JPH01174379A - Method for purifying virus using hydrophilic ultrafiltration membrane - Google Patents
Method for purifying virus using hydrophilic ultrafiltration membraneInfo
- Publication number
- JPH01174379A JPH01174379A JP33490487A JP33490487A JPH01174379A JP H01174379 A JPH01174379 A JP H01174379A JP 33490487 A JP33490487 A JP 33490487A JP 33490487 A JP33490487 A JP 33490487A JP H01174379 A JPH01174379 A JP H01174379A
- Authority
- JP
- Japan
- Prior art keywords
- membrane
- virus
- ultrafiltration membrane
- proteins
- polyvinyl formal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000012528 membrane Substances 0.000 title claims abstract description 77
- 241000700605 Viruses Species 0.000 title claims abstract description 61
- 238000000108 ultra-filtration Methods 0.000 title claims abstract description 8
- 238000000034 method Methods 0.000 title claims description 27
- 239000000725 suspension Substances 0.000 claims abstract description 22
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 19
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 19
- 229920002554 vinyl polymer Polymers 0.000 claims abstract description 11
- 239000011347 resin Substances 0.000 claims abstract description 9
- 229920005989 resin Polymers 0.000 claims abstract description 9
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 abstract description 6
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 abstract description 6
- 238000001914 filtration Methods 0.000 abstract description 6
- 239000012535 impurity Substances 0.000 abstract description 6
- 230000003612 virological effect Effects 0.000 abstract description 5
- 238000005194 fractionation Methods 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 description 11
- 238000000746 purification Methods 0.000 description 10
- 239000011550 stock solution Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000000926 separation method Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 229960005486 vaccine Drugs 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 239000011148 porous material Substances 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 4
- 229920002492 poly(sulfone) Polymers 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 230000001052 transient effect Effects 0.000 description 4
- 241000710842 Japanese encephalitis virus Species 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000012466 permeate Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000005199 ultracentrifugation Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 206010014596 Encephalitis Japanese B Diseases 0.000 description 2
- 201000005807 Japanese encephalitis Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 206010037742 Rabies Diseases 0.000 description 2
- 241000711798 Rabies lyssavirus Species 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 230000001524 infective effect Effects 0.000 description 2
- 239000004745 nonwoven fabric Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 229960003127 rabies vaccine Drugs 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000710843 Japanese encephalitis virus group Species 0.000 description 1
- 201000005505 Measles Diseases 0.000 description 1
- 241000712079 Measles morbillivirus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 241000710799 Rubella virus Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 210000001557 animal structure Anatomy 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 239000003760 tallow Substances 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明はウィルスの精製法に関するものである。[Detailed description of the invention] (Industrial application field) The present invention relates to a method for purifying viruses.
（発明の背景）
ウィルスはワクチンの原料であり、ウィルスの精製はこ
のワクチン製造における必須のプロセスである。(Background of the Invention) Viruses are raw materials for vaccines, and virus purification is an essential process in vaccine production.
すなわちワクチン用のウィルスは、生体臓器中での培養
、あるいは培養した動物細胞中での培養によって得られ
るものであり、このため得られたウィルス懸濁液中には
ウィルスの他に、組織、細胞由来のタンパク質やその他
の低分子、および培地由来の例えばウシ血清タンパク質
等が存在する。これらの不純物特にタンパク質は、これ
がワクチンに含まれて生体に投与されると抗原となり、
摂取を繰返して行なうと、免疫反応によるショックを起
す可能性がある。In other words, viruses for vaccines are obtained by culturing in living organs or cultured animal cells, and the resulting virus suspension contains tissues and cells in addition to the virus. There are proteins and other small molecules derived from the culture medium, such as bovine serum proteins, etc. derived from the culture medium. These impurities, especially proteins, become antigens when they are included in vaccines and administered to living organisms.
Repeated ingestion may cause shock due to immune response.
したがってこれらの不純物はワクチン製造の過程におい
て極力除去することが望まれる。Therefore, it is desirable to remove these impurities as much as possible during the vaccine manufacturing process.
