CN115141813A - Adenovirus purification method for efficiently removing residual proteins of host cells - Google Patents
Adenovirus purification method for efficiently removing residual proteins of host cells Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
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- C12N2710/10011—Adenoviridae
- C12N2710/10051—Methods of production or purification of viral material
Abstract
The invention discloses an adenovirus purification method for efficiently removing residual proteins of host cells, which is suitable for the purification production of adenovirus. The purification process comprises the following steps: the method comprises the steps of cracking the virus harvesting solution by Tween-80 and treating the virus harvesting solution by nuclease, clarifying the virus harvesting solution by a deep filter and a membrane filter, concentrating and washing and filtering the clarified solution by using a hollow fiber column, purifying the solution by using Source30Q anion exchange chromatography filler and CaptoQ anion exchange chromatography filler respectively, further purifying the ion exchange harvesting solution by using Sepharose4FastFlow chromatography filler, replacing the preservation solution, and sterilizing and filtering to obtain a stock solution. The purification method adopts two filters for clarification to remove most of Host Cell Protein (HCP), and two-step ion exchange chromatography in the purification process to further remove residual HCP, and purifying to obtain qualified stock solutionThe HCP residual quantity is less than 100 ng/1X 10 11 VP。
Description
Technical Field
The invention belongs to the technical field of virus purification, and particularly relates to an adenovirus purification method for efficiently removing residual proteins of host cells.
Background
Host Cell Proteins (HCPs) are major process-related impurities that constitute biopharmaceuticals. The risk associated with HCPs is primarily immunogenicity. HCPs are complex mixtures with a variety of physiochemical and immunological properties. Since immune responses may be triggered in humans, almost all HCPs are at clinical safety risk as foreign protein drugs. In addition, certain HCPs may also act as adjuvants to enhance the immune response of biopharmaceutical products. Certain HCPs with proteolytic activity, if not adequately removed or inactivated, can also affect the stability and efficacy of the drug product. Therefore, the amount of residual HCP is monitored to ensure quality and safety of the biological product during the purification and production of adenovirus by removing host cell proteins as much as possible.
Generally, in the conventional adenovirus purification method, under the condition that the culture solution of the adenovirus cultured in a large scale contains a large amount of impurities, the removal of the impurities is not thorough, particularly, the removal effect on HCP is not good, and qualified stock solution is difficult to obtain.
Disclosure of Invention
In view of the problems raised by the above background art, the present invention is directed to: aims at providing an adenovirus purification method for efficiently removing residual proteins of host cells.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
an adenovirus purification method for efficiently removing residual proteins of host cells comprises the following steps,
s1, cell lysis, wherein virus harvest liquid is subjected to Tween-80 lysis and nuclease treatment;
s2, clarifying and concentrating, namely clarifying the virus harvest liquid by using a deep filter and a membrane filter, and then concentrating and washing and filtering the clarified liquid by using a hollow fiber column;
s3, fine purification, namely, respectively using Source30Q anion exchange chromatography packing and Capto Q anion exchange chromatography packing to perform fine purification on the solution;
s4, performing replacement sterilization, further purifying the ion exchange harvest liquid by using Sepharose4Fast Flow chromatography packing, replacing the preservation liquid, and performing sterilization filtration to obtain a stock solution.
The invention has the beneficial effects that:
1. based on the existing purification method, the adenovirus purification method with two-step ion exchange chromatography scheme as the main technical characteristic is provided, so that the HCP content in the stock solution meets the standard (100 ng/1X 10) 11 VP)。
Drawings
The invention is further illustrated by the non-limiting examples given in the accompanying drawings;
FIG. 1 is a purification scheme of an embodiment of the present invention;
FIG. 2 is a flow chart of purification of a comparative example of the present invention;
FIG. 3 is a Source30Q ion exchange chromatogram of an embodiment of the present invention;
FIG. 4 is a Capto Q ion exchange chromatogram of an example of the invention;
FIG. 5 is a chromatogram of an example S4FF molecular sieve of the present invention;
FIG. 6 is a Capto Q ion exchange chromatogram of a comparative example of the present invention;
FIG. 7 is a comparative example S4FF molecular sieve chromatogram of the present invention.
Detailed Description
In order that those skilled in the art may better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below.
The experimental equipment is as follows:
an AKTApure purifier, a biological safety cabinet, an AKTAFlux 6 tangential flow filtration system and the like;
zeta Plus EZP depth filter, specification: E16A 07A 60SP02A,1.6m 2 。
Hollow fiber column: UFP-300-E-8A (Cytiva), 3600cm2, 300KDa.
