INFLUENZA VIRUS PRODUCTION METHOD USING SINGLE-USE CULTURE PROCESS SYSTEM AND RAPID CONFIRMATION TEST OF INFLUENZA VIRUS ANTIGEN PURIFICATION CONDITION
20230212230 · 2023-07-06
Inventors
- Hwan-ui JUNG (Gyeonggi-do, KR)
- Yun-hee KIM (Gyeonggi-do, KR)
- Hun KIM (Gyeonggi-do, KR)
- Yong Wook PARK (Gyeonggi-do, KR)
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2760/16134
CHEMISTRY; METALLURGY
C12M47/10
CHEMISTRY; METALLURGY
C12N2760/16122
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
C11D3/48
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to an influenza virus production method using a disposable culture process system, and a test for quickly checking conditions for influenza virus antigen purification. According to the present invention, conditions for influenza surface antigen obtainment (purification) may be quickly and reliably checked according to the unique method of the present invention, even without using the single radial immunodiffusion technique which is conventionally used as a standard test method when producing influenza vaccines, and thus the production time for an influenza surface antigen subunit vaccine is notably reduced, thereby enabling quick response as a result of rapid vaccine development/manufacturing, even in a rapid novel influenza pandemic situation. In addition, according to the influenza virus production method of the present invention, culture media exchange may be carried out in an airtight system by using a continuous low-speed centrifuge using a disposable bag, and thus the possibility of contamination occurring during the virus production process may be greatly reduced.
Claims
1. A method for purifying a surface antigen protein from an influenza virus using a detergent, wherein a detergent treatment concentration for treatment of a sampled influenza virus is determined by a value obtained by determining a total protein quantification value (TPQV) per unit dose of the sampled influenza virus and then substituting the total protein quantification value into Equation 1 as follows:
detergent treatment concentration=[{(a*TPQV(μg/mL))/(b μg/mL)}/c]*d [Equation 1] (where, a is 0.005% (v/v) to 0.100% (v/v), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and a value closest to the TPQV is selected, c is a concentration of detergent stock used for treatment (% (v/v)), and d is a dose of the sampled influenza virus to be treated with a detergent).
2. (canceled)
3. The method of claim 1, wherein a quantification process by SRID (single radial immunodiffusion) for an amount of hemagglutinin (HA) protein in a sample is not required in the determination of detergent treatment concentration.
4. The method of claim 1, wherein the detergent is a cationic detergent.
5. The method of claim 4, wherein the cationic detergent is CTAB (cetyl trimethyl ammonium bromide).
6. (canceled)
7. The method of claim 1, wherein the sampled influenza virus is obtained by a method including the following steps: (a) culturing a cell in a single-use bioreactor (SUB); (b) exchanging a portion of the medium in the cell culture solution of step (a) for a fresh medium using a low-speed continuous centrifuge using a single-use bag; (c) infecting the propagated cells with an influenza virus and culturing the cells under a condition permissive for replication of the influenza virus; and (d) isolating the influenza virus from the culture of step (c).
8. The method of claim 7, wherein the cell is Madin-Darby canine kidney (MDCK) cells.
9. The method of claim 7, wherein the medium is exchanged by a method in which the cells and the medium are continuously separated from each other using a low-speed continuous centrifuge, a portion of the medium is discarded, and a new medium is introduced into the cell incubator in step (b), without a process of recovering and reintroducing an entire amount of the culture solution.
10. The method of claim 9, wherein a portion of the medium is 50% to 80% of the entire culture solution.
11. The method of claim 7, wherein step (d) is performed including the following steps: (d-1) determining a purification condition based on a hemagglutination assay for influenza virus samples obtained through purification under different conditions to purify the influenza virus from the culture of step c); and (d-2) purifying the influenza virus from the culture of step (c) according to the condition determined in step (d-1).
12. The method of claim 1, wherein the total protein quantification value per unit dose of the sampled influenza virus is 50 μg/mL to 950 μg/mL.
13. A test method for rapid confirmation of influenza antigen purification conditions, the method comprising the following first purification condition determination method and second purification condition determination method: the first purification condition determination method, which is used in a step of purifying an influenza virus from an influenza virus culture, and in which a condition for the purification of an influenza virus is determined based on a hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained through purification under different conditions; and the second purification condition determination method, which is used in a step of purifying a surface antigen protein from an influenza virus using a detergent, and in which a detergent treatment concentration during the purification of a surface antigen protein is determined based on a total protein quantification assay for an influenza virus sample.
14. The method of claim 13, wherein the test method for rapid confirmation of influenza antigen purification conditions does not require a quantification process by SRID (single radial immunodiffusion) for an amount of hemagglutinin (HA) protein in an influenza virus.
15. The method of claim 13, wherein the step of purifying an influenza virus from the influenza virus culture is performed by chromatography.
16. (canceled)
17. The method of claim 13, wherein the conditions include one or more selected from the group consisting of buffer type, buffer concentration, pH, and conductivity.
18. (canceled)
19. The method of claim 13, wherein in the second purification condition determination method is characterized in that the detergent treatment concentration is determined by a value obtained by determining a total protein quantification value (TPQV) per unit dose of the influenza virus culture obtained through the first purification and then substituting the total protein quantification value into Equation 1 as follows:
detergent treatment concentration=[{(a*TPQV(μg/mL))/(b μg/mL)}/c]*d [Equation 1] (where, a is 0.005% (v/v) to 0.100% (v/v), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and a value closest to the TPQV is selected, c is a concentration of detergent stock used for treatment (% (v/v)), and d is a dose of the influenza virus culture to be treated with a detergent).
