CN111647075A - Method for removing viruses from target protein-containing sample by catalyzing bioceramic material - Google Patents
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Abstract
The invention discloses a method for removing viruses from a sample containing target proteins by catalyzing a bioceramic material, wherein positively charged porous ceramic particles prepared by utilizing the porous ceramic particles can specifically adsorb virus particles with negative charges in a virus-containing monoclonal antibody solution in a chromatography process, the removal effect reaches over 6logs, the protein concentration of the monoclonal antibody solution is not reduced, and a good virus removal effect is achieved.
Description
Technical Field
The invention relates to the field of biological product safety, in particular to a method for removing viruses from a sample containing target proteins by catalyzing a bioceramic material.
Background
The therapeutic biological product refers to protein, polypeptide and derivatives thereof prepared by engineering cells (such as bacteria, yeast, insect, plant and mammalian cells) with different expression systems, and comprises cytokines, plasminogen activator, recombinant plasma factor, growth factor, fusion protein, enzyme, receptor, hormone, monoclonal antibody and the like. The above target proteins (mainly monoclonal antibodies) include mainly extracellular secretion or intracellular expression, and a series of separation of the target proteins from various impurities such as cell debris, host intracellular proteins, enzymes, nucleic acid substances, etc. is required. Current applications in production mainly involve one or more chromatography steps to remove the above impurities. Typical monoclonal antibody purification methods are generally three steps: firstly, the target protein in the culture harvest liquid is captured by utilizing affinity chromatography, and then fine purification is carried out by utilizing two-step ion exchange chromatography or ion exchange and hydrophobic chromatography. Generally, each step has a certain amount of virus removal effect, but it is not always possible to completely remove the virus. Therefore, an additional purification step (nanofiltration) is typically added to the manufacturing process to ensure complete removal of the virus. However, the use of the existing chromatography step and nanofiltration step can ensure complete removal of the virus from the monoclonal antibody, but the time and material costs are enormous and the longer the monoclonal antibody is processed in the process, the higher the loss of the active components of the protein.
Disclosure of Invention
The invention aims to provide a method for removing viruses from a sample containing target proteins by catalyzing a bioceramic material, and aims to solve the problems that the existing chromatography step and the existing nanofiltration step can ensure that the viruses in a monoclonal antibody are completely removed, but the lost time cost and the material cost are huge, and the longer the processing time of the monoclonal antibody in the process is, the higher the loss of active ingredients of the proteins is.
To achieve the above object, the present invention provides a method for catalyzing a bioceramic material to remove viruses from a target protein-containing sample, comprising:
pretreating diatomite porous ceramic particles and then drying at room temperature;
soaking the dried diatomite porous ceramic particles in NaOH and YCl3·6H2In the sol solution prepared by O, ultrasonic coating reaction is carried out for 0.5 hour, and the sol solution is dried in a drying oven at 100 ℃, wherein Y is yttrium;
repeating the operation for three times, then placing the ceramic particles in a muffle furnace for dry baking at 700 ℃ and preserving heat for 1 hour to prepare positive charge porous ceramic particles;
screening positive charge porous ceramic particles through a screen;
loading the screened positive charge porous ceramic particles into a column in a hollow chromatographic column;
preparing a monoclonal antibody sample simulation solution, wherein the monoclonal antibody sample simulation solution comprises a bovine serum albumin standard solution and a virus, the ratio of the bovine serum albumin standard solution to the virus is 500:1, and the virus is a murine parvovirus or reovirus type 3;
enabling the monoclonal antibody sample simulation solution to flow through a chromatographic column taking positive charge porous ceramic particles as fillers;
collecting the flow-through liquid, the flushing liquid, the eluent and the cleaning liquid, and respectively determining the titer in the flow-through liquid, the flushing liquid, the eluent and the cleaning liquid.
In one embodiment, the screened positively charged porous ceramic particles are packed in a column in an empty chromatographic column, wherein the diameter of the column is 1cm to 1.5cm, and the packing density of the basis weight of the particles is 0.5 kg/l.
In one embodiment, the method for loading the screened positively charged porous ceramic particles into a column in an empty chromatographic column specifically comprises the following steps:
adding 10ml of settled positive charge porous ceramic particles into an empty chromatographic column;
rinsing with 50ml of deionized water was carried out to equilibrium.