（従来の技術）
ウィルスは通常直径約５００人の大きさをもち水中では
コロイド状になっている。本発明で対象とするウィルス
懸濁液は、通常ウィルス培養組織の破砕液あるいはウィ
ルス感作細胞の培養液を約１００００ Ｇで遠心分離し
た後の上滑液として得られる。該上滑液は、組織、細胞
、培地由来のタンパク質及び低分子物質とウィルスの混
合物である。その混合液中の物質のサイズは、通常のウ
ィルスが上述のごとく約５００人と最も大きく、他の分
子、例えはタンパク質は最大のものでも約１００人であ
る。(Prior Art) Viruses usually have a diameter of about 500 people and are colloidal in water. The virus suspension targeted by the present invention is usually obtained as the supernatant fluid after centrifuging a disrupted virus culture tissue or a culture fluid of virus-sensitized cells at about 10,000 G. The synovial fluid is a mixture of tissues, cells, media-derived proteins and low molecular weight substances, and viruses. As for the size of the substances in the mixture, as mentioned above, a normal virus is the largest, about 500 people, and other molecules, such as proteins, are the largest, about 100 people.
このウィルスを不純物のタンパク質と分離するには、従
来沈殿法あるいは超遠心法が用いられている。沈殿法は
ポリエチレングリコール等のウィルスに対する沈殿剤を
ウィルス懸濁液に加え、ウィルスを選択的に沈殿させる
方法であるが、沈殿操作によりウィルスの抗原活性の減
少が著しいという欠点を有する。また超遠心法はウィル
スと不純物タンパク質の僅かな比重差を利用して両者を
分離する方法であるが、ウィルス回収率が低く、長時間
を要すると共に大量処理が困難であり、また雑菌汚染の
恐れがあるという欠点をもつ。Conventionally, precipitation methods or ultracentrifugation methods have been used to separate this virus from impurity proteins. The precipitation method is a method in which a precipitant for viruses, such as polyethylene glycol, is added to a virus suspension to selectively precipitate viruses, but it has the drawback that the antigenic activity of the virus is significantly reduced by the precipitation procedure. In addition, ultracentrifugation is a method that uses the slight difference in specific gravity between viruses and impurity proteins to separate them, but it has a low virus recovery rate, requires a long time, is difficult to process in large quantities, and is also susceptible to bacterial contamination. It has the disadvantage that there is
上記のような沈殿法あるいは超遠心法とは別に、物質の
分離を分離膜を利用して行なう方法も従来知られており
、約１００人程度の物質の分離を行なう膜として一般に
限夕（ｆ過膜が知られている。Apart from the above-mentioned precipitation method or ultracentrifugation method, methods for separating substances using separation membranes are also known. Hypermembrane is known.
（発明が解決しようとする問題点）
しかし上記限外ｒ過膜として一般に使用されているポリ
サルフォン樹脂製の限外？濾過膜は、疎水性が強く、タ
ンパク質とウィルスが混合した上述したウィルス懸濁液
を処理しようとする場合、かかる液は疎水性が強いため
、？Ｐ Ａ ＩＩＵに対象液中の成分が吸着してしまい
、長時間にわたって詰腹のもつ本来の分離性能、透水性
能を発揮させることが困難である。(Problems to be Solved by the Invention) However, the ultraviolet ray membrane made of polysulfone resin, which is generally used as the ultraviolet ray membrane, is not suitable. Filtration membranes have strong hydrophobic properties, and when trying to process the above-mentioned virus suspension containing a mixture of proteins and viruses, because such liquids are highly hydrophobic, ? Components in the target liquid are adsorbed to the P A IIU, making it difficult to demonstrate the original separation performance and water permeability of the filler over a long period of time.
（問題点を解決するための手段）
本発明者等は、上記したような現状に鑑み、新たに開発
した親水性の限外？ＦＡ膜を用いることによって本発明
を完成するに至ったものである。(Means for Solving the Problems) In view of the above-mentioned current situation, the present inventors have newly developed a hydrophilic limit system. The present invention was completed by using an FA membrane.