20. A vaccine manufacturing method comprising purifying a surface antigen protein from an influenza virus, wherein the step is carried out under a condition of using a detergent for treatment in a value obtained by determining a total protein quantification value (TPQV) per unit dose of the sampled influenza virus and then substituting the total protein quantification value into Equation 1 as follows:
detergent treatment concentration=[{(a*TPQV(μg/mL))/(b μg/mL)}/c]*d [Equation 1] (where, a is 0.005% (v/v) to 0.100% (v/v), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and a value closest to the TPQV is selected, c is a concentration of detergent stock used for treatment (% (v/v)), and d is a dose of the sampled influenza virus to be treated with a detergent).
21. A method for rapid purification of an influenza surface antigen, the method comprising the following steps: a) infecting a cell with an influenza virus and culturing the cell to obtain a virus culture; b) determining a purification condition based on a hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained through purification under different conditions to purify the influenza virus from the culture of step a); c) purifying the influenza virus from the culture of step a) according to the condition determined in step b); d) determining a detergent treatment concentration during surface antigen protein purification based on a total protein quantification assay for an influenza virus to purify a surface antigen from the influenza virus purified in step c); and e) purifying the surface antigen protein from the influenza virus by performing detergent treatment according to the condition determined in step d).
22. The method of claim 21, wherein the method for rapid purification of an influenza surface antigen further comprises one or more step selected from (i) to (vii) as follows: (i) removing cells and cell debris from the virus culture prior to step b); (ii) an additional purification process by one or more methods selected from the group consisting of ultrafiltration and diafiltration after step c); (iii) a detergent removing process after step e); (iv) an additional chromatography performing process for removal of impurities after step e); (v) before a cell is infected with a virus in step a), a portion of the medium in the cell culture to be used for virus infection is exchanged for a fresh medium through a low-speed continuous centrifuge; (vi) a process of treatment with Benzonase, Exonuclease, Ribozyme, or a mixture thereof after step c); and (vii) a virus inactivation process after step c).
23. The method of claim 22, wherein the (i) removal of cells and cell debris from the virus culture is performed by one or more methods selected from the group consisting of filtration, dialysis and centrifugation; (iv) the chromatography is TMAE (trimethyl aminoethyl) anion exchange chromatography; or (vii) the virus inactivation is performed by treatment with a detergent, formaldehyde, β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), diethylamine, acetyl ethylenimine, or a combination thereof.
24-29. (canceled)
30. The method of claim 21, wherein a cell to be used for virus infection in step a) or a cell infected with a virus in step a) is cultured in a single-use bioreactor (SUB).
31-34. (canceled)
Description
BRIEF DESCRIPTION OF DRAWINGS
[0277]
[0278]
[0279]
[0280]
[0281]
DETAILED DESCRIPTION OF THE INVENTION
[0282] Hereinafter, the present invention will be described in detail.
[0283] However, the following Examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.
Example 1: Establishment of Novel Strategy for Influenza Virus Cultivation Process Using Single-Use Bag and Low-Speed Continuous Centrifuge Unique to the Present Invention
[0284] 1) Culture of MDCK Cell
[0285] The culture method of the present invention provides a method for efficiently culturing MDCK Sky-3851 cells using a single-use cell incubator using a single-use bag.
[0286] An aspect of the present invention is a culture method comprising culturing MDCK cells using a spinner flask in an incubator at 37° C. and 5% C02. The stirring speed in a 125 mL spinner flask is required to be set to 40 rpm to 150 rpm, and the stirring speed in 500 mL and 1 L spinner flasks is required to be set to 80 rpm to 150 rpm. As the medium used for culture, a chemically defined serum-free medium is used, and L-glutamine as a nutrient required for cell growth is added at a concentration of 3 mM to 6 mM. Culture using spinner flasks is carried out in a volume of 1 L or less.
[0287] An additional aspect of the present invention is culture through a cell incubator using a single-use bag subsequent to the flask culture, the scale of the culture solution may be increased by using a cell incubator of 10 L, 50 L, 200 L, or 2,000 L scale. Cell culture is carried out under favorable conditions and culture is performed while adjusting and maintaining culture conditions such as dissolved oxygen and pH to enable mass culture. The dissolved oxygen and pH are adjusted using the gas (air, oxygen, carbon dioxide, or nitrogen) and base (sodium bicarbonate) required for culture. In a specific aspect, the temperature, dissolved oxygen concentration, and pH during the culture step are maintained at 37° C.±3° C., 50%±10% DO, and pH 7.2±0.2, respectively, and the medium in the main culture contains L-glutamine at 3 mM to 6 mM.
[0288] 2) Virus Infection
[0289] The present invention is to obtain a virus culture solution by infecting the cell culture solution secured in the main culture step with an influenza virus. When a sufficient number of cells is secured through the main culture, the culture medium is exchanged before the cells are infected with the virus.
[0290] The medium exchange before virus infection is performed by a method in which the cells and the medium are continuously separated from each other using a low-speed continuous centrifuge using a single-use bag without a process of recovering and reintroducing the culture solution, a certain amount (70% to 80%) of the medium is discarded, and a new medium is introduced into the cell incubator again. Unlike a general method in which the entire amount of the culture solution is recovered and then divided into smaller portions to be put in containers and centrifugation is performed, when a low-speed continuous centrifuge using a single-use bag is used, medium exchange can be performed in a closed system and the possibility of contamination can be greatly diminished. Contamination generated in the process step leads to waste of the medium used in the culture step and extension of the process period, resulting in time and economic loss.
