal/inactivation rate is greater than 4log, preferably greater than 5log.
In another preferred embodiment, the first or second purified solutions have a pseudorabies virus (PRV) and murine leukemia virus (X-MuLV) removal/inactivation rate of greater than 4log, preferably greater than 5log.
In another preferred embodiment, the first or second purified solution has a mouse adenovirus (MVM) removal/inactivation ratio of greater than 4log, preferably greater than 5log.
In another preferred embodiment, the first or second purified solution reovirus type III (Reo 3) removal/inactivation rate is greater than 4log, preferably greater than 5log.
In another preferred embodiment, the HCP removal rate of the second purified Fc fusion protein is 99% or more.
In another preferred embodiment, the second purified Fc fusion protein has an HCD removal rate of 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 understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
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Detailed Description
The present inventors have made extensive and intensive studies, and have unexpectedly developed a method for separating and purifying proteins using low pH incubation for the first time, which can not only achieve the purpose of removing/inactivating viruses in Fc fusion proteins, but also retain the activity of the target protein, fc fusion protein, thereby producing high-purity and high-concentration proteins. On this basis, the present invention has been completed.
Terminology
Fc fusion protein: that is, the Fc fragment fusion protein of Ig G1 refers to a recombinant protein in which VEGFR1 and VEGFR2 genes are genetically linked to the Fc fragment gene of immunoglobulin Ig G1 and expressed in a eukaryotic expression system. The VEGFR1 and VEGFR2 genes are linked to the Fc fragment gene of immunoglobulin Ig G1 and are expressed in eukaryotic expression systems. A total of 2-3 extracellular regions 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 of the present invention comprises the sequence shown in SEQ ID No. 1, more preferably, the amino acid sequence of the Fc fusion protein of the present invention is the sequence shown in SEQ ID No. 1:
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No.:1)。
preferably, the Fc fusion protein of the present invention is expressed by CHO-DG44 cell line and the Fc fusion protein may be selected from fermentation supernatants or obtained by any initial purification step. Preferably, the Fc fusion protein concentration is 0.1-8g/L (mass concentration).
HCP: i.e., host protein residues. Preferably, it refers to host proteins from CHO-DG44 cell lines.
HCD: i.e., host cell DNA. Preferably, it refers to host DNA from a CHO-DG44 cell line.
SEC-HPLC Size Exclusion Chromatography (SEC) is a liquid chromatography method that exploits the unique properties of porous gel stationary phases to produce a separation based primarily on differences in molecular size. The SEC-HPLC is used for measuring the purity, and the method is recommended by a high performance liquid chromatograph (Agilent, 1260) by referring to a four-part <0514> molecular exclusion chromatography of China pharmacopoeia 2015.
Fluorescent quantitative PCR method: referring to the method for measuring exogenous DNA residual quantity in the fourth edition <3407> of China pharmacopoeia 2015, the fluorescence quantitative PCR instrument is ABI company (Stepone Plus). In the present invention, HCD was measured using CHO cell residual DNA detection kit from the biotechnology company Shen Ke, lake.
Enzyme-linked immunosorbent assay (ELISA): HCP was assayed by reference to the method recommended by the microplate reader (molecular Devices, model spectromax i3 x). In the present invention, the kit used was Cygnus CHO Host Cell Proteins ELISA Kit.
The virus removal/inactivation reduction is defined according to the virus inactivation evaluation rule of the national drug administration of the 2002 'blood product removal/inactivation Virus technical method and verification guidelines', and the virus reduction is used as the evaluation of the virus inactivation/removal effect. If the virus reduction is 4logs, this means that the inactivated/removed virus reduction in this step is 4.
Deep filtration: when the particle size is smaller than the diameter of the media pore channel, a filter cake cannot be formed on the surface of the filter media, and the particles enter the interior of the media, approach the pore channel wall by inertia and diffusion, 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 use a Millipore deep filtration membrane (Saidoles model MX0 SP) for the deep filtration of a sample (recombinant human antibody fusion protein nanofiltration solution).
Virus inactivation assay method: reference is made to "methods for removing/inactivating viruses in blood products" and guidelines for validation (national drug administration [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 chromatographic medium is not particularly limited, and may generally be 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 specifically and effectively separate Fc fusion proteins from other impurities.
The cation exchange resin suitable for use in the present invention is not particularly limited, and in the present invention, a column having a medium particle diameter of less than 60. Mu.m, preferably Nuvia HR-S, is used for cation chromatography.