本発明は、可溶性タンパク質を含むウィルス懸濁液を、
ウィルスの通過は阻止するが可溶性タンパク質は通過さ
せ、かつどちらに対しても吸着性の低い限外ｆ過膜で一
過することにより、ウィルスと可溶性タンパク質の分離
・回収を好適に実現する方法を提供することを目的とす
る。The present invention provides virus suspensions containing soluble proteins.
A method for appropriately separating and recovering viruses and soluble proteins by blocking the passage of viruses but allowing soluble proteins to pass through, and passing through an ultraf membrane that has low adsorption to either. The purpose is to provide.
而して、かかる目的の実現のためになされた本発明より
なるウィルス精製法の特徴は、可溶性タンパク質を含む
ウィルス懸濁液を、分画分子量２００万〜５００万のポ
リビニルホルマール樹脂よりなる非対称構造の膜で一過
するようにしたところにある。Therefore, the feature of the virus purification method according to the present invention, which has been made to achieve this purpose, is that a virus suspension containing soluble proteins is purified by an asymmetric structure made of polyvinyl formal resin with a molecular weight cut-off of 2 million to 5 million. This is where the water passes through the membrane.
上記構成における限外ｒ過膜は概ね′）００人〜２００
０人の微細孔径を有する。The ultraviolet membrane in the above configuration is approximately 00 to 200 people.
It has a micropore size of 0.
以下本発明の詳細な説明する。The present invention will be explained in detail below.
本発明で対象とされるウィルスとしＣは、例えば日本脳
炎、狂犬病、インフルエンリゞ、麻疹あるいは風疹ウィ
ルス等を例示することができる。これらのウィルスを懸
濁した液としては、例えばウィルスに感染した動物臓器
をホモジナイズし緩衝液で希釈したもの、あるいは動物
細胞を培養し、この細胞をウィルスの感染させたものな
どが例示される。Examples of viruses targeted by the present invention include Japanese encephalitis, rabies, influenza, measles, and rubella viruses. Examples of solutions in which these viruses are suspended include those obtained by homogenizing animal organs infected with viruses and diluting them with a buffer solution, or those obtained by culturing animal cells and infecting the cells with viruses.
これらのウィルス懸濁液には予め遠心分離により不溶物
を除去したものを用いるのが望ましい。なぜなら不溶物
の存在は膜の目詰まりを著しく促進し、透水性能の低下
を引き起こすからである。It is desirable to use these virus suspensions after previously removing insoluble materials by centrifugation. This is because the presence of insoluble matter significantly promotes membrane clogging and causes a decrease in water permeability.
本発明において使用される限外ｒ過膜としては、具体的
には湿式製膜法により製膜された非対称構造を有するポ
リビニルホルマール樹脂膜が上げられる。本発明におい
て膜が非対称構造であるとは、分離を行なう膜表面で最
も孔径が小さく、膜の裏側でその孔径が大きくなる構造
を言う。このような膜の非対称構造は湿式製膜法による
製膜によって自然に形成することができ、上記膜表面の
孔径が膜の分画性能を決める。このような非対称構造の
膜を使用することで、膜表面から膜内に侵入した粒子は
、膜内部で詰まることがなく、よって膜表面の近傍を適
当に攪拌することて膜の目詰まりが防止できる。A specific example of the ultrafiltration membrane used in the present invention is a polyvinyl formal resin membrane having an asymmetric structure formed by a wet film forming method. In the present invention, a membrane having an asymmetric structure refers to a structure in which the pore size is smallest on the surface of the membrane where separation is performed, and the pore size becomes larger on the back side of the membrane. Such an asymmetric structure of the membrane can be naturally formed by membrane formation using a wet membrane formation method, and the pore size of the membrane surface determines the fractionation performance of the membrane. By using a membrane with such an asymmetric structure, particles that enter the membrane from the membrane surface do not become clogged inside the membrane, and therefore, by appropriately stirring the vicinity of the membrane surface, clogging of the membrane can be prevented. can.