Ion exchange chromatography column: 100mm diameter column (sharp ear), source30Q packing (Cytiva), about 1000ml.
Ion exchange chromatography column: column (sharp ear) 100mm in diameter, capto Q packing (Cytiva), about 800ml.
Molecular sieve chromatographic column: column chromatography (spiked) 72mm in diameter, sepharose4Fast Flow packing (Cytiva), approx.3000 ml.
Ion exchange chromatography column: 26mm diameter column (Cytiva), capto Q packing (Cytiva), about 30ml.
Molecular sieve chromatographic column: 26/40 standard column (Bogelong), sepharose4Fast Flow pad (Cytiva), about 160ml.
Experimental conditions:
in this experiment, two experiments, namely an example experiment and a comparative experiment, are carried out, and samples are obtained from harvest liquid cultured by 50LWAVE cell bags produced in one department.
Examples
The method for adenovirus purification of this example, comprising the steps of:
1. cell lysis
Obtaining adenovirus cell harvest liquid by virus-infecting amplification culture in a 50L WAVE cell bag, wherein the cell lysis condition is that 0.5 percent Tween-80 is used for lysis for 2h at 37 ℃, then nuclease with the concentration of 20U/ml is added for continuous treatment for 3 h, and then NaCl with the final concentration of 300mM is added to terminate enzyme digestion for 30min.
2. Deep filtration
Filtering the treated cell harvest liquid by a 3M deep layer filter, firstly washing the deep layer filter by 300mM sodium chloride solution, putting the cell harvest liquid into a liquid inlet end pipeline of the deep layer filter, putting a liquid outlet end of the deep layer filter into a dry baking container, and adjusting a peristaltic pump to set a proper flow rate to be 0.5mL/cm 2 Regulating the pressure of the pump every minute, performing deep filtration, cleaning the filter with a small amount of 300mM sodium chloride solution after the deep filtration is finished, and emptying the filter to obtain a deep filtration collection solution filter.
3.0.5/0.2 μm filtration
The filtering sleeve and the silicone tube are assembled with the filter element after being washed by soda, and the inlet and the outlet are respectively connected with the silicone tube. Inserting a tube at the sample injection end into the solution to be filtered, washing the outlet end with injection water, and putting the washed outlet end into a dried bottle. Starting a peristaltic pump, setting the flow rate to be 600-1000ml/min, and collecting the filtrate.
4. Concentration washing filter
Selecting hollow fiber column (3600 cm) 2 300 KD), connected tangential flow ultrafiltration concentration systemAnd (3) carrying out uniform AKTAFlux 6, flushing the hollow fiber column with injection water for 5min, circulating 0.5M NaOH for 30min, then cleaning the hollow fiber column with the injection water until the pH values of a backflow end and a permeation end are neutral, and emptying. Setting the shear force to be less than 2000, controlling TMP to be less than 15, ultrafiltering and concentrating 5-10 times, washing with liquid B, stopping concentrating, controlling concentration to be 2 × 10 12 Below VP/ml, avoid virus aggregation. The concentrate was then washed 3 more times with dilution pair a. A small portion of the sample was left for comparative experiments.
5.Source 30Q (S30Q for short) ion exchange chromatography
Ion exchange chromatography column: column (sharp ear) 100mm in diameter, source30Q packing (Cytiva), about 1000ml.
The loading amount is 1.0X 10 according to the column loading capacity 12 VP/ml loading, sample adding liquid B to 30% of liquid B, loading at a flow rate of 30cm/h, performing ion exchange chromatography, washing with liquid B at a flow rate of 45cm/h for about 4-6CV, eluting with liquid B at 30-60% of 4-6CV, observing peak condition, collecting target peak, and inspecting.
6.Capto Q (short for CPTQ) ion exchange chromatography
Ion exchange chromatography column: a100 mm diameter column (spikelet), capto Q packing (Cytiva), about 800ml.
The sample loading is 1.5X 10 12 VP/ml packing, after regeneration of the ion exchange column, 30% of the B solution balance, the sample obtained in the above 5 was directly loaded, 45cm/h loaded, 35% of B solution was washed at 45cm/h to 2-4CV,50% of B solution was eluted at 45cm/h to 2CV, the peak condition was observed, the target peak was collected, and the sample was sent for examination.
7.Sepharose 4fast Flow (S4 FF) molecular sieve chromatography displacement and filtration sterilization
Molecular sieve chromatographic column: a column (spiked) 72mm in diameter, sepharose4Fast Flow packing (Cytiva), approximately 3000ml.