Method for removing and/or inactivating virus in Fc fusion protein (fermentation broth)
The present invention provides methods for removing and/or inactivating viruses in an Fc fusion protein comprising any combination of any two, three, or all steps including pH incubation, from low pH incubation, depth filtration, anion chromatography, cation chromatography.
Specifically, the method comprises the following steps:
s 1) providing a raw material liquid, wherein the raw material liquid comprises Fc fusion proteins generated by fermentation of CHO cell strains;
s 2) incubating the raw material liquid for 0.5-8 hours at low pH, thereby preparing a first purified liquid;
wherein the pH value of the low pH incubation is 3.0-4.0, and citric acid or salt thereof is used for adjusting the pH, preferably the pH value is 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 time is 1-7 hours, preferably 2-4 hours, more preferably 2 hours, 3 hours, 4 hours.
Preferably, the low pH incubation is carried out at-28 ℃ to 28 ℃, preferably-5 ℃ to 25 ℃.
Preferably, the method further comprises the step of:
s 3) loading the first purified solution on a deep filter for deep filtration to obtain a deep filtrate;
s 4) loading the deep filtrate on an anion chromatographic column for anion chromatography to obtain anion chromatographic liquid;
s 5) loading the anion chromatographic liquid on a cation chromatographic column for cation chromatography to obtain cation chromatographic liquid; and
optionally, s 6) nanofiltration of said cationic layer-separated solution to obtain a second purified solution.
Preferably, in step s 4), the washing liquid of the anion chromatography is 10-60mmol/L citric acid, pH 5.00-6.0; preferably 25mmol/L citric acid.
Preferably, in step s 5), the eluate of the cationic chromatography is 10mmol/L phosphoric acid, 0.5mol/LN aCl, pH 6.2.+ -. 0.2.
Preferably, in step s 3), depth filtration is carried out with a filter flux of 10-250L/m 2 Preferably 40-180L/m 2 Preferably 60-150L/m 2 More preferably 100-150L/m 2 Wherein the depth filtration uses a filter with a pore size of 0.1-5. Mu.m, preferably 0.15-4. Mu.m, more preferably 0.15-3.5. Mu.m.
Preferably, in step s 4), anion chromatography is performed using chromatography column Bestarose Diamond MIX-A, preferably citric acid, tris, more preferably citric acid, at a concentration of 25mmol/L, equilibrated with an organic acid.
Preferably, in step S5), the cation chromatography is carried out using a chromatography column with a medium having a particle size of less than 60 μm, preferably Nuvia HR-S; the chromatography column in which the cation chromatography is equilibrated with an organic acid is preferably citric acid or Tris, more preferably citric acid.
Preferably, in step s 6), the nanofiltration membrane pore size is less than 20nm.
Preferably, the concentration of the citric acid is 0.5-3mol/L; preferably 1 to 2mol/L, more preferably 1mol/L to 1.5mol/L.
Preferably, the first purified Fc fusion protein has a SEC-HPLC assay purity of greater than 99.5%, preferably greater than 99.7%.
Preferably, the second purified Fc-fusion protein has a SEC-HPLC assay purity of 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, more preferably the Fc fusion protein is a recombinant protein in which recombinant human vascular endothelial growth factor receptor 1 and/or receptor 2 is linked to an Fc fragment gene of Ig G1, preferably VEGFR1 and VEGFR2 genes are linked to an Fc fragment gene of immunoglobulin Ig G1, and expressed in a eukaryotic expression system. A total of 2-3 extracellular regions 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 as shown in SEQ ID No.: 1.
Preferably, the removed/inactivated virus is selected from: mouse picovirus (MVM), reovirus type III (Reo 3), pseudorabies virus (PRV), murine leukemia virus (X-MuLV), or combinations thereof.
Preferably, the first or second purified Fc fusion protein virus removal/inactivation rate is greater than 4log, preferably greater than 5log.
Preferably, the first or second purified liquid Fc fusion protein pseudorabies virus (PRV) removal/inactivation rate is greater than 4log, preferably greater than 5log.
Preferably, the first or second purified Fc fusion protein has a murine leukemia virus (X-MuLV) removal/inactivation rate of greater than 4log, preferably greater than 5log.
Preferably, the first or second purified Fc fusion protein pseudorabies virus (PRV) and murine leukemia virus (X-MuLV) removal/inactivation rates are both greater than 4log, preferably greater than 5log.
Preferably, the first or second purified Fc fusion protein has a mouse adenovirus (MVM) removal/inactivation rate of greater than 4log, preferably greater than 5log.
