GB2348886A

GB2348886A - Virus or viral antigen purification method and vaccine producing method

Info

Publication number: GB2348886A
Application number: GB0006716A
Authority: GB
Inventors: Akira Yamamoto; Yae Kurosawa
Original assignee: Asahi Kogaku Kogyo Co Ltd
Current assignee: Pentax Corp
Priority date: 1999-03-18
Filing date: 2000-03-20
Publication date: 2000-10-18
Also published as: JP2000262280A; DE10013017A1; GB0006716D0; FR2791698A1

Abstract

A method for purifying a virus or viral antigen is disclosed, by which a virus or viral antigen can easily and quickly be separated and purified from a culture solution or cellular emulsion containing the virus or viral antigen without lowering the activation of the virus or viral antigen. The purification method is characterised by comprising a process in that a sample containing a virus or viral antigen is contacted with hydroxyapatite as a solid carrier which is sintered at 400 through 1300{C and capable of absorbing a virus or viral antigen, and a process in that the virus or viral antigen absorbed by the solid carrier is eluted by using an eluant in a neutral pH range. In this case, it is preferable that the hydroxyapatite is formed of spherical porous particles. It is also preferable that a phosphoric acid-based buffer solution is used as an eluate. Further, a vaccine producing method using such a purification method is also disclosed.

Description

23 48886 VIRUS OR VIRAL ANTIGEN PURIFICATION METHOD AND VACCINE PRODUCING

METHOD The present invention relates to a virus or viral antigen purification method and vaccine producing method, and in particular to a method for easily separating and puri f ying a virus or viral antigen from a culture solution containing the virus or viral antigen, and a vaccine producing method using the purification method.

Separation and purification of a virus or viral antigen (hereinafter, also referred to as "virus or the liken) from a culture solution, culture medium and host cells are important steps in the fields of genetic engineering, clinical diagnosis and vaccine production.

Generally, in the culture solution or the like, a virus or viral antigen does not exist alone, but exists together with cell culture cells and impure proteins. Thus, it is necessaryto separate and purify, only a virus or viral antigen from the culture solution or the like.

Conventionally, separation and purification of a virus or viral antigen have been performed by means of an ultra- centrifugal separating method or a density inclination centrifugal method or the like.- However-, since these methods require expensive and large-scale apparatus, - and complicated operations, work involving these methods is very troublesome.

-An alternative method for extracting a virus employing column chromatography using hydroxyapatite has been proposed (Sumiaki Tsuru et al., B:Lo-Medical Materials and Engineering Vol. 1, pp. 1-5, 1991).

According to this method, an eluant of substantially pH 7 is used, to extract a virus or the like from a sample at a high extracting rate.

However, this method leads to an unsatisfactorily separation of the virus or the like from impure proteins. Therefore, when the extracted virus is used for producing a vaccine, ultra- centrifugal separation must be further carried out. Thus, there is a problem in that it is necessary to carry out further purification process in order to obtain a virus of high purity.

A further method for purifying a virus employing column chromatography in which silica is used as a solid carrier has been proposed. In this method, there is an advantage that a virus can be extracted by one step f rom a biological material such as a culture solution. However, since an eluant whose pH is high is used, activitY Of the virus is liable to be lowered. Therefore, in this method, it I is very difficult to efficiently purify the virus without lowering activation.

An object of the invention is to provide a virus or viral antigen purification method by which a virus or the like can be easily and quickly separated and purified from a culture solution or cell emulsified solution or the like containing the virus or viral antigen, without losing or lowering activation of the virus or viral antigen, A further object of the invention is to produce a vaccine using said purification method.

According to one embodiment of the present invention there is provided a virus or viral antigen purification method comprising,. the steps of: contacting a sample containing a virus or viral antigen with hydroxyapatite as a solid carrier, said hydroxyapatite being sintered at between substantially 400 to 1300'C and capable of absorbing the virus or viral antigen; and eluting the virus or viral antigen absorbed by the solid carrier using an eluant within a neutral pH range.