[0291] In the present invention, there is established a culture process capable of minimizing the possibility of contamination and maximizing the reduction of production period and production cost by culturing cells using a single-use bag, and exchanging the medium using a low-speed continuous centrifuge and then infecting the cells with a virus in the step infecting cells with a virus. This type of cell culture and virus infection process is referred to as a “virus cultivation process using single-use bioreactor and low-speed continuous centrifuge (abbreviation: SBCC)”.
[0292] The medium exchange and virus infection step is a step of removing the medium in which the nutrients have been consumed during the main culture and introducing a new medium and a virus from the outside, is a step having the highest risk of contamination. If contamination occurs in the medium exchange step, damage to the production period occurs. It takes about 13 days from the cell thawing step of thawing the MDCK-Sky3851 cell line for vaccine production that is stored frozen to the medium exchange in the case of performing virus infection in a 10 L cell incubator. In the case of 2,000 L commercial production, it takes about 24 days to the medium exchange for virus infection. The production cost also increases because of the additional purchase of medium and single-use bags.
[0293] After medium exchange using the SBCC process, a virus strain for manufacture is prepared, the virus in the amount calculated to have a predetermined multiplicity of infection (MOI) and trypsin at a concentration of 2.5 μg/mL to 20 μg/mL are aseptically introduced into the cell incubator, the cells are infected and cultured for about 2 to 4 days, virus cultivation is terminated when the virus titer, cell number, and viability reach appropriate levels, and the culture solution is recovered. In Example of the present invention, MDCK cells having a secured cell number of 3×10.sup.6 cells/mL or more were treated with a virus in an amount calculated to have an MOI of 0.0001 to 0.01 and trypsin at a concentration of 1 μg/mL to 20 μg/mL, and cultured for about 3 days.
Example 2: Novel Establishment of Rapid Search Strategy for Purification Condition During Purification Process of Surface Antigen from Influenza Virus
[0294] During the purification process of a surface antigen from an influenza virus, a detergent (ionic or non-ionic) may be added to the influenza virus, and the surface antigen protein may be isolated and purified through fractionation means such as centrifugation. At this time, as an example, it is common to use an ionic detergent such as cetyl trimethyl ammonium bromide (CTAB).
[0295] As for the detergent treatment concentration for treatment of an influenza virus, it is preferable to determine the treatment concentration depending on the content of hemagglutinin, a virus surface antigen. However, it takes about 2 to 3 days to perform the standard HA content test method SRID (single radiation immunodiffusion) generally used at present, thus SRID is not suitable as an in-process confirmation test, and has a disadvantage that the test cannot be performed particularly when the supply of SRID standard products from NIBSC and the like is not made or delayed.
[0296] Meanwhile, during the purification process of a surface antigen from an influenza virus, the detergent treatment concentration that can selectively isolate only hemagglutinin and neuraminidase, surface antigens without destroying the viral core is different for each virus. A surface antigen vaccine is a high-quality vaccine with few side effects and high purity, and it is greatly important to set an appropriate detergent treatment concentration. When an excessive amount of detergent is used for treatment, the whole virus is destroyed, and the protein inside the viral core, which is an impurity, is exposed to the outside, resulting in a decrease in purity of the surface antigen in the purified product. When the detergent treatment concentration is insufficient, most surface antigens are present in the pellet and the yield of surface antigens decreases. It is an important factor in the production of influenza vaccines to confirm the appropriate detergent treatment concentration in a short time.
[0297] The present inventors have first invented the method of the present invention to be described later after various comparative experiments utilizing various analysis techniques, and confirmed that it is possible to set the conditions for detergent treatment suitable for the rapid isolation of surface antigens without depending on SRID, as well as the method has a significant relationship with the method based on the practically existing standard test method SRID, and that reliability and reproducibility are supported. The following Examples support that the effect according to the method unique to the present invention is a special effect that is hardly predicted from the existing conventional processes. The following Examples typically show an example of using CTAB as a detergent, and this is abbreviated as CTCCT (CTAB treatment concentration confirmation test).
[0298] 2-1. Novel Establishment of SRID-Independent Surface Antigen Purification Process Condition Setting Method
[0299] The present inventors have derived an equation relationship as shown in Equation 1 below by implementing an algorithm for estimating the optimized detergent (here, CTAB as an example) treatment concentration during surface antigen purification using the total protein quantification value (TPQV) per unit dose of influenza virus sampled from an inactivated influenza virus concentrate. The total protein quantification value (total protein content value) was able to be measured within 1 hour by performing BCA assay (or Lowry assay, Bradford assay or the like). The reference total protein quantification value may vary depending on the strain of the virus, but the protein content in the inactivated virus concentrate used in the experiment of the present study was confirmed to be 200 μg/mL to 600 μg/mL through analysis. After measurement of the protein content in the inactivated virus solution, among the values of 100 μg/mL, 200 μg/mL, 300 μg/mL, 400 μg/mL, 500 μg/mL, 600 μg/mL, 700 μg/mL, 800 μg/mL, and 900 μg/mL, the value corresponding to the approximate value of the measured TPQV was set as the reference value of the protein content for CTAB treatment. The CTAB treatment concentration was determined according to Equation 1 below, and the CTAB treatment concentration (%) with respect to the protein content in the inactivated virus solution was determined within 0.005% (v/v) to 0.100% (v/v).
detergent treatment concentration=[{(a*TPQV(μg/mL))/(b μg/mL)}/c]*d [Equation 1]
[0300] (where,
[0301] a is 0.005% (v/v) to 0.100% (v/v),
[0302] b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and a value closest to the TPQV is selected,
[0303] c is a concentration of detergent stock used for treatment (% (v/v)), and
[0304] d is a dose of the sampled influenza virus to be treated with a detergent).