In one embodiment, after rinsing to equilibrium with 50ml of deionized water, the method further comprises:
the packed column was washed with 50ml of 0.5M NaOH solution at a rate of 1.0 ml/min.
In one embodiment, after washing the packed column with 50ml of 0.5M NaOH solution at a rate of 1.0ml/min, the method further comprises:
and (3) balancing the filled chromatographic column by using 120ml of balance buffer solution at the flow rate of 2.0ml/min, and setting the ultraviolet absorbance value to zero when the baseline of the absorbance value at the ultraviolet 280nm wavelength is stable.
According to the method for removing the virus from the sample containing the target protein by catalyzing the bioceramic material, the positively charged porous ceramic particles prepared by the porous ceramic particles can specifically adsorb the virus particles with negative charges in the virus-containing monoclonal antibody solution in the chromatographic process, the removal effect reaches over 6logs, the protein concentration of the monoclonal antibody solution is not reduced, the good virus removal effect is achieved, compared with the existing method for removing the virus by anion chromatography or affinity chromatography and then nanofiltration, the time and nanofiltration consumables are saved, the total time of the protein solution on chromatographic equipment and nanofiltration equipment is greatly reduced, and the activity of the protein solution is reduced and the total amount of the protein solution is increased.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a schematic flow chart of a method for catalyzing a bioceramic material to remove viruses from a target protein-containing sample according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Referring to fig. 1, fig. 1 is a schematic flow chart illustrating a method for catalyzing a bioceramic material to remove viruses from a target protein-containing sample according to an embodiment of the present invention. Specifically, the method for catalyzing the bioceramic material to remove viruses from the target protein-containing sample can comprise the following steps:
s101, pretreating diatomite porous ceramic particles and then drying at room temperature;
in the embodiment of the invention, the diatomite porous ceramic particles are made of porous ceramic materials, and the porous ceramic materials are porous ceramic products formed by controlling the size and distribution of pore diameters in the material forming and sintering processes. Due to the covalent and complex ionic bonding and the complex crystal structure, proteins and other substances can be specifically adsorbed when a fluid flows through pores. The method comprises the following steps of pretreating diatomite porous ceramic particles and drying at room temperature, wherein the steps comprise: soaking diatomite porous ceramic particles in 1mol/L HNO3Wherein the diatomite porous ceramic particles have the specification of 123mm × 10mm, the pore diameter of 0.3-1.0 micron, the particles with large pore diameter are easy to escape viruses with small particle diameter in use, the diatomite porous ceramic particles with the specification and the size of the pore diameter can adsorb the viruses with smaller particle diameter, and 1mol/L HNO is used3Completely soaking the diatomite porous ceramic particles in the diatomite porous ceramic particles, and performing acidification treatment to ensure that NaOH and YCl in the next step3·6H2O reacts more readily on the particle surface to form a film.
S102, soaking the dried diatomite porous ceramic particles in NaOH and YCl3·6H2In the sol solution prepared by O, ultrasonic coating reaction is carried out for 0.5 hour, and the sol solution is dried in a drying oven at 100 ℃;
in the embodiment of the invention, the positive charge coating layer mainly made of yttrium hydroxide material is formed on the surface of the particle, so that the virus particles can be adsorbed conveniently. Yttrium, chemical symbol Y, is a rare earth metal element, is a grey black metal, and is ductile. YCl3·6H2O is yttrium (III) chloride hexahydrate.
S103, repeating the operation for three times, then placing the mixture in a muffle furnace for dry baking at 700 ℃ and preserving heat for 1 hour to prepare positive charge porous ceramic particles;
in the embodiment of the invention, the operation is repeated for three times, and the operation refers to drying at room temperature after pretreatment, and drying after ultrasonic coating reaction after soaking.
S104, screening positive charge porous ceramic particles through a screen;
in the embodiment of the invention, the diameter of the positive charge porous ceramic particle can be determined according to the size of the virus particle which is accurately eliminated, the virus particle can be captured to the maximum extent, the outer diameter of the positive charge porous ceramic particle is not more than 40 micrometers, the particle size can be screened by a 400-mesh screen, and the diameter of the positive charge porous ceramic particle is preferably 20nm to 100 nm.