上記限外？Ｐ Ａ ＩＩＵ ハ、 ５００人〜２０００
人程度の微細孔径な有するものであることが好ましい。Is it outside the above limit? P A IIU Ha, 500 to 2000 people
It is preferable that the pore size be as small as that of a human being.
微細孔径が５００人未満では可溶性タンパク質が膜を自
由に通過できなくなり、反対に２０００Å以上の孔径で
はウィルスのリークが生ずるからである。但し膜の微細
孔径がウィルスの直径より小さい必要はなく、実質的に
微細孔径がウィルスの直径の５倍以内であればウィルス
のリークは無視することかできる。This is because if the micropore diameter is less than 500 Å, soluble proteins will not be able to freely pass through the membrane, whereas if the pore diameter is 2000 Å or more, virus leakage will occur. However, it is not necessary that the micropore diameter of the membrane is smaller than the diameter of the virus, and if the micropore diameter is substantially within 5 times the diameter of the virus, leakage of the virus can be ignored.
本発明で使用する限外ｒ過膜は平膜型のものであること
が好ましい。中空糸型、スパイラル型のものでは一般に
膜透過流速分布が大きく、膜の目詰まりが激しい傾向が
あることから上記のように平膜型のものが好ましく採用
されるのである。なおチューブラ型の膜は一般に大量の
被処理液を必要とすることから、通常被処理液が数に〜
数十℃であるウィルス懸濁液を処理するのには適当でな
い場合が多い。本発明において上記の如く好ましく採用
される平膜型の限外ｒ過膜にあっては、耐雑菌汚染性に
優れると共に、膜−枚当たりの圧力損失が少なく、この
ため膜の目詰まりが少ない状態で装置を操作で籾る。The ultraviolet ray membrane used in the present invention is preferably of a flat membrane type. Hollow fiber type and spiral type membranes generally have a large membrane permeation flow velocity distribution and tend to have severe membrane clogging, so flat membrane type membranes are preferably employed as described above. In addition, since tubular membranes generally require a large amount of liquid to be treated, the number of liquids to be treated is usually large.
It is often not appropriate to treat virus suspensions at temperatures of several tens of degrees Celsius. The flat membrane type ultrafiltration membrane preferably employed in the present invention as described above has excellent bacterial contamination resistance, and has low pressure loss per membrane, resulting in less membrane clogging. In this state, operate the device to paddy rice.
上記した平膜型のポリビニルホルマール樹脂製の限外ｒ
過膜は、例えば次のようにして作製することができる。The above-mentioned flat film type polyvinyl formal resin ultra-r
The membrane can be produced, for example, as follows.
すなわちポリビニルアルコールにホルムアルデヒドを常
法に従って作用させて、分子内ホルマール化された好ま
しくは重合度６００〜９５０程度のポリビニールホルマ
ール樹脂を得、これを水と相溶性のある極性溶媒好まし
くはＮ−メチル−２−ピロリドン（あるいはこれにアセ
トンを該ピロリドンに対し７：３〜８；２の割合で添加
）で溶解してドープを作り、これを例えばポリエステル
不織布上に均一に塗布し、５〜４５℃の水を入れた凝固
浴に導入して凝固させる湿式製膜法に従って製膜するこ
とができる。That is, polyvinyl alcohol is reacted with formaldehyde according to a conventional method to obtain an intramolecularly formalized polyvinyl formal resin with a degree of polymerization of preferably about 600 to 950. This is then mixed with a polar solvent compatible with water, preferably N-methyl -2-pyrrolidone (or acetone is added to the pyrrolidone at a ratio of 7:3 to 8:2) to make a dope, and this is uniformly applied onto, for example, a polyester nonwoven fabric at 5 to 45°C. A film can be formed according to a wet film forming method in which the film is introduced into a coagulation bath containing water and coagulated.