Preferably, the first or second purified Fc fusion protein reovirus type III (Reo 3) removal/inactivation rate is greater than 4log, preferably greater than 5log.
Preferably, the HCP removal rate of the first or second purified Fc fusion protein is 99% or more.
Preferably, the HCD removal rate of the first or second purified Fc fusion protein is 99% or more.
Pharmaceutical composition
Preferably, the pharmaceutical composition comprises i) an Fc fusion protein made 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 recombinant protein in which the VEGFR1 and VEGFR2 genes are linked to the Fc fragment genes of immunoglobulin Ig G1 and expressed in a eukaryotic expression system. More preferably 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 as shown in SEQ ID No.: 1.
The main advantages of the invention include:
(1) The purification method has good effect of removing/inactivating viruses, and can prepare the Fc fusion protein with high purity, and the purity of the Fc fusion protein is up to more than 99.8 percent.
(2) In the purified product, the mouse adenovirus (MVM), reovirus III (Reo 3) and host HCP and HCD are effectively removed/inactivated, and the requirements of medicines 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 operation of gel chromatography, dialysis and the like which are difficult to amplify, and is beneficial to large-scale production.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Examples
Experimental materials
An Fc fusion protein sample was obtained from Shanghai Jing Ze Biotechnology Inc., expressed by the CHO-DG44 cell line, wherein the amino acid sequence of the resulting Fc fusion protein is shown as SEQ ID No.: 1.
Assay HCP: cygnus company CHO Host Cell Proteins ELISA Kit.
Determination of HCD: huzhou Shen Ke biotechnology limited CHO cell residual DNA detection kit.
1. Incubation at low pH
Buffer selection: directly regulating the pH of a sample to 5.5 by using 1mol/L Tris, and placing the sample at 2-8 ℃ for detection; the same batch of samples is taken, the pH of the Fc fusion protein is regulated to 3.0 by an acidic titration solution (the concentration is 1 mol/L), the sample is kept at room temperature (18-26 ℃) for 3 hours, and then the pH is regulated to 5.5 by an alkaline titration solution. The purity of the monomer was measured and the results are shown in Table 1
TABLE 1 influence of different acids or buffers of acids on the purity of Fc fusion protein monomers
The results show that: the obtained Fc fusion protein monomer has lower purity than citric acid and citrate buffer solution under the condition of obtaining the same pH value (3.0) by using 1mol/L hydrochloric acid, phosphoric acid and buffer solution titration solution thereof with the same concentration.
The pH value of the Fc fusion protein is regulated to 3.0 by citric acid and citrate buffer solution, the purity of the Fc fusion protein is maintained to be more than 99.1% at room temperature (18-26 ℃) for 3 hours, the fusion protein is stable, and the biological activity detection results are all in the range of 80-120%. Thus, the product was initially considered stable by incubation for 3 hours at pH3.3-3.7, with an acidic titration of 1mol/L citric acid and citrate (pH 3.0).
Incubation time and pH selection: the pH value of the control sample is directly regulated to 5.5 by citric acid and citrate buffer solution, and the control sample is placed at 2-8 ℃ for detection. The pH is adjusted to 3.2, 3.4, 3.6, 3.8 and 4.0 by citric acid or citrate buffer solution, and the pH is adjusted back to 5.5 after incubation for 2 hours, 4 hours, 6 hours and 8 hours at room temperature (18-26 ℃). The effect of low pH incubation on the product is shown in Table 2 below
Biological Activity: reference was made to the "chinese pharmacopoeia" 2015 edition three <3531> nitobuzumab anti-biological activity assay, based on the amount of Fc fusion protein required to inhibit half of VEGF.
TABLE 2
The above results indicate that: the purity of the sample is lower than 99.7% after incubation for 8h under five pH conditions (pH3.2, 3.4, 3.6, 3.8 and 4.0), and the purity of the sample is basically unchanged after incubation for 1h, 2h, 4h and 6h, and is higher than 99.5%. The purity of the incubation under five pH conditions (pH3.2, 3.4, 3.6, 3.8 and 4.0) is between 99.5 and 99.8 percent, and the purity tends to decrease along with the extension of the incubation time. The biological activity detection results have a decreasing trend along with the extension of the incubation time, but are all in the range of 75% -115%.