Hydroxyapatite sintered at the above-mentioned temperature range has a porous structure having fine pore diameters and a porous rate that can exhibit excellent virus absorbing capacity. In this connection, only a virus or viral antigen will be efficiently separated and purified from impure protein or the like which is coexisting with the virus or viral antigen in the sample. Further, the hydroxyapatite which has been sintered at the above temperature range has an increased strength, so that the hydroxyapatlte does not collapse even while having sufficient pores to absorb the virus or viral antigen. - -j Furthermore, such hydroxyapatite is suf f iciently durable against any pressure applied in the purification process by means of column chromatography- By using an eluant within a neutral pH range, a virus whose activation is maintained can be obtained at a high purity. Here, the neutral range means a range of substantially 6 through 7.5 pH Preferably, the hydroxyapatite is formed of spherical porous particles. In a preferred embodiment, the average particle diameter of the hydroxyapatite is 10 through 1009m. Convenieritly, the hydroxvepatite is formed of porous particles.

Preferably, the eluant is a buffer solution.

Conveniently, the buffer solution is a phosphoric acid-based buffer solution.

In the present invention, it is preferred that the size of the virus or viral antigen is between substantially 10 through 500nm.

Preferably the virus or viral antigen is obtained from a living animal or cell culture cells. In a preferred embodiment, the virus or viral antigen is propagated in brain cells or neurons of marmals. Conveniently the virus or viral antigen may be propagated in an egg.Preferably the virus is propagated in cell culture cells.

In the present invention,'it is I preferred that the virus is the Japanese encephalitis virus or an influenza virus.

Further, in the present invention, it is preferred that the virus or viral antigen is Purified by means of colunul chromatography.

Furthermore, it is also preferred that the eluting speed in the eluting process Is 0. 05 through 2 ml/min.

Claims

According to a further embodiment of the present invention there is

provided a method for producing a vaccine comprising: purifying the virus or viral antigen by means of the method of purification as set forth in any one of Claims 1 to 15; and a process for inactivating the virus or viral antigen purified in the purification process.-

These and other objects, structures and advantages of the present invention will be apparent more clearly from the following description of the Invention based on the examples.

Fig. 1 is a diagram showing the eluting pattern of the Japanese encephalitis virus from a mouse brain emulsion according to the virus or viral antigen purification method of the invention (hydroxyapatite was sintered at 400C with a particle size of 40ILm is used; the eluting speed was 0.5 ml/min).

Fig. 2 is a diagram showing the eluting pattern of the Japanese encephalitis virus according to the virus or viral antigen purification method of the invention.

Fig. 3 is a diagram showing the eluting pattern of the 6 Japanese encephalitis virus according to the virus or viral antigen purification method of the invention.

Fig. 4 is a diagram showing the eluting pattern of an influenza virus or viral antigen from a sample of infected egg according to the virus or viral antigen purification method of the invention (hydroxyapatite was sintered at 400'C; the eluting speed was 0.1 ml/min).

Fig. 5 is a diagram showing the eluting pattern of an influenza virus or viral antigen from a sample of infected egg according to the virus or viral antigen purification method of the invention (hydroxyapatite was sintered at 700OC; the eluting speed was 0.1 ml/min).

Fig.6 is a diagram showing the eluting pattern of an influenza virus or viral antigen from a sample of infected egg according to the virus or viral antigen purification method of the invention (hydroxyapatite was sintered at 1000C; the eluting speed was 0.1 ml/min).

Fig. 7 is a diagram showing the eluting pattern of the Japanese encephalitis virus from a sample of infected mosquito cells according to the virus or viral antigen purification method of the invention (hydroxyapatite was sintered at 1000'C; the eluting speed was 0.5 ml/min).

"AU" in the Figures 1 to 7 means "arbitary units".

The present invention comprises a process in the which a sample containing a virus or viral antigen is contacted with hydroxyapatite sintered at between substantially 400 to 1300'C, wherein the hydroxyapatite is a solid carrier capable of absorbing the virus or viral antigen, and a process in that the virus or viral antigen absorbed by the solid carrier is eluted by using an eluant wi thin a neutral pH range.

Here, the "neutral pH range" means a range of substantially pH 6 to pH 7. 5. By using an eluant within this neutral pH range, a virus whose activation is maintained can be obtained at a high purity.

In the invention, as a solid carrier, hydroxyapatite which is sintered at a sIntering temperature of substantially 400 to 13000C is used, and more preferably, hydroxyapatite sintered at between substantially 950 to 1050C is used.