以上の構成の限外ｒ過膜を使用して行なうウィルスの一
過精製においては、−船釣なりロスフローｒ過方式によ
って一過を行なうことにより目的を達成することができ
る。すなわち被処理液を適当な線速度で膜表面を流すこ
とにより、膜面に対する適当な一過圧力を与え、被処理
液を連続的に一過する方法である。特に次のような条件
でクロスフロー一過を行なうことが好ましい。すなわち
被処理液の膜面線速を３０ｃｍ／ｓｅｃ〜８０ｃｍ／ｓ
ｅｃとすることで不純物タンパク買は比較的効率よく膜
を通過してウィルスと分離され、ウィルスを効率よく回
収することができる。In the transient purification of viruses using the ultraviolet filtration membrane having the above configuration, the purpose can be achieved by carrying out the transient purification using the loss-flow r-filtration method. That is, this is a method in which the liquid to be treated is caused to flow over the membrane surface at an appropriate linear velocity, thereby applying an appropriate transient pressure to the membrane surface, and causing the liquid to be treated to pass through the membrane surface continuously. In particular, it is preferable to carry out the cross flow transient under the following conditions. In other words, the film surface linear velocity of the liquid to be treated is 30 cm/sec to 80 cm/s.
By using EC, impurity protein particles can pass through the membrane relatively efficiently and be separated from the virus, and the virus can be efficiently recovered.
また膜分離時の目詰まりを防ぎつつ安定した透水量を長
時間保持するためには、膜透過流速は３０４１７ｍ２・
時以下であることが望ましい。In addition, in order to prevent clogging during membrane separation and maintain a stable water permeation amount for a long time, the membrane permeation flow rate is 30417 m2.
It is desirable that it be less than 1 hour.
（発明の効果）
本発明によれば、ウィルス懸濁液から除タンパクを行な
うに当たり、他の分離膜例えばポリサルフオン膜などを
利用した時に比べ、ウィルスの活性を高度に保持するこ
とが可能であり、比較的短時間に精製を行なうことがで
きるという効果が得られる。(Effects of the Invention) According to the present invention, when removing proteins from a virus suspension, it is possible to retain virus activity to a higher degree than when other separation membranes such as polysulfon membranes are used. The effect is that purification can be carried out in a relatively short time.
また本発明は、ウィルスを材料とするワクチン製造の全
ての対象に適用可能であり、ワクチン製造を行なう分胃
におけるその有用性は極めて大なるものがある。Furthermore, the present invention is applicable to all targets for vaccine production using viruses, and is extremely useful in the production of vaccines.
（実 施 例）
以下本発明を実施例に基づいてさらに説明するが、これ
らの実施例により本発明が限定されるものではない。(Examples) The present invention will be further described below based on Examples, but the present invention is not limited to these Examples.
第１図は本発明方法の実施に用いる装置の構成概要を示
したものであり、この図で示した装置は、有効面積６５
ｃｍ２の平膜を装着できる公知のクロスフロー式の膜分
離装置（例えばｒ ＴＯ５Ｑ）ｌ ＬＩＦ−Ｌａｂｏシ
ステム」商品名：東ソー■社製」）を示している。FIG. 1 shows an outline of the configuration of an apparatus used for carrying out the method of the present invention, and the apparatus shown in this figure has an effective area of 65
A known cross-flow type membrane separation device (for example, ``TO5Q'' LIF-Labo System'' (trade name: manufactured by Tosoh Corporation)) that can be equipped with a cm2 flat membrane is shown.
この図において、１は第２図の構成を有する膜モジュル
であり、処理原液槽２からポンプ３を介して処理原液流
路４により処理原液が供給される。膜モジュル１に供給
された処理原液はその一部は限外ｒ過膜を通って透過液
となり、透過液流路管６を通り透過液槽７に集められる
。また上記処理原液の残りは循環液流路５を通って処理
原液流路４に戻る。In this figure, reference numeral 1 denotes a membrane module having the configuration shown in FIG. 2, to which a processing stock solution is supplied from a processing stock solution tank 2 through a processing stock solution channel 4 via a pump 3. A part of the treated stock solution supplied to the membrane module 1 passes through the ultraviolet filtration membrane and becomes a permeate liquid, which passes through a permeate flow path pipe 6 and is collected in a permeate tank 7 . Further, the remainder of the processing stock solution returns to the processing stock solution channel 4 through the circulating fluid channel 5 .