Passing the collected sample after secondary ion exchange chromatography through molecular sieve at flow rate of 15cm/h, collecting sample, sterilizing and filtering with 0.2 μm capsule filter to obtain stock solution, and freezing at-80 deg.C refrigerator.
Comparative example
The adenovirus purification method of this comparative example, comprising the steps of:
1. steps 1-4 As in the examples, a small portion of the filter-washed sample was used in a pilot experiment to compare the HCP removal performance of different chromatographic protocols.
CPTQ ion exchange chromatography
Ion exchange chromatography column: 26mm diameter column (Cytiva), capto Q packing (Cytiva), about 30ml.
The sample loading is 1.5X 10 12 After the VP/ml packing and ion exchange column were regenerated, 30% of the B solution was equilibrated, and the sample of 1 above was supplemented with the B solution until the content of the B solution was 30% by volume, the sample was loaded at a flow rate of 10ml/min, and then eluted at a flow rate of 10ml/min with 30% of the B solution at about 4-6CV, and the sample was eluted at about 30-60% of the B solution at about 4-6CV, and the peak was observed, and the target peak was collected and examined.
3.S4FF molecular sieve chromatography
Molecular sieve chromatographic column: 26/40 column size (Bogelong), sepharose4Fast Flow packing (Cytiva), about 160ml. And (4) enabling the collected sample after the secondary ion exchange chromatography to pass through a molecular sieve at the flow rate of 5ml/min, collecting the sample, and inspecting.
Experimental results and analysis:
1. example Host Cell Protein (HCP) assay results for samples
Taking samples of each purification step, detecting HCP by ELISA method and calculating single-step HCP removal rate, the results are shown in Table 1, table 1, HCP residual quantity detection result table of example purified samples
* The HCP residue was 32 ng/1X 10 by conversion of the starting solution 11 VP. (qualification standard: ≤ 100ng/1 × 10) 11 VP)
2. Host Cell Protein (HCP) assay results for comparative samples
The purification methods of samples 1 to 4 of the comparative example were identical, and the results are shown in Table 1, and the samples of the subsequent purification steps were subjected to the detection of HCP by ELISA and the single-step HCP removal rate was calculated, and the results are shown in Table 2
Table 2 HCP residual amount test results of comparative example purified samples table
* The HCP residue was 374 ng/1X 10 by conversion of the stock solution 11 VP
3. Example purification preparation statistics
Taking samples of each purification step, detecting the number of virus particles by HPLC (UV method for stock solution sample) and calculating the recovery rate of each step, the results are shown in Table 3
Table 3 example preparation statistics of purified samples
4. Analysis of
According to the test results in table 1, the following purification methods were used in this example: harvesting solution → cytolytic enzyme digestion → two-step filtration → ultrafiltration concentration and washing → two-step ion exchange chromatography → molecular sieve chromatography → sterilization filtration, the HCP content of the raw solution sample is 583.26ng/ml, if converted according to the dosage, the residual of HCP is 32ng/1 × 10 11 VP and HCP content of the stock solution are qualified. According to the results of the measurements in Table 2, the present comparative example employed the following purification method: harvesting solution → cytolytic enzyme digestion → two-step filtration → ultrafiltration concentration and washing → one-step ion exchange chromatography → molecular sieve chromatography, the HCP content of the stock solution sample is 583.26ng/ml, if converted according to the dosage, the HCP residue is 374ng/1 × 10 11 VP, stock HCP content failed. From the two experiments, the HCP removal rate of the ion exchange chromatography in the fine purification step is the highest, and the single-step HCP removal rate of the ion exchange chromatography is as high as more than 95%. The two-step ion exchange chromatography protocol of the example showed better HCP removal than the one-step ion exchange chromatography protocol of the comparative example.
As can be seen from Table 3, the total recovery of 38% and total VP of 5.12X 10 were achieved after each purification step 14 The yield is higher. Although more one-step chromatography step is added for treatment, the recovery rate is better, the loss is small, and HThe CP removing effect is greatly increased, and the HCP content of the stock solution can be qualified.