Hydroxyapatite sintered at such a temperature range has a porous structure having f ine pore diameters and a porous rate that can exhibit excellent virus absorbing capacity, In this respect, a virus or viral antigen can be ef f iciently separated and purified from impure protein or the like which coexist5 with the virus or viral antigen in - a "sample. Further, a hydroxyapatite which has been sintered at the above temperature range has an increased strength, so that the hydroxyapatite does not collapse even while having sufficient pores to absorb the virus or viral antigen. I F urthermore, such hydroxyapatite Is suf f iciently durable against any pressure applied in the purification process by means of column chromatography.

In a case where the sintering temperature of the hydroxyapatite Is less than 4000C, the absorbing capacity of the hydroxyapat:Lte becomes too high, thus leading to the case where elution of the virus or viral antigen in the eluting process described later becomes difficult, or impure protein is also absorbed. On the other hand, when the sintering temperature is higher than 1300-C, the hydroxyapatite is apt to be heat-decomposed so that it becomes unavailable as a solid carrier.

Particularly, in hydroxyapatite sintered between substantially 950 to 1050c, the number of pores to be formed is limited such that absorption of impure protein can be restricted.

Preferably, the hydroxyapatite used in this invention is formed of spherical porous. 'particles. By using a porous structure the ef f ectIve surface area per unit volume of the hydroxyapatJ-te can be maximized, which makes it possible to improve the virus absorbing capacity.

Hydroxyapatite can be effectively filled into a chromatograph column at a high density, allowing the virus separation to be carried out in a stable manner. In addition, even though the hydroxyapatite is f illed in the column at a high density, spaces are f ormed between the particles due to spherical shapes of the particles, which makes it possible to satisf actor:Lly maintain liquid permeation of an eluant such as a buf f er solution.

Preferably, the hydroxyapatite has an average particle diameter of 10 through 100jAm, and more preferably, 40 through 80pm. In a case where the particle diameter is less than 10pm, it becomes difficult for the virus or viral antigen to pass through the spaces between the hydroxyapatite particles, 9 Accordingly, the amount of virus or viral antigen absorbed by the hydroxyapatite may be -lowered. On the other hand, if the particle diameter exceeds 10OWn, the particle spaces become too large, and the speed that the virus or viral antigen passes through the particle spaces increases,. makes it dif f icult for the virus or viral antigen to be absorbed by the hydroxyapatite. Also, if the particles are too large, the interstitial space between each particle (packed beads) become large. The void space between the packed beads in the column may induce the diffusion of the separated fractions (or bands). Accordingly, the resolution of the column is affected.

Furthermore, it is preferable that the hydroxyapatite is formed of porous particles. If the hydroxyapatite is formed of dense particles which have few pores the surface area per unit volume is small, which results in the case where a sufficient virus absorbing capacity cannot be obtained.

With regard to the - porosity of the hydroxyapatite particle, 50 to 80% porosity is preferable and 65 to 75% is more preferable. If the porosity is too high, it is not possible to achieve sufficient absorptivity due to relatively small specific surface area, while if the porosity is too low, mechanical strength is liable to be lowered.

The spherical porous particles of the hydroxyapatite can be produced by the following methods. First, hydroxyapatite slurry obtained by a generally-known wet method is formed into particles by being directly spray-dried, thereby forming spherical porous particles. Alternatively, spherical porous particles can be formed by adding an additive such as a viscosity regulator, heat-decomposable organic compound particles or fiber or the like to the hydroxyapatite slurry, and then spray-drying. Next, the spherical porous particles - produced by either of the above methods are sintered at between substantially 400 to 1300C to produce porous spherical particles.

The size of the virus or viral antigen to be purif iedis not particularly limited, but preferably, approximately 10 through 500nm. The virus or viral antigen within this range can be satisfactorily separated and purified by the method according to the present invention.

Preferably, examples of the virus to be purified include an influenza virus having a size of approximately 100 through 120nm or the Japanese encephalitis virus of approximately 40 through 50nm. Further, examples of the viral antigen include structural proteinS of the J-nf luenza virus or Japanese encephalitis virus or the like.