第２図における符合１１は限外ｒ過膜を示し、下側ハウ
ジング１２の上に載置された多孔板１３を覆うように組
み付けられ、シールリング１４により原液側と処理液側
を液密的にシールするように構成されている。１５は上
側ハウジングである。Reference numeral 11 in FIG. 2 indicates an ultraviolet filtration membrane, which is assembled to cover a perforated plate 13 placed on the lower housing 12, and is sealed liquid-tight between the raw liquid side and the processing liquid side by a seal ring 14. Configured to seal. 15 is an upper housing.
実施例１［動物用狂犬病ワクチンの精製コ（ウィルス懸
濁液の調製）
ハムスター由来の株化細胞（ＢＨＫ−２１）にウィルス
（西力原株）を感作し、イーグルＭＥＭを用いて１０９
６牛脂児血清を添加し組織培養を行なった。Example 1 [Purification of animal rabies vaccine (preparation of virus suspension) Hamster-derived cell line (BHK-21) was sensitized with virus (Nishi Rikihara strain), and 109
6 tallow serum was added and tissue culture was performed.
３７℃、５日間の培養後、培養液を５ＱＱＯＧ、３０分
間遠心し、上清をウィルス懸濁液として得た。After culturing at 37°C for 5 days, the culture solution was centrifuged at 5QQOG for 30 minutes to obtain a supernatant as a virus suspension.
（膜の作製及び装置の構成） 使用した膜は以下のようにして得た。(Membrane preparation and device configuration) The membrane used was obtained as follows.
ホルマール化率８０％、平均重合度７５０のポリビニル
フォルマール樹脂をＮ−メチル−２−ピロリドンを溶媒
として８重量％濃度のドープを調製した。このトープを
ポリエステル不織布上に均一に塗布し、続いて水温４５
℃の純水を満たした凝固浴中に導入する湿式製膜法によ
り限外ｒ過膜を作製した。A dope having a concentration of 8% by weight was prepared from a polyvinyl formal resin having a formalization rate of 80% and an average degree of polymerization of 750 using N-methyl-2-pyrrolidone as a solvent. This taupe was applied evenly onto a polyester nonwoven fabric, followed by water temperature 45°C.
An ultra-permeable membrane was produced by a wet membrane forming method in which the membrane was introduced into a coagulation bath filled with pure water at .degree.
得られた限外ｆ過膜の特性は次の通りである。The properties of the obtained ultraf film are as follows.
分画分子量 ３ Ｘ １０６
微細孔径 １５００人（推定値）この膜を第１図
の装置に装着してシステムを構成した。Molecular weight cut off: 3 x 106 Micropore diameter: 1500 people (estimated value) This membrane was attached to the apparatus shown in Figure 1 to construct a system.
（ウィルスの事前製）
予め５０００Ｇの遠心分離で粗大な不溶物を除いた上述
したウィルス懸濁液の２００ｍｆｔを、フラスコに入れ
、循環式−適法により ５１１ｍβまで濃縮し、その後
１３４ｍｆｔの１７５０Ｍリン酸緩衝液をウィルス濃縮
液に加え、全量を再び２００ｍＪ２とした。(Pre-preparation of virus) 200 mft of the above-mentioned virus suspension, which had been centrifuged at 5000 G to remove coarse insoluble matter, was placed in a flask and concentrated to 511 mβ using a circulation method, and then added to 134 mft of 1750 M phosphate buffer. The solution was added to the virus concentrate, and the total volume was again brought to 200 mJ2.
この操作を３回行ない、計３９６ｍＪＺのｒ液を得た。This operation was performed three times to obtain a total of 396 mJZ of r liquid.
？過操作中、膜面のウィルス懸濁液の線速は５０ｃｍ／
ｓｅｃとし、膜透過流速は２５ｊ２／ｍ２・時に調節し
た。? During over-operation, the linear velocity of the virus suspension on the membrane surface was 50 cm/
sec, and the membrane permeation flow rate was adjusted to 25j2/m2·hr.