[0305] The CTAB treatment concentration (%) based on the protein content varies depending on the strain and virus properties, and SDS-PAGE was performed in an arbitrary range to confirm and determine the appropriate CTAB treatment concentration (%). In other words, the inactivated virus solution was divided into small portions and treated with CTAB in an arbitrary range, and then ultracentrifugation was performed at 30,000 rpm or more according to the Sky Cell Flu manufacturing process to confirm the optimal CTAB treatment concentration at which the surface antigen protein was recovered as a supernatant and the viral core protein was isolated as a precipitate.
[0306] The confirmed optimal CTAB treatment concentration was applied to the manufacturing process. SDS-PAGE was performed using the sample (B/Maryland/15/2016 virus) obtained by performing the above-described process, and the same result as in
[0307] 2-2. Confirmation of Reliability and Reproducibility of Surface Antigen Purification Process Condition Setting Method According to the Present Invention—Correlation with Method Based on Existing SRID
TABLE-US-00001 TABLE 1 CTAB treatment HA HA CTAB amount concentration Volume of 5% CTAB content in content in (g) Protein to protein inactivated treatment inactivated supernatant reacted content content virus solution amount virus solution after CTAB with HA Virus (μg/mL) (%) (mL) (mL) (μg/mL) treatment (1 g) NYMCX-275 418 0.08 160,000 2,675 75 50 11.15 (A/Michigan/45/2015 402 0.08 160,000 2,573 68 57 11.82 (H1N1)pdm09-like virus) IVR-190 459 0.06 160,000 2,350 55 44 13.35 (A/Brisbane/02/2018 456 0.06 160,000 2,345 56 42 13.09 (H1N1pdrn09-like virus) IVR-195 567 0.06 200,000 2,268 103 77 5.50 (A/Kansa/14/2017 554 0.06 200,000 2,216 102 73 5.43 (H3N2)-like virus) B/Phuket/3073/2013 543 0.04 200,000 1,738 128 95 3.39 B/Maryland/15/2016 542 0.04 200,000 1,734 134 100 3.24 (B/Colorado/06/2017- 450 0.04 200,000 1,800 158 114 2.85 like virus) 369 0.04 200,000 1,476 130 108 2.84
[0308] Table 1 is a summary of 2,000 L scale commercial production data. According to Equation 1, the detergent treatment concentration was calculated based on the protein content, and the CTAB treatment process was performed.
[0309] In addition, the HA content in the inactivated virus solution and the HA content in the ultracentrifugation supernatant obtained by performing the ultracentrifugation process after treatment of the inactivated virus solution with CTAB were obtained and compared with each other, as a result, the HA content in the ultracentrifugation supernatant was a level of 70% to 80% of the HA content in the inactivated virus solution, and sufficient commercial feasibility was confirmed. When CTAB is excessively added for the treatment in order to increase the HA recovery rate, the vaccine may be manufactured in the form of a split vaccine rather than the intended subunit vaccine, and it is thus greatly important to determine a proper CTAB treatment concentration.
[0310] Accordingly, it has been confirmed that the detergent treatment concentration calculation method according to Equation 1 established in the present invention is a method that can rapidly and accurately confirm the CTAB treatment concentration required in the purification process of surface antigens of influenza viruses.
[0311] 2-3. Comparison of Execution Time by Purification Condition Setting Based on Existing SRID (Comparative Example 1) and Execution Time by Purification Condition Setting According to the Present Invention
[0312] As carried out in Example 2-2, the process execution time by the surface antigen purification condition setting method by the conventional SRID (single radial immunodiffusion) and the process execution time by the purification condition setting method of the present invention (see Example 2-1 and Example 2-2) were compared with each other, as a result, the execution time by the method of the present invention was much shorter than that by the conventional single radial immunodiffusion as shown in Table 2 below, and the method of the present invention was confirmed to be more efficient in confirming the influenza surface antigen purification condition.
TABLE-US-00002 TABLE 2 Base analytical method for condition Time for standard Time for sample Division setting antibody fabrication analysis Total time required Comparative SRID 2 to 3 months 2 to 3 days (2 to 3 months) + Example 1 (2 to 3 days) Present invention Total protein N/A 1 hour 3 hours quantification assay (e.g., BCA assay) SDS-PAGE N/A 2 hours
Example 3: Establishment of Rapid Influenza Virus Antigen Purification Condition Test Set (Abbreviated RIVPCT) after Virus Cultivation
[0313] In the production process of a subunit vaccine containing an influenza surface antigen, the purification process requires largely two process steps of a purification process of an influenza virus from a cell culture infected with the influenza virus and an isolation process of a surface antigen protein from the influenza virus. In the existing vaccine production process, the process conditions may be optimized using the standard test method single radial immunodiffusion (SRID) in the two process steps, but SRID has several disadvantages in vaccine production as described above. Accordingly, the present inventors have searched for a novel method that can set suitable process conditions without single radial immunodiffusion (SRID), and developed a rapid influenza virus antigen purification condition test set including the following first purification condition determination method and second purification condition determination method (based on the new strategy established in Example 2) as a set. In the present invention, the set of the first purification condition determination method and the second purification condition determination method is herein referred to as RIVPCT.