Biological Activity (Cell based bioassay) and purity (SEC-HPLC (%))
At 25 ℃, 4g/L Fc fusion Protein solution purified by Protein A affinity chromatography 5L,1mol/L citric acid, pH3.4 and incubation for 2h were taken to determine biological activity (Cell based bioassay) and purity (SEC-HPLC (%))
The incubation results are shown in table 3:
TABLE 3 Table 3
After incubation, the pH was adjusted back to 5.5 with citric acid, and the sample stability was measured and the results are shown in Table 4:
TABLE 4 Table 4
| Temperature (temperature) | Time | Purity SEC-HPLC (%) |
| Room temperature | 0 | 99.8 |
| Room temperature | 28D | 99.7 |
| 2～8℃ | 56D | 99.8 |
The results show that: incubation at pH 3.6 for 2h virus inactivation, pH adjustment to 5.5, the sample was stable at room temperature (18-26 ℃) for 14 days, monomer purity (SEC-HPLC) 99.8%; the monomer purity (SEC-HPLC) was 99.8% when stored at 2-8deg.C for 28 days, consistent with the first day of storage. This means 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 the virus removal/inactivation effect (residual indicator virus titer (LgTCID 50/0.1 ml)) of the sample mouse leukemia virus (X-Mulv) and pseudorabies virus (PRV) are shown in Table 5, with reference to the blood product virus removal/inactivation technical method and the validation guidelines (national drug administration [2002]160 ]
TABLE 5-1
| Virus (virus) | Viral reduction |
| X-Mulv | ≥4.78 |
| PRV | ≥4.83 |
The results show that the low pH incubation method can effectively remove the mouse leukemia virus (X-Mulv) and the pseudorabies virus (PRV).
2. Deep filtration
Low pH incubations were performed with a flux of 120L/m through depth filter NP7PDE21 (PALL) (0.2-3.5 um) equilibrated with 25mmol/L citrate buffer 2 The HCP (ELISA) was determined to be 192ppm and the HCD (fluorescent quantitative PCR method) was determined to be 8pg/mg.
TABLE 5-2
HCP and HCD removal rates reached 54% and 91%, respectively.
3. Anion chromatography
The filtrate from the previous step of depth filtration was applied to a "25mmol citric acid buffer, pH 5.3-5.7" equilibrated Bestarose Diamond MIX-A column (packing from Boglabron Shanghai Biotechnology Co.). Peaks containing the recombinant human antibody fusion protein of this example were detected in the collected fluid by ELISA.
The virus removal/inactivation effect was verified by reference to the methods of blood product virus removal/inactivation techniques and guidelines for verification (national drug administration [2002] 160), and the removal/inactivation effects of mouse adenovirus (MVM) and reovirus type III (Reo 3) of the samples (residual indicated virus titer (LgTCID 50/0.1 ml)).
The virus reduction was calculated by the Karber method using a 96-well cytopathic method. The assay was repeated twice for each batch of samples. The formula of the Karber method is: lgtcid50=l-d (S-0.5), where l=the logarithm of the highest dilution; d = difference between log dilutions; s = positive well ratio sum. The amount of virus reduction is the difference between the zero control sample and the sample virus titer (LgTCID 50/0.1 mL) after the inactivation process.
The results are shown in Table 6 below
TABLE 6
Experimental results show that the method has the advantages that the reduction of the mouse leukemia virus (X-Mulv) virus is 4.48log, the reduction of the mouse Micro Virus (MVM) virus is 4.45log, and the method has good removal effects on HCP (ELISA) and HCD (fluorescence quantitative PCR method), and the results are respectively 90ng/mg and 0.1pg/mg through measurement.
4. Cationic chromatography
The flow-through obtained in the previous step was applied to a Nuvia HR-S chromatography column (Bio-Rad Co.) and subjected to gradient elution with 0.01M/L phosphate buffer (pH 6.0-6.4) and 0.01M/L phosphate buffer (pH 6.0-6.4) containing 0.5M/L NaCl, respectively, and the eluate was collected.
5. Nanofiltration
And (3) virus removal and filtration are carried out on the collected cation eluent by using an SV4 filter membrane of Pall company, a nanofiltration sample is recombinant human antibody fusion protein nanofiltration liquid, and the purified fusion protein is verified by SEC-HPLC (Agilent), and the peak position is consistent with a predicted theoretical value.
Product purity was determined by SEC-HPLC (Agilent), HCP was determined by enzyme-linked immunosorbent assay (ELISA), and purities were determined by SEC-HPLC (Agilent) for 7 days and 28 days at 2-8deg.C.
The virus removal/inactivation effect was verified by reference to the methods of blood product virus removal/inactivation techniques and guidelines for verification (national drug administration [2002] 160), and the removal/inactivation effects of mouse adenovirus (MVM) and reovirus type III (Reo 3) of the samples (residual indicated virus titer (LgTCID 50/0.1 ml)).