分離回収されたウィルスについて活性測定を行なった。The activity of the separated and recovered virus was measured.
活性測定は、ウィルス懸濁液をイーグルＭＥＭでｌθ倍
段階希釈し、その１ ｍｌｔを予め調製したマイクロ
プレート上の狂犬病感受性細胞に各希釈５ウエルに接種
し、−週間観察した。To measure the activity, the virus suspension was serially diluted lθ times with Eagle MEM, and 1 ml of the diluted dilution was inoculated into 5 wells of rabies-susceptible cells on a microplate prepared in advance, and observed for 1 week.
狂犬病ウィルス特有のＣ，ＰＥ （細胞変性効果）の出
現をもってプラスとし、Ｂｅｈｒａｎｓ −Ｋａｒｂｅ
ｒ法により、ｆＬ ｏｇ ＴＣＩＤ ｓｏを算定した。The appearance of C, PE (cytopathic effect) peculiar to rabies virus is considered a plus, and
fL og TCID so was calculated by the r method.
その結果を下記表１に示した。なお回収率は原液を１０
０として百分率で表示した。The results are shown in Table 1 below. The recovery rate is 10% for the stock solution.
It is expressed as a percentage with 0 being taken as a percentage.
本例によれば、僅か３回の回分子過で感染価（活性）は
、４倍に精製され、また感染価の損失はなかった。According to this example, the infective titer (activity) was purified four times after only three passes, and there was no loss in the infective titer.
表 １
実施例２［日本脳炎ウィルスの精製コ
（ウィルス懸濁液の調製）
４週令マウス（ｄ、ｄ系）の脳内にウィルス（中山予研
株）を接種し、４口径脳を採取した。ホモジナイズ後１
７５０Ｍリン酸ｉ衝液を加え２０＊懸濁液とした。この
懸濁液を１８００Ｇで３０分間遠心し、上清にボルタミ
ンサルフェートを終点濃度０１５零となるように添加し
、更に１８００Ｇで１５分間遠心した上清をウィルス懸
濁液として得た。Table 1 Example 2 [Purification of Japanese encephalitis virus (preparation of virus suspension)] Virus (Nakayama Yoken strain) was inoculated into the brains of 4-week-old mice (d, d lines), and 4-caliber brains were collected. . After homogenization 1
A 750M phosphoric acid solution was added to make a 20* suspension. This suspension was centrifuged at 1800G for 30 minutes, voltamine sulfate was added to the supernatant to give an end point concentration of 015, and the supernatant was further centrifuged at 1800G for 15 minutes to obtain a virus suspension.
（ウィルスの精製）
第１図の装置に次の特性を有する限外ＷＡ膜を用いてウ
ィルスの精製を行なった。(Purification of virus) Virus was purified using the apparatus shown in FIG. 1 and an ultra-WA membrane having the following characteristics.
分画分子量 ３ Ｘ １０６
微細孔径 １５００人（推定値）膜分離による精
製の操作は実施例１と同様に３倍濃縮の回分法で３回行
なった。Molecular weight cut off: 3 x 106 Micropore diameter: 1500 people (estimated value) Purification by membrane separation was performed three times in the same manner as in Example 1 using the batch method of 3-fold concentration.
ウィルス活性（感染価）は次のようにして測定した。原
液１．０ｍＪＺに希釈液１．０ｍＩＬを加え、これを１
０−１とする。その後、更にｌＯ倍段階希釈を行ない、
１Ｏ−３〜１０−８まで調整した。マウスは４週令マウ
ス（ｄ、ｄ系）を用い、各希釈ウィルス液の０．０３ｍ
ｕを１希釈当たり１０匹のマウス脳内に与えた。日本脳
炎特有の症状を示すマウスをプラスとし、Ｂｅｈｒａｎ
ｓ−Ｋａｒｂｅｒの方法でＩｌｏｇＬＤ５゜を算出し、
感染価を求めた。Viral activity (infectious titer) was measured as follows. Add 1.0ml of diluted solution to 1.0mJZ of stock solution, and add 1.0ml of diluted solution to 1.0mJZ of stock solution
The score shall be 0-1. After that, further serial dilution was carried out by 10 times,
It was adjusted to 10-3 to 10-8. The mice used were 4-week-old mice (d, d lines), and 0.03 m of each diluted virus solution was used.