In the present invention, it is possible to use the virus or viral antigen which is obtained by being propagated in a living animal or cell culture cells. More specifically, for example, a virus propagated in the brain cells or neurons of mammals, a virus propagated in an egg, and a virus propagated in cell culture cells, etc., can be preferably used. By propagating the virus in such a system, it is possible to obtain a large quantity of the virus constantly. Further more, in the case where cell culture cells are used for propagating the virus, the virus can be cultured in a condition of relatively few impurities, which result in a relatively easy purification of the virus.

As for the method for contacting the solid carrier formed f rom hydroxyapatite with the virus or viral antigen in the contacting process, various methods can be selectively used depending on the formation of the solid carrier to be used and the volume of the sample and the like. For example, a batch method could be used whereby hydroxyapatite is thrown into a sample to absorb the virus, or a method whereby a sample is added into a column filled with hydroxyapatite can be used.

Next, the virus absorbed by hydroxyapatite as a solid carrier is eluted. In a method of the present invention, an eluant within a neutral range is used to separate and collect a desired virus from the solid carrier in the eluting process. For example, in the case of column chromatography, fractions containing the desired virus are separately extracted.

By using such an eluant in the neutral range, the virus can be easily eluted. In addition, lowering of activation or denaturation of the virus can be suppressed.

An eluant in such a neutral pH range, for example, a buffer solution having a buffering capacity in a range of pH6 through pH7.5 can be used. Examples of such a buffer qolution include a phosphoric acld-based buffer solution, a sodium acetate-acetic acid-based buffer solution, a sodium acetate -hydrochloric acid-based buffer solution or tris aminomethane -based buffer solution or the like. Among these solutions, the phosphoric acid-based buffer solution is particularly preferable. The phosphoric acid-based buffer 12 - solution can be used as a buf f er solution that exhibits a buf f er action at substantially pH7, whereby separation and purification of the virus can be performed while maintaining the activation of the virus in stable condition.

In this connection, it is preferred that, before proceeding to the eluting process from the absorbing process of the virus by hydroxyapatite, the hydroxyapatite is washed several times using an eluant with a low concentration of salt In order to remove as many as possible of the impurities.

which are trapped in the column and are not absorbed by the hydroxyapatite. Examples of such -impurities include the crushed matter of the culture cells, partS of the virus particles, and impure protein and the like which exist In the sample.

Elution of the virus can also be performed by increasing the concentration of salt in the buf f er solution, f or example. The concentration of salt in the buf f er solution can be Increased in accordance with either a straight inclination or curved Inclination. According to this method, a substance having low absorptive such as protein is first eluted from the hydroxyapatite. This means that it is possible to selectively separate a desired virus, which has high absorption capacity with respect to the hydroxyapatitef from the hydroxyapatite.

A batch method or column I I chromatography or the like can be used to purify the virus or viral antigen. Among these methods, the column chromatography is particularly preferable, because the virus or viral antigen can be purified at a high extracting rate with high purity, and reproductivity thereof is excellent.

In a case where the virus or viral antigen is separated by column chromatography, the velocity of the flowing fluid in the eluting process is properly selected in accordance with the capacity of the sample and capacity of the column. In this case, it is pref erable that the velocity is 0. 0 5 through 2ml /min, and more preferably, 0.1 through Iml/min.

If the velocity of - th e-- f luid is too slow, it takes a relatively long time to carry out the elution of the virus, which results in lowering the activity of the virus. On the other hand, if the velocity of the f luid is too f ast, reproductivity of the separating efficiency of the virus may be deteriorated.

Further, the vaccine producing method of the invention is characterized by carrying out a virus or viral antigen purifying process according to the present invention, and a process for inactivating the virus or viral antigen purified by said purifying process. In this connection, since the virus ir viral antigen is purified at a high purity, a danger of contamination due to other microbes can be reduced, thereby allowing a highly - 14 safe vaccine to be produced.

In the process for inactivating' the virus or viral antigen purifiedby the method of the present invention, inactivation can be performed by various methods in accordance with the type of vaccine to be produced.

The virus or viral antigen purifying method and vaccine, producing method of the present invention are described in the above. However, the present invention is not limited thereto. For example, for refining of the virus or viral antigen, it is possible to use a solid carrier in which a ligand having biological specific interaction with a target virus are carried on the surface of hydroxyapatite particles.

Hereinbelow, detailed examples of the present invention will be described.

