Application Of Ultrafiltration In The Production Of Human Rabies Vaccine
In order to promote the application of ultrafiltration in the production of human rabies vaccine, 300kDa ultrafiltration membrane cassette was used to concentrate the rabies virus harvest solution and detect the antigen recovery, endotoxin removal rate and host protein removal rate after ultrafiltration. The results showed that under the appropriate pressure, the rabies virus harvest solution was concentrated 80 times, eluted 25 times, the antigen recovery rate was 86.2%, the bacterial endotoxin removal rate was 87.5% ~ 88.7%, and the host protein removal rate was 91.6%.
Foreword
Rabies is a traditional, ancient zoonotic disease caused by the Rabies Virus (RV), with a fatality rate of up to 100%. At present, there is no effective treatment for post-exposure (i.e. damage to the skin and mucous membranes) other than emergency vaccine and immunoglobulin vaccination. The main source of rabies in humans is bites from diseased or potentially infected animals (mainly dogs). The mortality rate of rabies in China ranks second in the world, and rabies is one of the problems threatening the public health security in our country. The capsular glycoprotein of rabies virus, which is the only key antigen that induces the production of protective neutralizing antibody, has been the focus of research and development of new vaccines and new diagnosis and treatment technologies of rabies virus.
The rabies virus belongs to the genus rabies virus of the rhabdovirus family. The shape of the nucleocapsid is elasticated, the nucleocapsid is helically symmetrical, and the surface has an envelope containing single-stranded RNA. It's the pathogen that causes rabies. Rabies virus has two main antigens: one is the glycoprotein antigen on the outer membrane of the virus, which can bind to the acetylcholine receptor to make the virus neurotoxic, and produce neutralizing antibodies and hemagglutination inhibiting antibodies in the body, and the neutralizing antibodies have a protective effect; The other is the inner riboprotein antigen, which can make the body produce complement binding antibodies and precipitin, and has no protective effect.
Endotoxin is a kind of lipopolysaccharide substance (LPS), which has heat resistance and chemical stability and is not easy to be destroyed. If there is a certain concentration of endotoxin in the vaccine, it will cause severe fever and even death after vaccination, so the content of endotoxin in the vaccine should be reduced as much as possible. The 2005 edition of the Chinese Pharmacopoeia (Part III) determined that the control value of the qualified index of rabies vaccine endotoxin should not be higher than 100EU/ dose. With the rapid development of membrane separation technology, the application of ultrafiltration membrane separation technology to reduce the content of endotoxin in vaccines is becoming more and more widespread in pharmaceutical industry.
A rabies vaccine is a vaccine against rabies. There are usually three types of rabies vaccines, including purified Vero cell vaccine, human diploid cell vaccine, and primary cell culture vaccine. Rabies vaccine is made by inoculating rabies virus fixed virus into cellular matrix, after culture, harvest, concentration, inactivation, purification, and adding appropriate stabilizer. The main function of rabies vaccine is to stimulate the body to produce a rapid immune response and produce protective antibodies against rabies virus, so as to prevent infection caused by the virus and reduce the risk of disease.
The production process of vaccine plays an important role in ensuring the quality of vaccine, especially the quantification of some indicators in the production process is more important. In the production process of human rabies vaccine, ultrafiltration concentration technology is a necessary means to produce human rabies vaccine. Using reasonable aperture ultrafiltration membrane for ultrafiltration concentration can effectively remove endotoxin and host protein, and retain RV antigen, which is conducive to vaccine production and quality improvement. The relative molecular mass of RV molecules is 350kDa ~ 460kDa, and in theory, RV can be completely trapped by ultrafiltration concentration using membrane cassette with interception molecular weight of 100kDa and 300kDa. Endotoxin often forms aggregate structure in different aqueous solutions, because of the degree of aggregation, the molecular size is different. The relative molecular mass of endotoxin monomer is 10kDa ~ 20kDa, and the relative molecular mass of its polymerization form is 300kDa ~ 1000kDa or more than 1000kDa. Based on the difference in molecular weight, the RV antigen in human rabies vaccine was retained and the endotoxin and host protein in human rabies vaccine were removed by 300kDa ultrafiltration membrane.