S105, placing the screened positive charge porous ceramic particles into a column in a hollow chromatographic column;
in the embodiment of the invention, the diameter of the hollow chromatographic column is 1 cm-1.5 cm, and the packing density of the basis weight of the particles is 0.5 kg/l. Putting the screened positive charge porous ceramic particles into a column in a hollow chromatographic column, and specifically comprising the following steps: adding 10ml of settled positive charge porous ceramic particles into an empty chromatographic column; rinsing with 50ml of deionized water was carried out to equilibrium. The packed column was washed with 50ml of 0.5M NaOH solution at a rate of 1.0 ml/min. And (3) balancing the filled chromatographic column by using 120ml of balance buffer solution at the flow rate of 2.0ml/min, and setting the ultraviolet absorbance value to zero when the baseline of the absorbance value at the ultraviolet 280nm wavelength is stable.
S106, preparing a monoclonal antibody sample simulation solution, wherein the monoclonal antibody sample simulation solution comprises a bovine serum albumin standard solution and a virus, the ratio of the bovine serum albumin standard solution to the virus is 500:1, and the virus is a murine parvovirus (MVM) or a reovirus type 3 (ReoV-3);
in the embodiment of the invention, the titer of the viruses is more than 7.0logs, and the concentration of the monoclonal antibody sample simulation solution is 2.0 mg/ml.
S107, enabling the monoclonal antibody sample simulation solution to flow through a chromatographic column taking positive charge porous ceramic particles as fillers;
in the examples of the present invention, the column flow rate was 0.2 ml/min.
S108, collecting the flow-through liquid, the flushing liquid, the eluent and the cleaning liquid, and respectively determining the titer of the washing liquid, the flushing liquid, the eluent and the cleaning liquid.
In the embodiment of the invention, the collecting of the flow-through liquid, the washing liquid, the eluent and the cleaning liquid, and the respective determination of the titer comprise: loading the monoclonal antibody sample simulation solution at the flow rate of 2.0ml/min, and collecting the flow-through liquid when the ultraviolet absorbance value (UV value) begins to rise; after the sample loading is finished, washing the filled chromatographic column by using 60ml of equilibrium buffer solution at the flow rate of 2.0ml/min, and finishing collecting the washing liquid when the ultraviolet absorbance value (UV value) begins to drop to be level; eluting with 50ml of eluent at a flow rate of 2.0ml/min, collecting the eluent when the ultraviolet absorbance value (UV value) begins to rise, and collecting the eluent when the ultraviolet absorbance value (UV value) drops to a level value; cleaning the filled chromatographic column with 50ml of deionized water at the flow rate of 2.0ml/min, then cleaning the filled chromatographic column with 50ml of 0.5M NaOH solution at the flow rate of 1.0ml/min, then cleaning the filled chromatographic column with 120ml of deionized water at the flow rate of 2.0ml/min, and collecting the cleaning solution; the chromatographic column is preserved by 25ml of 20 percent ethanol; diluting the flow-through liquid, the flushing liquid, the eluent and the cleaning liquid by a factor of 10, and adding the diluted liquid into a 96-well cell culture plate prepared on the previous day for detection.
Example 1: and (3) a verification process for removing MVM in the monoclonal antibody by catalyzing the bioceramic material.
Column assembling: adding 10ml of settled diatomite porous ceramic particles (the particles are obtained by the method in the technical scheme) into an organic glass chromatographic column, wherein the diameter of the column body is 1cm, the height of the column body is 20cm, assembling the diatomite porous ceramic particles onto a full-automatic AKTA protein purification system, and washing the column body with 50ml of deionized water until the column body is balanced. And (3) cleaning the filler: the column was washed with 50ml of 0.5M NaOH solution at a rate of 1.0 ml/min. Ion exchange chromatography equilibrium: the column was equilibrated with 120ml of equilibration buffer at a flow rate of 2.0ml/min, and after the baseline of A280 (absorbance at 280nm ultraviolet) had stabilized, the UV value (absorbance of ultraviolet) was set to zero. Loading: 100ml of a commercial BSA (bovine serum albumin) standard solution (0.48 mg/ml) was prepared as follows: the MVM virus solution is added in the proportion of 500, after mixing uniformly, the PH is adjusted to 4.0 (the BSA protein is enabled to be in positive charge), a sample is marked as a sample 1 to be detected, and the rest samples are loaded at the flow rate of 2.0 ml/min. When the UV value starts to rise, the flow through