[0314] 3-1. First Purification Condition Determination Method—Case of Isolation/Purification/Concentration of Influenza Virus from Influenza Virus Culture
[0315] There is provided a determination method/discrimination method for rapidly setting optimal conditions for each virus without SRID measurement in setting the purification conditions used in the step of purifying the influenza virus from an influenza virus culture. Specifically, there is provided a test method/judgment method for determining the conditions for purification of the influenza virus from the influenza virus samples obtained through purification under different conditions based on the hemagglutination assay, SDS-PAGE, and discrimination criteria unique to the present invention, which will be described later.
[0316] In the following Examples, an example using chromatography is shown as a representative virus purification means. Before the virus culture solution is introduced into the chromatography column, a pretreatment in which cells and cell debris are primarily removed from the virus culture solution may be performed, such pretreatment may be performed by, for example, centrifugation, dialysis, filtration, or the like.
[0317] In order to perform the chromatography process, it is necessary to set different conditions for each influenza virus, and it is necessary to rapidly confirm the purification conditions for each virus strain because of the nature of influenza viruses, where the recommended strain for vaccine production is changed every year. In order to set the chromatography purification condition, the condition under which the influenza virus can bind to the resin most favorably is confirmed, and then a test for the elution condition under which the bound virus can be isolated and purified with optimal yield and purity is performed. Hereinafter, a method for determining influenza virus purification conditions (binding conditions and elution conditions of the target substance to the CS chromatography column resin) in the case of using Cellufine Sulfate (CS) chromatography is described as a representative example, which is abbreviated as CSPCT (CS chromatography purification condition test).
[0318] The principle of virus binding to the resin of the chromatography column is ionic bonding due to the affinity of the surface protein of influenza virus for the sulfate group of resin, and the binding capacity is generally determined depending on conductivity and pH. In the present Example, it is exemplarily shown that conditions under which the maximum number of viruses can bind to the resin are searched while maintaining the pH of the buffer used in the process at a neutral pH of 7.0 to 7.4 to increase the protein stability and lowering the conductivity. The conductivity of a generally obtained influenza virus culture (solution) is about 10.0 mS/cm to 15.0 mS/cm, and the binding capacity is confirmed while lowering the conductivity of the sample to be loaded on the column by diluting the influenza virus culture (solution) with a sodium phosphate buffer having a low conductivity. In the present Example, it is exemplarily shown that the time and variables required for confirming the purification conditions are diminished in a simple manner of adjusting only the conductivity, except for the pH, which is generally set as one of the purification conditions in order to rapidly confirm the process conditions.
[0319] In general, it is known that the influenza virus binds to the CS resin more favorably as the conductivity is lower, but the volume of the sodium phosphate buffer to be introduced to lower the conductivity and the time for the CS chromatography process increase, this increases the production period and production cost, and it is thus important to confirm the proper conductivity. Therefore, while lowering the conductivity to 10.0 mS/cm or less in a manner of diluting the virus culture solution 0.5 to 4 times using a sodium phosphate buffer, the hemagglutination assay (HA assay) was performed on the column flowthrough to confirm whether the binding capacity was proper.
[0320] Specifically, the HA assay was performed according to the protocol described in the “Manual for the laboratory diagnosis and virological surveillance of influenza” prepared and distributed by the WHO. According to the method, the proper conductivity of the loading sample was confirmed based on the trend of decreasing the HA titer of the column flowthrough, and SDS-PAGE was also performed at the same time to confirm whether the influenza virus was lost into the column flowthrough for each condition.
[0321] After the optimal conditions for virus binding to the CS column resin have been determined as described above, conditions suitable for virus elution are searched. The elution of the influenza virus bound to the resin was performed in a manner of searching for the optimal conductivity using a buffer solution containing sodium chloride at an appropriate concentration to cleave binding to the resin. When virus culture solution is introduced into the column while increasing the salt concentration with 2 M to 5 M sodium chloride buffer under a linear gradient condition, a UV peak appears as the viruses, proteins and the like are eluted. These fractions were recovered and the conductivity and salt concentration in the fraction containing the virus were confirmed by performing HA assay and SDS-PAGE. Based on the virus elution conditions confirmed here and the conductivity conditions under which the virus binding capacity was proper confirmed above, the virus was eluted under a step gradient condition using sodium chloride buffer and sodium phosphate buffer to confirm whether most influenza viruses were eluted at the corresponding elution concentration, and then virus elution conditions were established. In order to confirm whether the virus is eluted with high yield and high purity, the HA assay was performed by the same method and criteria as those described above. SDS-PAGE was also performed for the confirmation.
[0322] The CS chromatography purification condition test (CSPCT) for purifying the virus from the recovered virus culture solution, which is performed in a series of processes as described above, can be completed within about 6 to 12 hours, and such time reduction is also related to the reduction of the overall production period because of the nature of influenza vaccine, where the strain for vaccine production is changed every year.
[0323] When the chromatography conditions are set, the most accurate method is to confirm the hemagglutinin content in the column flowthrough by performing the standard test method, single radial immunodiffusion technique (hereinafter, SRID), but it takes 2 to 3 days to confirm one purification condition, greatly increasing the condition test period, and the SRID technique has a disadvantage that the test cannot be performed when the supply of SRID standard products from NIBSC (National Institute for Biological Standards and Control), TGA (Therapeutic Goods Administration) and the like is not made or delayed. When the HA assay and SDS-PAGE are performed according to the present invention, the test result can be acquired within 3 hours, thus the purification condition test for each virus is rapidly done within 1 day, and there are few restrictions on test execution since there are no issues such as the supply of standard products. It can be said that the characteristics of above-described CSPCT are the method in which the binding capacity of an influenza virus is confirmed by appropriately lowering the conductivity and the conditions for eluting the virus with high yield and high purity are rapidly confirmed within 12 hours, and the principles and procedures thereof are the same as those described above.