In this experiment, ultrafiltration membrane cassette with 300kDa aperture and 0.11m2 membrane area was used as small sample treatment to concentrate human rabies vaccine intermediate products, and membrane flux, treatment efficiency, concentration multiple, antigen interception, endotoxin removal, and host protein removal were tested.
2 Material Method
2.1 Experimental materials
Rabies virus fixed virus CTN-IV strain; Vero cells; 0.5M sodium hydroxide; 300kDa ultrafiltration membrane cassette (PES material, 0.11m² membrane area).
2.2 Experimental methods
2.2.1 Preparation of virus harvesting solution
Frozen Vero cells were resuscitated in warm water at 37℃~ 39℃, cell suspension was sipped out, and then added to 199 culture medium containing 10% inactivated calf serum. Uniform monolayer cells were cultured at 37℃. The CTN-IV strain was inoculated with rabies virus at a ratio of 0.1mol/L and cultured at 37℃. After 48h, the culture medium was discarded and 199 culture medium containing 0.1% human blood albumin was added to maintain the culture at 33℃ ~ 35℃. Harvest the disease venom once every 3d-5d, and combine the multiple harvest fluids.
2.2.2 Installing the Membrane Cassette
Install the membrane cassette and connect the wiring as shown in the figure below.
2.2.3 Water Washing
Close the inlet valve, return valve and through valve, and insert the return end and through end pipe into the waste liquid tank. Fill the intake tank with 1L of water for injection.
Open the inlet and return valves, close the through valves, open the pump, clean the return line with 10% volume of purified water or water for injection, and maintain the inlet pressure at 0.35bar (5psi).
Open the filter valve, close the return valve, and use the remaining injection water to clean the filter pipe, maintaining TMP at 0.35bar.
2.2.4 Disinfection
Close the inlet valve, return valve and transmission valve, and insert the return end and transmission end lines into the waste liquid tank. Fill the intake tank with 2L of cleaning solution.
Open the inlet and return valves, close the through valves, open the pump, clean the return line with 200mL of 0.5M sodium hydroxide, and maintain the inlet pressure at 0.35bar (5psi).
Open the through valve, close the return valve, and clean the through pipe with a 200mL volume of cleaning solution, maintaining TMP at 0.35bar.
Then the return end and the through end pipe are inserted into the liquid tank for cyclic cleaning. Cycle cleaning with 1 ~ 1.5 times the process tangential flow rate for 30min.
Drain 0.5M of sodium hydroxide and rinse with water for injection according to 2.2.4 until neutral.
2.2.5 Water flux test
Add purified water to the intake tank, measure and record the water temperature in the intake tank. Insert the return end into the tank. Start the pump and adjust the pump speed and return valve to achieve a 0.35bar (5psi) transmembrane pressure difference. Using a cylinder, measure and record the flow rate at the through-end in mL/min. Adjust pump speed and return valve to obtain 1bar (15psi) transmembrane differential pressure. Using a cylinder, measure and record the flow rate at the through-end in mL/min.
To standardize the water flow value, divide the calculated water flow value by the transmembrane pressure difference and multiply the temperature correction factor in the following table.
2.2.6 Ultrafiltration concentration
Wash the water out of the system with the buffer top. Cycle for 5min to adjust the pH and ion stability of the system. If the temperature needs to be adjusted, continue the cycle until the system temperature is stable.
Add the feed liquid to the intake tank, set a feed flow rate (e.g. 400LMH), and test the through flow rate at different TMP values. According to the test data, curves were drawn with TMP as the abscissa and Flux as the ordinate.
Direct the through-end pipe into a suitable container or drain, such as through-end collection tanks, waste liquid tanks, and process drains.
Record the initial weight of the feed liquid in the feed tank, start the feed pump, and slowly circulate the feed liquid for 3min to 4min. Recirculation can help remove the residual air in the flow path, thereby maximizing the performance of the film.
Open the through valve, slowly increase the pump speed until the optimal tangential flow rate is reached, and adjust the return valve to achieve the optimal TMP of the system.
Then put the tube into the collection container, start timing when the liquid enters the container, and record the inlet and return pressure at appropriate intervals, and record the quality of the liquid over time at the return end and the through end to calculate the instantaneous flow rate.
The ultrafiltration concentration of the sample is completed when the liquid volume of the feed is reduced to the target concentration volume.