Example 1 Hydroxyapatite slurry obtained by a wet method. (molar ration of Ca/P is 1.67) was formed into particles by being directly spray-dried, thereby producing spherical secondary particles of hydroxyapatite. Next, the spherical secondary particles were sintered at the respective temperatures of 4000C, 7000C, and 10000C, and then the sintered spherical Porous particles were classified to obtain I - hydroxyapatite particles having a particle diameter of 40pm. By using thus obtained hydroxyapatite particles as a solid carrier, the Japanese encephalitis virus was purified by column chromatography.

Japanese encephalitis virus was propagated in a mouse's brain, and then the brain was extracted, and a solution of the emulsif ted brain (Japanese encephalitis virus infected mouse brain emulsion) was used as the sample.

0.45g of hydroxyapatite particles obtained by being sintered at 4000C, 0. 45g hydroxyapatite particles obtained by being Sintered at 7000C of and 0. 9g of hydroxyapatite obtained by being sintered at 1000'C were placed onto separate filters with pores having a size of 35ALm installed in a syringe having an inner diameter of 8mm and a height of 20mm. Into each column thus prepared, O.lml of the Japanese encephalitis virus infected mouse brain emulsion was added.

Next, a buffer solution of phosphoric acid of pH7 was used as an eluant to elute the Japanese encephalitis virus. The buffer solution of phosphoric acid having a linear-gradient of a phosphoric acid concentration of lOmM through 400mM was supplied Into the column at a velocity of fluid of O.5ml/min, whereby elution was performed.

The solution passed through the column was extracted by each fraction of Iml, and biological activity of the Japanese encephalitis virus in each fraction Was calculated in 16 - accordance with a plaque assay in Vero cells (obtained from the kidney of the Af rican Green Monkey Also, protein content in each fraction was inspected by means of the UV absorbing spectrum (280nm). The results are shown in Fig. 1 to Fig. 3.

Furthermore, protein in each fraction was detected by SDS-PAGE.

As clearly understood f rom Fig. 1 to Fig. 3 and the results of the SDS-PAGE (not illustrated), in all cases, most of the impure protein was not absorbed by hydroxyapatite, or washed out by the buffer solution of phosphoric acid of a low concentration. On the other hand, the Japanese encephalitis virus was securely absorbed by the hydroxyapatite, and washed out by the buffer solution of phosphoric acid of a high concentration, thereby being satisfactorily separated from the impure protein.

Also, the Japanese encephalitis virus collected the fractions No. 8 through No. 10 maintained in viral activity, while having a high purity and a high collecting rate.

Example 2 As is the same with the Example 1, an influenza virus (viral antigen) was purifiedby column chromatography using hydroxyapatite particles having a particle diameter of 40pm obtained by being sintered at 4000C, 7000C, and 10000C, respectively.

I I 17 - The influenza virus was propagated in a chicken embryo egg which was subsequently processed for use as a sample.

The same buf f er solution of phosphoric acid as in Example 1 was supplied at a velocity of fluid of 0. Iml/min, and the solution passed through the column was -collected in fractionsof 2ml, and the biological activity of the influenza virus in each fraction was calculated by the plaque assay in MDCK (Madin-Darby Canine Kidney cells).

Also, the protein content in each fraction was inspected by means of a UV absorbing spectrum (280nm).

Furthermore, hemagglutinatIon assay was performed for each fraction, and HA protein which was one of the viral antigens was detected..

The results are shown in Fig. 4 through Fig. 6.

As clearly understood from Fig. 4 through Fig. 6, in all cases, most of the impure protein was not absorbed by hydroxyapatite, or washed out by the buffer solution of phosphoric acid of a low concentration. On the other hand, the influenza virus (viral antigen) was securely absorbed by the hydroxyapatite, andwashed outby the buffer solution of phosphoric acid of a high concentration, thereby being satisfactorily separated from impure protein.

Also, the influenza virus collected in the fractions No. 4 -and No. 5 had a high purity and high - 18 collecting rate, and it was found that such an influenza virus had biological activity.

Furthermore, the results of the hemagglutination assay were also the same as the results of the plaque assay.

Example 3 The Japanese encephalitis virus was purified in the same way as in Example 1 except that a sample comprising the top clear layer of culture cells obtained by propagating Japanese encephalitis virus in C6/36 (mosquito's cells) was used.