[0324] 3-2. Second Purification Condition Determination Method—Isolation and Purification of Surface Antigen from Influenza Virus
[0325] For the isolation and purification of surface antigens from an influenza virus, an example of using CTAB as a detergent is representatively presented in the present Example. CTAB treatment concentration was rapidly confirmed by performing CTCCT in RIVPCT in the same manner as in Example 2.
[0326] 3-3. Purification Effect of Surface Antigen Protein According to First Purification Condition Determination Method and Second Purification Condition Determination Method
[0327] In the case of determining the necessary conditions for the purification processes required for vaccine production based on the HA content confirmed by performing SRID, more accurate purification conditions can be set. However, according to the RIVPCT of the present invention, there is an advantage that conditions necessary for the purification processes required for vaccine production can be identified more rapidly. When performing the method using SRID, it takes 2 days or longer even if the possession of standard products and the period of stock are not taken into consideration. In order to shorten the time required, the RIVPCT method using SDS-PAGE, HA assay and the like has been invented.
TABLE-US-00003 TABLE 3 Setting of condition for purifying virus from Setting of condition for isolating surface virus culture solution (CSPCT) antigen from virus (CTCCT) Base analytical Base analytical method for condition Total time required method for condition Total time required setting for condition analysis setting for condition analysis Setting method SRID (2 to 3 months) + SRID (2 to 3 months) + using SRID (2 to 3 days) (2 to 3 days) Present HA assay, SDS- 6 to 12 hours Total protein 3 hours invention PAGE quantification assay (RIVPCT) (e.g., BCA assay), SDS-PAGE
Example 4: Influenza Subunit Vaccine Production Process with Reduced Production Period
[0328] 4-1. Infection of Cell with Influenza Virus and Culture of Cell
[0329] 1) Culture of MDCK (Madin-Darby Canine Kidney) Cells
[0330] One vial of the MDCK-Sky3851 cell line for vaccine production kept frozen was thawed in a 37° C. constant temperature water bath and diluted with a culture medium. Next, the diluted cells were centrifuged to separate the cells from the medium, and then the cell pellet was again released with a fresh medium, a sample was taken, and the cell number and viability were measured. The cells were inoculated in a 125 mL spinner flask at an inoculation concentration of 1×10.sup.5 cells/mL to 5×10.sup.5 cells/mL, and cultured for 3 to 4 days at 37° C., 5% CO.sub.2 with stirring at 80 rpm.
[0331] Then cell number and viability of MDCK cells being cultured in the 125 mL spinner flask were measured, the cell culture solution was inoculated sufficiently to start culture on a 250 mL scale in a 500 mL spinner flask, and the primary seed culture was performed for 3 to 4 days at 37° C. and 5% CO.sub.2 with stirring at 100 rpm. The cells were cultured in a batch process, a chemically defined medium was used as a cell culture medium, and L-glutamine was added at a concentration of 3 mM/L to 6 mM/L. After the primary seed culture, the cell number and viability were measured, and then cells were inoculated sufficiently to start culture on a 500 mL scale in two 1 L spinner flasks, and cultured for 3 to 4 days under the same conditions as in the primary seed culture.
[0332] A sample was taken from the 1 L spinner flask in culture on a 500 mL scale, and the number of cells and viability were measured, and then the cells of the culture solution in the 1 L spinner flask were aseptically inoculated into the cell incubator sufficiently to start culture on a 5 L scale in a 10 L single-use cell incubator, and cultured for 3 to 4 days. The necessary instruments and pH and DO probes were prepared and sterilized in advance to inoculate the cell culture solution in the spinner flask into the cell incubator. The pH and DO probes were installed, the culture medium was introduced, then the temperature was set to 37° C.±1° C., and the DO value was corrected for inoculation of cells.
[0333] In the same manner, cell culture was sequentially performed up to a 50 L scale, 100 L scale, 500 L scale, and 2,000 L scale.
[0334] 2) Virus Infection
[0335] When the number of cells of 1×10.sup.6 cells/mL to 3×10.sup.6 cells/mL or more was secured by taking the culture solution in the cell incubator being cultured on the 2,000 L scale and measuring the number of cells and the viability, the existing culture medium was replaced with a fresh medium. The culture medium was exchanged by removing 70% to 80% of the medium based on the cell culture solution using a low-speed continuous centrifuge and introducing a fresh medium again into the single-use bag for cell culture.
[0336] During the medium exchange and virus infection processes, the inside of the cell incubator is maintained in a closed environment, the material used for medium exchange is a single-use bag in a sterilized state, and thus the possibility of contamination is significantly low. The process time required for the corresponding process was 3 to 4 hours when the process was performed on a 10 L scale, and 7 to 12 hours for commercial production on a 2,000 L scale.
[0337] Virus infection and propagation was carried out under the conditions of DO 50%±10%, pH 7.2±0.2, the virus strain for manufacture of the influenza virus to be manufactured was thawed, the virus in an amount calculated to have an MOI of 0.0001 to 0.002 and trypsin at a concentration of 2.5 μg/mL to 20 μg/mL were aseptically introduced into the cell incubator, the cells were infected and cultured for about 3 to 4 days, the culture was terminated when the virus titer, number of cells, and viability reached proper levels, and the culture solution was recovered.