2.2.7 Dialysis
Put the intake pipe, replenishment pipe and return pipe into the intake tank, put the transmission pipe into the collection container, and put the collection container on the balance to clear.
Open the return valve, start the inlet pump, open the through valve, adjust the pump speed and TMP to the appropriate value. Open the replenishment pump, keep the liquid volume in the intake tank unchanged, and start the equal volume dialysis. Start timing when the liquid enters the container, and record the pressure at the inlet end, the return end and the mass of the liquid at the appropriate time interval, and obtain the instantaneous flow rate by calculation.
2.2.8 CIP
First rinse with buffer, then rinse with water for injection (operation 2.2.3), then clean with cleaning agent (operation 2.2.4), and finally rinse with water for injection (operation 2.2.3).
2.2.9 Water flux test
The operation is as follows: 2.4.5.
2.2.10 Preservation of membrane cassette
Short-term storage (≤3 days), keep the membrane cassette in the fixture and use the storage solution to cycle for 10min to 15min, close the system valve, cut off the power supply to the liquid pump, and ensure that the liquid inlet tank is correctly sealed.
If the storage time is 3 days to 1 month, please remove the membrane cassette from the fixture and seal it into a plastic bag or other airtight container. Add about 50mL to 100mL of the storage solution to a plastic bag or airtight container and seal it. Or it can be immersed directly in the storage solution. The following table recommends storage conditions.
3 Results and analysis
3.1 Investigation of influencing factors of pyrogen removal efficiency
Ultrafiltration operation pressure: 15 test batches were run continuously. In each batch, the filtration pressure was maintained at 5, 10, 15 and 20psi during ultrafiltration, and the virus harvest liquid was concentrated 30 times and eluted with buffer liquid (10 times the volume) in continuous flow. Results as shown in the table below, the removal rate of endotoxin was less than 87.0%, the recovery rate of antigen was stable at 86.0%, and the removal rate of host protein was stable at 90.0%, indicating that different operating pressures had little influence on the effect of antigen recovery, endotoxin removal and host protein removal.
Ultrafiltration concentration ratio: 15 test batches were run continuously. In each batch, the virus harvest liquid was concentrated 30 times, 50 times, 80 times and 100 times respectively during ultrafiltration. The filtration pressure was maintained at 20psi, and the buffer liquid (10 times the volume) was eluted in continuous flow. Results as shown in the following table, the removal rate of endotoxin ranged from 53.3% to 86.9%, the antigen recovery rate remained stable at 86.0%, and the removal rate of host protein was stable at 91.0%. In segmented sampling, no antigen was detected in the transmission solution, and less than 10% of the host protein remained in the concentrated virus solution. The concentration of endotoxin in the concentrated solution of virus increased with the increase of the concentration times, and the endotoxin removal rate was the highest in the concentrated sample of 80 times, and the manufacturer could also consider the concentration of 100 times, which not only improved the production efficiency and reduced the cost, but also met the requirements of vaccine production.
3.2 Membrane cassette flux depletion and cleaning regeneration
After treatment, the membrane flux recovery rate was 92.6%. The first recovery rate of the new membrane cassette is normal, according to the current observation properties of the feed liquid, the subsequent prediction of the second and NTH times, the membrane cassette flux recovery rate will be close to the data after this treatment, and tend to be stable. Within 2h of single use, the membrane cassette depletion was ideal, and only 10% of the membrane flux was depleted under overall operation.
3.2.1 Flux depletion in the process of membrane cassette treatment
3.2.2 Result of cleaning and regeneration of membrane cassette
About Guidling
Guidling Technology is a national high-tech enterprise focusing on biopharmaceuticals, cell culture, purification and concentration of biomedicine, diagnosis and industrial fluids. We have successfully developed centrifugal filter devices, ultrafiltration & microfiltration cassettes, virus filter, TFF system, depth filter, hollow fiber, etc. Which fully meet the application scenarios of biopharmaceuticals, cell culture, and so on. Our membranes and membrane filters are widely used in concentration, extraction and separation of pre-filtration, microfiltration, ultrafiltration and nanofiltration. Our many product lines, from small, single-use laboratory filtration to production filtration systems, sterility testing, fermentation, cell culture and more, meet the needs of testing and production. Guidling Technology is looking forward to cooperating with you!