The virus reduction was calculated by the Karber method using a 96-well cytopathic method. The assay was repeated twice for each batch of samples. The formula of the Karber method is: lgtcid50=l-d (S-0.5), where l=the logarithm of the highest dilution; d = difference between log dilutions; s = positive well ratio sum. The amount of virus reduction is the difference between the zero control sample and the sample virus titer (LgTCID 50/0.1 mL) after the inactivation process.
The results are shown in Table 7 below
TABLE 7
Experimental results show that the reduction of the mouse picovirus (MVM) virus is up to 5.01log and the reduction of reovirus type III (Reo 3) is up to 5.12log by using the method. In addition, the method of the present invention has good removal effect on HCP (ELISA) and HCD (fluorescence quantitative PCR method), and the results are 2ng/mg and 0.07pg/mg respectively after measurement. The removal rates of HCP (ELISA) and HCD (fluorescence quantitative PCR) before low pH incubation are respectively 99.52% and 99.92%. Further, the obtained Fc fusion protein was stored at 2-8deg.C for 7D and 28D without change in purity.
In conclusion, compared with the prior art, the method has excellent effect of removing/inactivating viruses, and simultaneously has the advantages of HCP and HCD removal and excellent effect.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
<110> Jiangsu JING biological medicine Co., ltd
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
Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu
1 5 10 15
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
35 40 45
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
225 230 235 240
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
245 250 255
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
340 345 350
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
355 360 365
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
370 375 380
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
385 390 395 400
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
405 410 415
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425 430
Claims (11)
1. A method for removing/inactivating a virus comprising the steps of:
s 1) providing a raw material liquid, wherein the raw material liquid comprises Fc fusion proteins generated by fermentation of CHO cell strains; and
s 2) incubating the raw material liquid for 2-6 hours at low pH, thereby preparing a first purified liquid;
wherein the pH value of the low pH incubation is 3.4+/-0.1 or 3.5+/-0.1, and citric acid or a salt thereof is used for regulating the pH, wherein the concentration of the citric acid is 1mol/L-1.5mol/L;
wherein the method further comprises the steps of:
s 3) loading the first purified solution on a deep filter for deep filtration to obtain a deep filtrate;
s 4) loading the deep filtrate on an anion chromatographic column for anion chromatography to obtain anion chromatographic liquid, wherein the anion chromatographic column is Bestarose Diamond MIX-A;
s5) loading the anion chromatographic liquid on a cation chromatographic column for cation chromatography to obtain cation chromatographic liquid, wherein the cation chromatographic column is Nuvia HR-S; and
optionally, s 6) nanofiltration of the cationic layer-separated solution to obtain a second purified solution;
the removed/inactivated virus is selected from the group consisting of: mouse adenovirus (MVM), reovirus type III (Reo 3), pseudorabies virus (PRV), murine leukemia virus (X-MuLV), or combinations thereof;
the amino acid sequence of the Fc fusion protein is a sequence shown in SEQ ID No. 1;
the detection purity of the first or second purified solution Fc fusion protein SEC-HPLC is more than 99.5%;
the removal rate of the HCP of the Fc fusion protein of the second purified solution is more than 99 percent;
the removal rate of HCD of the second purified solution Fc fusion protein is more than 99 percent.
2. The method of claim 1, wherein the low pH incubation is at a temperature of-5 ℃ to 25 ℃.
3. The method according to claim 1, wherein in step s 3), depth filtration is performed using a filter flux of 10 to 250L/m 2 。
4. The method according to claim 1, wherein in step s 5), the cation chromatography is performed using a column having a medium particle size of less than 60 μm.
5. The method of claim 1, wherein the first purified Fc-fusion protein has a SEC-HPLC assay purity of greater than 99.7%.
6. The method of claim 1, wherein the second purified Fc-fusion protein has a SEC-HPLC assay purity greater than 99.7%.
7. The method of claim 1, wherein the second purified solution has a mouse adenovirus (MVM) removal/inactivation rate of greater than 5log and a reovirus type III (Reo 3) removal/inactivation rate of greater than 5log.
8. The method of claim 1, wherein the low pH incubation is for a period of 2 to 4 hours.
9. The method of claim 1, wherein the first or second purified solution has a virus removal/inactivation rate of greater than 4log.
10. The method of any one of claims 1-9, wherein the citrate is selected from the group consisting of: sodium citrate, potassium citrate, calcium citrate, and ammonium citrate.
11. The method of any one of claims 1-9, wherein the low pH incubation has a pH of 3.4±0.1.
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