[0338] Microcarriers were not used in the cell culture and virus infection method. In particular, cells were cultured and viruses were propagated by a batch cell culture system rather than a perfusion system. After the MDCK-Sky3851 was infected with the influenza virus, the influenza virus was propagated in the cell, burst the cell, and was ejected to the outside, and thus the perfusion system was not suitable for the system of the present invention since the number of cells gradually decreased when cells were cultured by the perfusion system.
[0339] 4-2. Setting of Condition for Purification of Influenza Virus from Virus Culture and Virus Purification
[0340] 1) Centrifugation and Filtration
[0341] When the virus cultivation in the cell incubator was completed, cells and cell debris were removed by centrifuging the virus culture solution at a speed of 8,000 rpm to 20,000 rpm using a low-speed continuous centrifuge and recovering the supernatant. The recovered supernatant was filtered through a 1.0/0.5 μm pre-filter and then filtered again through a 0.45 μm filter to remove large particles of a certain size or more.
[0342] 2) Purification Condition Setting and Influenza Virus Purification Using Cellufine Sulfate (CS) Affinity Resin
[0343] The filtered virus culture solution was subjected to a primary chromatography process using a resin (Cellufine Sulfate) having affinity for influenza virus. After the column was equilibrated using a sodium phosphate buffer for equilibration, the virus filtrate was introduced. Purification conditions were rapidly set by the CSPCT method as described in Example 3-1 above. Purification condition setting test was performed using IVR-190 (A/Brisbane/02/2018(H1N1) pdm09-like virus), and the result by the CSPCT method and the result by the method using SRID were compared with each other and analyzed.
[0344] First, in order to determine the optimal conditions for virus binding to the CS column resin, the linear gradient purification process was performed and SDS-PAGE and HA assay were performed to confirm the conductivity conditions under which the loss of hemagglutinin into the column flowthrough was minimized (
TABLE-US-00004 TABLE 4 IVR-190 virus, Linear gradient purification - HA assay result Flowthrough fraction Peak fraction HA titer HA titer (HAU/ (HAU/ No. Description 50 μL) No. Description 50 μL) 1 Flowthrough 0 1 0.1M eluate-1 0 fraction 1 2 Flowthrough 0 2 0.1M eluate-2 8 fraction 2 3 Flowthrough 0 3 0.1M eluate-3 64 fraction 3 4 Flowthrough 0 4 0.1M eluate-4 256 fraction 4 5 Flowthrough 0 5 0.2M eluate 123 fraction 5 6 Flowthrough 0 6 0.2M eluate-1 32 fraction 6 7 Flowthrough 0 7 0.2M elbate-2 32 fraction 7 8 Flowthrough 0 8 0.2M eluate-3 16 fraction 8 9 Flowthrough 0 9 0.2M eluate-4 16 fraction 9 10 Flowthrough 0 10 0.3M eluate 16 fraction pooling 11 Regeneration 256
[0345] The conductivity was lowered from 13.03 mS/cm to 7.54 mS/cm by diluting the IVR-190 virus culture solution with a sodium phosphate buffer for equilibration at 1:1, and a linear gradient purification condition test was performed. It was confirmed that on SDS-PAGE, there was no HA lost into the column flowthrough, and impurities except HA were eluted. The result of HA assay using 0.5% chicken RBC was also confirmed to be 0 HAU/50 μL in all column flow fractions, and thus it was confirmed that the conductivity of the loading sample used for the test was suitable for the primary chromatography purification.
[0346] Regarding the confirmation of the virus elution concentration, a sodium phosphate buffer for elution containing sodium chloride and a sodium phosphate buffer were mixed at an appropriate ratio and introduced, and the eluted virus fraction was collected while confirming the UV280 peak, as a result, the highest peak (UV280 value) and HA titer on the chromatogram were confirmed at the 0.1 M to 0.2 M NaCl concentration as well as the peak and HA titer were confirmed to gradually decrease until the 0.3 M NaCl concentration condition, and thus it was confirmed that 0.4 M NaCl was suitable as the virus elution concentration in order to increase the yield. Considering the test results, the appropriate conductivity of the sample to be loaded on the CS column was judged to be a level of 7.54 mS/cm, and 0. M NaCl was judged to be suitable as the virus elution concentration.
[0347] In order to compare the CSPCT method with the method based on SRID, after the conductivity condition of the loading sample was set according to the CSPCT method described above, the step gradient purification process was performed by setting the virus elution concentration to 0.3 M, 0.35 M. and 0.4 M NaCl, and then the HA contents in the virus eluates of the respective batches were compared with each other. In addition, by performing SDS-PAGE and HA assay, the correlation between the results acquired by the CSPCT method of the present invention and the results acquired by the method using SRID was confirmed.
[0348] As a result of performing SRID, it was confirmed that the yield and purity were the most favorable when the virus was eluted at a 0.4 M NaCl concentration (Table 5).
TABLE-US-00005 TABLE 5 IVR-19 virus, Step gradient purification -SRID result (virus eluate) Virus elution concentration 0.3M NaCl 0.35M NaCl 0.4M NaCl HA content in virus eluate 3 μg/mL 8 μg/mL 10 μg/mL Total protein content 129 μg/mL 130 μg/mL 142 μg/mL Purity (HA/protein) 2.3% 6.2% 7.0%
[0349] The HA assay and SDS-PAGE used in the CSPCT method were performed using the sample used for SRID to analyze the virus eluate under each condition.