starts to collect. Washing: after the end of the sample loading, the column was washed with 60ml of equilibration buffer at a flow rate of 2.0ml/min, and collection was terminated when the UV value began to drop to a plateau, the flow-through collected at this time being sample 2 to be tested. And (3) elution: eluting with 50ml of eluent at the flow rate of 2.0ml/min, starting to collect the eluent when the UV value starts to rise, and ending the collection when the UV value is reduced to be flat, wherein the collected eluent is the sample 3 to be detected. Cleaning: the column was washed with 50ml of deionized water at a flow rate of 2.0ml/min, then with 50ml of 0.5M NaOH solution at a flow rate of 1.0ml/min, then with 120ml of deionized water at a flow rate of 2.0ml/min, and finally with 25ml of 20% ethanol for storage of the column. And (3) detection: diluting the sample to be detected by 10 times, adding the diluted sample into a 96-well cell culture plate prepared on the previous day for detection, and calculating according to a karber method. And (4) observing results: the result shows that the titer of the MVM in the sample before sampling is 5logs, the titer of the MVM in the flow-through liquid is not detected, the titer of the MVM in the eluent is 4logs, and the virus is not detected in the flow-through liquid after large-volume determination and blind third-generation determination, so that the MVM is specifically adsorbed by the porous ceramic material in the chromatographic verification process, the virus removal effect exceeds 4logs required by the general evaluation principle of virus safety evaluation technology of biological tissue extraction products and eukaryotic cell expression products, and the biological safety requirement of the process is ensured.
Example 2: and (3) a ReoV-3 removal verification process for removing the monoclonal antibody by catalyzing the bioceramic material.
Column assembling: adding 10ml of settled diatomite porous ceramic particles (the particles are obtained by the method in the technical scheme) into an organic glass chromatographic column, wherein the diameter of the column body is 1cm, the height of the column body is 20cm, assembling the diatomite porous ceramic particles onto a full-automatic AKTA protein purification system, and washing the column body with 50ml of deionized water until the column body is balanced. And (3) cleaning the filler: the column was washed with 50ml of 0.5M NaOH solution at a rate of 1.0 ml/min. Ion exchange chromatography equilibrium: the column was equilibrated with 120ml of equilibration buffer at a flow rate of 2.0ml/min, and after the baseline of A280 (absorbance at 280nm ultraviolet) had stabilized, the UV value (absorbance of ultraviolet) was set to zero. Loading: 100ml of a commercial BSA (bovine serum albumin) standard solution (0.48 mg/ml) was prepared as follows: 500, adding ReoV-3 virus solution, mixing uniformly, sampling and marking as a sample 1 to be detected, and loading the rest samples at the flow rate of 2.0 ml/min. When the UV value starts to rise, the flow through starts to collect. Washing: after the end of the sample loading, the column was washed with 60ml of equilibration buffer at a flow rate of 2.0ml/min, and collection was terminated when the UV value began to drop to a plateau, the flow-through collected at this time being sample 2 to be tested. And (3) elution: eluting with 50ml of eluent at the flow rate of 2.0ml/min, starting to collect the eluent when the UV value starts to rise, and ending the collection when the UV value is reduced to be flat, wherein the collected eluent is the sample 3 to be detected. Cleaning: the column was washed with 50ml of deionized water at a flow rate of 2.0ml/min, then with 50ml of 0.5M NaOH solution at a flow rate of 1.0ml/min, then with 120ml of deionized water at a flow rate of 2.0ml/min, and finally with 25ml of 20% ethanol for storage of the column. And (3) detection: diluting the sample to be detected by 10 times, adding the diluted sample into a 96-well cell culture plate prepared on the previous day for detection, and calculating according to a karber method. And (4) observing results: the result shows that the titer of ReoV-3 in a sample before sampling is 5.7logs, the titer of ReoV-3 in a flow-through liquid is not detected, the titer of ReoV-3 in eluent is 4.3logs, and viruses are not detected in the flow-through liquid after large-volume determination and blind third-generation determination, so that the ReoV-3 in the chromatographic verification process is specifically adsorbed by the porous ceramic material, and the virus removal effect exceeds 4logs required by the general technical evaluation principle of virus safety evaluation of biological tissue extraction products and eukaryotic cell expression products, and the requirement of the biological safety of the process is ensured.