In this time, the harvest solution in this embodiment is cracked by Tween-80 to release virus, nuclease is used to cut and degrade cellular nucleic acid, the viscosity of the harvest solution is reduced, and the original HCP content of the harvest solution is up to 1.97 × 10 6 ng/ml, therefore, a two-step filtration method was used to retain most of the cell debris and impurities, and a depth filter was used to remove most of the HCP by electrostatic and hydrophobic mechanisms. Wherein the average removal rate of the two-step ion exchange HCP can reach more than 98 percent, most HCP is further removed, the residual amount of HCP in the sample is reduced, the final HCP removal effect is expected, and the HCP residual amount of the stock solution is reduced to 32ng/1 multiplied by 10 11 VP, and the HCP removal effect of only one step of ion exchange chromatography in the comparative example was not expected, demonstrating the high feasibility of the two-step ion exchange chromatography protocol of the examples of the present invention. The improved adenovirus purification method of the present example is demonstrated to be highly effective in removing residual proteins from host cells.
The foregoing embodiments are merely illustrative of the principles of the present invention and its efficacy, and are not to be construed as limiting the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims (9)
1. An adenovirus purification method for efficiently removing residual proteins of host cells is characterized in that: the adenovirus purification method comprises the following steps,
s1, cell lysis, wherein virus harvest liquid is subjected to Tween-80 lysis and nuclease treatment;
s2, clarifying and concentrating, namely clarifying the virus harvest liquid by using a deep filter and a membrane filter, and then concentrating and washing and filtering the clarified liquid by using a hollow fiber column;
s3, fine purification, namely, respectively using Source30Q anion exchange chromatography packing and Capto Q anion exchange chromatography packing to perform fine purification on the solution;
s4, performing replacement sterilization, further purifying the ion exchange harvest liquid by using Sepharose4Fast Flow chromatography packing, replacing the preservation liquid, and performing sterilization filtration to obtain a stock solution.
2. The method for purifying adenovirus with high efficiency of removing residual proteins of host cells according to claim 1, wherein: the cell lysis conditions were 0.5% Tween-80, lysis at 37 ℃ for 2h, treatment with 20U/ml nuclease for 3 hours, and termination with 300mM NaCl for 30min.
3. The method for purifying adenovirus with high efficiency of removing residual proteins of host cells according to claim 1, wherein: the membrane filter specification was 0.5/0.2 μm.
4. The method for purifying adenovirus with high efficiency of removing residual proteins of host cells according to claim 1, wherein: the pore diameter of the hollow fiber column is 300kDa.
5. The method for purifying adenovirus with high efficiency of removing residual proteins of host cells according to claim 1, wherein: and liquid A is adopted in the washing and filtering treatment process, the liquid A is 50mM Tris-HCl and 2mM MgCl2 (pH 8.0), and the washing and filtering times are more than or equal to 3.
6. The method for purifying adenovirus with high efficiency of removing residual proteins of host cells according to claim 1, wherein: source30Q anion exchange chromatography comprises the following conditions, the sample pretreatment is carried out by adding the washed and filtered sample to a final concentration of 30% B solution by 1M NaCl,50mM Tris-HCl,2mM MgCl 2 (pH 8.0), loading: control of the load 5X 10 11 ～1.5×10 12 VP/ml packing, flow rate: 30cm/h, elution: the flow rate is 45cm/h, and the column bed is cleaned by 30 percent of liquid B for 2-4 CV;30 to 60 percent, the volume of the solution B is 4 to 6CV, and the target peak is collected.
7. The method for purifying adenovirus with high efficiency of removing residual proteins of host cells according to claim 6, wherein: capto Q anion exchange chromatography involves the following conditions, the sample is directly loaded with the ion exchange harvest obtained in claim 6, at a flow rate: 20-50 cm/h, elution: the flow rate is 30cm/h, and the column bed is cleaned by 35 percent of liquid B for 2-4 CV;50% by volume of B solution, and collecting the desired peak.
8. The method for purifying adenovirus with high efficiency of removing residual proteins of host cells according to claim 1, wherein: further purifying the ion exchange harvest liquid by Sepharose4Fast Flow chromatography packing and replacing a preservation liquid, wherein the preservation liquid is as follows: 10mM Tris,75mM NaCl,5% Sucross, 1mM MgCl 2 10mM L-Histidine,0.5% absolute ethanol, 0.01% Tween80, pH 7.5.
9. The method for purifying adenovirus with high efficiency of removing residual proteins of host cells according to claim 1, wherein: and finally, sterilizing and filtering the sample by a 0.2-micron bag filter to obtain a stock solution, and freezing the stock solution in a refrigerator at the temperature of-80 ℃.
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| CN116769736A (en) * | 2023-08-17 | 2023-09-19 | 赛奥斯博生物科技(北京)有限公司 | Production process for rapidly purifying lentivirus |
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