[0350] Referring to the SDS-PAGE result of
TABLE-US-00006 TABLE 6 HA titer of virus eluate at each virus elution concentration Virus elution concentration 0.3M NaCl 0.35M NaCl 0.4M NaCl HA titer Sample loaded on 512 512 512 (HAU/50 column μL) Column 0 0 0 flowthrough Virus eluate 1,024 1,024 2,048 Regeneration 1,024 1,024 512
[0351] Meanwhile, the pH of the equilibration buffer used in the purification process was maintained at a neutral pH of 7.0 to 7.4, and the virus filtrate was diluted with an equilibration buffer to lower the conductivity to the value at which the maximum number of viruses could bind. At the termination of virus cultivation, the conductivity of the virus culture solution was about 10.0 mS/cm to 15.0 mS/cm, the binding capacity of the virus to the resin was confirmed while lowering the conductivity by diluting the virus culture solution with an equilibration buffer having a low conductivity, as a result, the virus exhibited an appropriate level of binding capacity in the condition of about 4.0 mS/cm to 10.0 mS/cm although there is a difference depending on the type of influenza virus.
[0352] After the virus filtrate diluted with the equilibration buffer was introduced into the CS column, the column flowthrough that did not bind to the column but flowed out was sampled and discarded, the column was sufficiently washed by introducing the equilibration buffer by 2 to 3 CV, then a sodium phosphate buffer for elution containing sodium chloride and a sodium phosphate buffer were diluted at an appropriate ratio and introduced, and the eluted virus fraction was collected while confirming the UV280 peak. The conditions for elution of influenza virus were established by confirming the elution buffer containing sodium chloride at an appropriate concentration at which binding between virus and resin could be cleaved by performing CSPCT (refer to Example 3-1) in RIVPCT, and most viruses were eluted at a sodium chloride concentration of 1 M or less.
[0353] 4-3. Concentration of Virus and Desalting
[0354] Primary ultrafiltration is performed to concentrate the virus eluate and exchange the virus eluate for PBS buffer. The virus eluate was concentrated to 10% or less based on the volume of cell culture solution using an ultrafiltration filter having a size of 300 kDa to 800 kDa, and diafiltration was performed with PBS buffer in a volume equal to or more than 8 times the concentrated volume until the conductivity was the same as that of the PBS buffer.
[0355] 4-4. Removal of MDCK Cell-Derived DNA in Virus Concentrate
[0356] In order to remove cell line-derived DNA from the recovered virus concentrate, the recovered virus concentrate was treated with Benzonase (Merck, Inc.), a DNA cleaving enzyme at 0.5 U/mL to 50 U/mL and the reaction was conducted with slow stirring at room temperature for 30 minutes to 24 hours. To increase the activity of Benzonase, 0.5 mM to 5 mM magnesium chloride hexahydrate was added as a cofactor of Benzonase.
[0357] 4-5. Virus Inactivation
[0358] Infectivity was eliminated by inactivating the influenza virus concentrate subjected to the Benzonase reaction. As the chemical means for inactivating viruses, any known means can be used, and examples thereof include a detergent, formaldehyde, beta-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), and the like. In the present Example, the virus was inactivated by treatment with a formaldehyde solution at a level of 0.01% to 0.1%.
[0359] 4-6. Isolation of Surface Antigen Using Detergent
[0360] Surface antigen proteins can be isolated and purified by treating an influenza virus with a detergent (ionic or nonionic) or solvent, and as a representative example, it is common to use an ionic detergent such as cetyl trimethyl ammonium bromide (CTAB).
[0361] In the present Example as well, the virus was treated with CTAB based on the protein content in the inactivated virus concentrate, the CTAB treatment concentration was confirmed by performing CTCCT in RIVPCT as in Example 3-2. After the reaction is conducted in refrigeration or at room temperature for 1 hour or longer, centrifugation is performed at 30,000 rpm or higher using a continuous ultracentrifuge, then the supernatant is recovered, and only the surface antigen protein is isolated.
[0362] In order to remove CTAB in the ultracentrifugation supernatant, resin for adsorption (Amberlite XAD-4) was added to the supernatant to remove CTAB. Next, the resin for adsorption was removed by filtration through a filter.
[0363] 4-7. Residual DNA Removal Using TMAE Anion Exchange Resin
[0364] In order to further remove host cell-derived DNA, a secondary chromatography process was performed using an anion exchange chromatography resin. After the column was equilibrated using a sodium phosphate buffer for equilibration, the virus surface antigen recovery filtrate is introduced. At this time, the filtrate is diluted with a sodium phosphate buffer containing 1 M to 6 M sodium chloride and having a high conductivity, the chromatography process is performed at a conductivity at which the surface antigen does not bind to resin but DNA binds to resin, and the column flowthrough not bound to the resin is recovered as a surface antigen fraction.
[0365] 4-8. Concentration of Surface Antigen Fraction and Desalting
[0366] The surface antigen fraction was concentrated using an ultrafiltration filter having a size of 30 kDa to 50 kDa. When the concentration process was completed, buffer exchange was performed using PBS buffer until the conductivity of the concentrate was the same as that of the PBS buffer, and the concentrate was recovered. The recovered concentrate was sterilized and filtered through a 0.5/0.2 μm filter to produce a stock solution, and the stock solution was stored at 2° C. to 8° C.
INDUSTRIAL APPLICABILITY
[0367] It is possible to rapidly and reliably confirm the conditions for obtaining (purifying) the influenza surface antigen according to the method unique to the present invention without using the single radial immunodiffusion technique, which has been used as a standard test method in the production of influenza vaccines, thus the production period of influenza surface antigen subunit vaccines is significantly shortened, in turn, it is possible to quickly cope with a situation where the new influenza is rapidly spreading by the rapid development/manufacture of vaccine, and the industrial applicability of the method is significantly high.