As a result, the virus titer is not detected in the flow-through liquid, and higher virus titer can be detected in the flushing liquid and the cleaning liquid, because the non-enveloped virus with negative charge can be well adsorbed on the filler prepared by the porous ceramic particles with positive charge, and because the inert material is adopted, the protein solution is not easily adsorbed on the filler, and the concentration of the protein in the flow-through liquid is not reduced. Wherein the method for determining virus titer is 50% tissue cell infection amount (TCID)50) End point dilution titration method of (1). The positive charge porous ceramic particles prepared by the porous ceramic particles can specifically adsorb virus particles with negative charges in a virus-containing monoclonal antibody solution in a chromatography process, the removal effect reaches over 6logs, the protein concentration of the monoclonal antibody solution is not reduced, a good virus removal effect is achieved, compared with the existing method for removing viruses by anion chromatography or affinity chromatography and then nanofiltration, the time and nanofiltration consumables are saved, the total time of the protein solution on chromatography equipment and nanofiltration equipment is greatly reduced, and the activity and the total amount of the protein solution are reduced and increased. The column of the invention is compared to the anion exchange columns present in the prior art processes as shown in table 1:
table 1 comparison of the chromatography column of the present invention with the anion exchange chromatography column present in the prior art process
In the prior art, protein in a protein solution is generally adsorbed on a chromatographic column, then equilibrium washing is carried out, and finally elution is carried out to elute the protein from the chromatographic column, but inevitable virus in the solution is also adsorbed on the chromatographic column together with the protein, and a part of the virus is removed during washing, but the eluted protein solution often contains the virus. The virus particles in the protein solution are specifically adsorbed onto the chromatographic column, and the protein does not combine with the inert material on the chromatographic column and flows down along with the flowing-through liquid, so that the effect of removing virus particles is achieved.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (5)
1. A method of catalyzing the removal of a virus from a sample containing a protein of interest from a bioceramic material, comprising:
pretreating diatomite porous ceramic particles and then drying at room temperature;
soaking the dried diatomite porous ceramic particles in NaOH and YCl3·6H2In the sol solution prepared by O, ultrasonic coating reaction is carried out for 0.5 hour, and the sol solution is dried in a drying oven at 100 ℃, wherein Y is yttrium;
repeating the operation for three times, then placing the ceramic particles in a muffle furnace for dry baking at 700 ℃ and preserving heat for 1 hour to prepare positive charge porous ceramic particles;
screening positive charge porous ceramic particles through a screen;
loading the screened positive charge porous ceramic particles into a column in a hollow chromatographic column;
preparing a monoclonal antibody sample simulation solution, wherein the monoclonal antibody sample simulation solution comprises a bovine serum albumin standard solution and a virus, the ratio of the bovine serum albumin standard solution to the virus is 500:1, and the virus is a murine parvovirus or reovirus type 3;
enabling the monoclonal antibody sample simulation solution to flow through a chromatographic column taking positive charge porous ceramic particles as fillers;
collecting the flow-through liquid, the flushing liquid, the eluent and the cleaning liquid, and respectively determining the titer in the flow-through liquid, the flushing liquid, the eluent and the cleaning liquid.
2. The method for catalyzing removal of viruses from a target protein-containing sample according to claim 1, wherein the screened positively charged porous ceramic particles are loaded into a column in which the diameter of the column is 1cm to 1.5cm and the packing density of the particle basis weight is 0.5 kg/l.
3. The method for catalyzing a bioceramic material to remove viruses from a target protein-containing sample as recited in claim 1, wherein the step of loading the screened positively charged porous ceramic particles into a hollow chromatography column comprises:
adding 10ml of settled positive charge porous ceramic particles into an empty chromatographic column;
rinsing with 50ml of deionized water was carried out to equilibrium.
4. The method of catalyzing removal of a virus from a sample comprising a protein of interest according to claim 3, wherein after rinsing to equilibrium with 50ml of deionized water, the method further comprises:
the packed column was washed with 50ml of 0.5M NaOH solution at a rate of 1.0 ml/min.
5. The method for catalyzing removal of viruses from a sample containing target proteins of claim 4, wherein after washing the packed column with 50ml of 0.5M NaOH solution at a rate of 1.0ml/min, the method further comprises:
and (3) balancing the filled chromatographic column by using 120ml of balance buffer solution at the flow rate of 2.0ml/min, and setting the ultraviolet absorbance value to zero when the baseline of the absorbance value at the ultraviolet 280nm wavelength is stable.
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Application publication date: 20200911 |