The results in respect of the hydroxyapat-ite s - intered at 1000'C are shown in Figure 7; similar results were obtained when hydroxyapatite sintered at 4000C and 7000C, was used.

As clearly understood from Fig. 7 and the results of the SDS-PAGE (not illustrated), in all cases, most of the impure protein was not absorbed by the hydroxyapatite, or washed out by a buf f er solution of phosphoric acid of a low concentration. On the other hand, the Japanese encephalitis virus was securely absorbed by the hydroxyapatite and washed out by a buffer solution of phosphoric acid of a high concentration, thereby being satisfactorily separated from the impure protein.

The Japanese encephalitis virus -. collected in the fraction No. 9 had a high purity and a high collection rate, and it was f ound that such a Japanese encephalitis virus had biological activity.

I I Furthermore, in comparison with the results of Example 1, the eluting pattern of the virus- was sharp, and thereby it was found that the separating capacity was excellent.

Also, when the virus was puri f ied in the same way as In Examples I through 3 using 'a particle diameter of 80pn of hydroxyapatite obtained at the respective sintering temperatures, the same results as those in Examples I through 3 were obtained.

As described above, according to the virus or viral antigen purification method-of the invention, a highly pure virus or viral antigen can be efficiently obtained by simple operations.

Furthermore, since the virus or viral antigen separated and purified by the method of the present invention satisfactorily maintains the biological activity, it Is possible to produce an effective and safe vaccine.

Finally, it is to be understood that many changes and additions may be made to the embodiments described above without departing from the scope and spirit of the invention as defined in the following claims.

Further, it is also -to be- understood that the present disclosure relates to subject matter contained in Japanese Patent Application No. 11- 073915 (filed on March 18, 1999) which is expressly incorporated herein by reference in its entirely.

- CLAIMS 1. A virus or viral antigen purification method, comprising the steps of: 5 contacting a sample containing a virus or viral antigen with hydroxyapatite as a solid carrier, said hydroxyapatite being sintered at between substantially 400 to 1300C and capable of absorbing the virus or viral antigen; and eluting the virus or viral antigen absorbed by the solid carrier using an eluant within a neutral pH range.
2. A virus or viral antigen purification method according to Claim 1, wherein the hydroxyapatite is formed of spherical porous particles.
3. A virus or viral antigen purification method according to either Claim 1 or 2, wherein the average particle diameter of the hydroxyapatite is 10 through 100ym.
4. A virus or viral antigen purification method according to any preceding claim, wherein the hydroxyapatite is formed of porous particles.
5. A virus or viral purification method according to any 25 preceding claim, wherein the eluant is a buffer solution.
6. A virus or viral antigen purification method according to Claim 5, wherein the buffer solution is a phosphoric acidbased buffer solution.
7. A virus or viral antigen purification method according to any preceding claim, wherein the size of the virus or viral antigen is between substantially 10 to 500nm.
8. A virus or viral antigen purification method according to any preceding claim, wherein the virus or viral antigen is obtained from a living animal or cell culture cells.
9. A virus or viral antigen purification method according to any preceding claim, wherein the virus or viral antigen is propagated in brain cells or neurons of mammals.
10. A virus or viral antigen purification method according to any preceding claim, wherein the virus or viral antigen is propagated in an egg.
11. A virus or viral antigen purification method according to any preceding claim, wherein the virus is propagated in cell culture cells.
12. A virus or viral antigen purification method according to any preceding claim, wherein the virus is the Japanese encephalitis virus.
13. A virus or viral antigen purification method wherein the virus is an influenza virus.
14. A virus or viral antigen purification method as set forth in Claim 1, wherein the virus or viral antigen is refined by means of column chromatography.
15. A virus or viral antigen purification method as set forth in Claim 1, wherein the eluting speed in the eluting process is 0.05 through 2 ml/min.
16. A virus or viral antigen purification method as set forth in Claim 1, wherein the "neutral range" is a range of - 22 approximately pH 6 through pH 7.5.
17. A method for producing a vaccine comprising: purifying the virus or viral antigen by means of the 5 method of purification as set forth in any one of the Claims I to 16; and a process for inactivating the virus or viral antigen purified in the purification process.

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