u was given intracerebrally in 10 mice per dilution. Mice showing symptoms specific to Japanese encephalitis were considered positive, and Behran
Calculate IlogLD5° using s-Karber's method,
The infectious titer was determined.
分離結果は下記表２に示した。僅か３回の回分濃縮操作
にもかかわらずタンパク性窒素量は２０零まで低下し、
活性回収率は９０＊であった。The separation results are shown in Table 2 below. Despite only three batch concentration operations, the amount of protein nitrogen decreased to 20 zero,
Activity recovery rate was 90*.
表 ２
比較例１
ポリビニルホルマール製限外ｒ過膜のかわりに、同等の
分画分子量をもつポリサルフオン製限外ＦＡＩＩｉを使
用した以外は実施例１と同様の方法で動物用狂犬病ワク
チンの精製を行なった。その結果を下記表３に示した。Table 2 Comparative Example 1 A veterinary rabies vaccine was purified in the same manner as in Example 1, except that polysulfone ultraFAIIi having the same molecular weight cut-off was used instead of the polyvinyl formal ultrafilter membrane. Ta. The results are shown in Table 3 below.
表 ３
比較例２
ポリビニルホルマール製限外−過膜のかわりに、ポリサ
ルフオン製限外ｒ過膜を使用した以外は実施例２と同様
の方法で日本脳炎ウィルスの精製を行なった。その結果
を下記表４に示した。Table 3 Comparative Example 2 Japanese encephalitis virus was purified in the same manner as in Example 2 except that a polysulfon ultrafiltration membrane was used instead of the polyvinyl formal ultrafiltration membrane. The results are shown in Table 4 below.
表 ４
以上の表１〜４の結果から明らかであるように、親木性
ポリビニルホルマール樹脂製の限外？ｊ’ Ａ ＷＡを
使用してウィルスの精製を行なう本発明の方法が有効で
あることが分かる。Table 4 As is clear from the results in Tables 1 to 4 above, the limiter made of wood-philic polyvinyl formal resin is It can be seen that the method of the present invention for virus purification using j'A WA is effective.
図面第１図は本発明方法の実施に用いられるクロスフロ
一方式の一過装置の構成概要−例を示した図、第２図は
同ｒ過装置の膜モジュルの構造を説明するための図であ
る。Figure 1 is a diagram showing an outline of the configuration of a cross-flow one-type transit device used to carry out the method of the present invention, and FIG. 2 is a diagram for explaining the structure of a membrane module of the same transit device. be.
Claims (1)
２００万〜５００万のポリビニルホルマール樹脂よりな
る非対称構造の膜でろ過することを特徴とする親水性限
外ろ過膜を用いたウィルスの精製法。A method for purifying viruses using a hydrophilic ultrafiltration membrane, characterized in that a virus suspension containing soluble proteins is filtered through a membrane with an asymmetric structure made of polyvinyl formal resin with a molecular weight cutoff of 2 million to 5 million.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33490487A JPH01174379A (en) | 1987-12-28 | 1987-12-28 | Method for purifying virus using hydrophilic ultrafiltration membrane |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33490487A JPH01174379A (en) | 1987-12-28 | 1987-12-28 | Method for purifying virus using hydrophilic ultrafiltration membrane |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01174379A true JPH01174379A (en) | 1989-07-10 |
Family
ID=18282538
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP33490487A Pending JPH01174379A (en) | 1987-12-28 | 1987-12-28 | Method for purifying virus using hydrophilic ultrafiltration membrane |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01174379A (en) |
-
1987
- 1987-12-28 JP JP33490487A patent/JPH01174379A/en active Pending
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