CN1036961A - 高纯度治疗上有用的人类白细胞干扰素的工业规模制备方法 - Google Patents
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Abstract
本发明涉及一种制备人干扰素的新方法,其包括
分离血液白细胞、除去红细胞、将白细胞悬浮于营养
溶液中、用干扰素预处理并用诱导剂诱导、由细胞中
分离含干扰素的液体并纯化粗制干扰素,其特征在于
使用氯化铵溶液除去红细胞、用人血清补充营养溶
液、将白细胞以0.5×107-1.5×107细胞/ml的浓度
悬浮于营养溶液中并结合使用可控制的微孔玻璃栓
层析法及乙醇分级分离法进行纯化。
Description
本发明涉及一种制备人白细胞干扰素的方法,其包括由血液中分离白细胞、从中除去红细胞、将白细胞悬浮于合适的营养液中,用干扰素预处理、用适当诱导物诱导、由细胞中分离出含干扰素的液体、并结合使用可控制的微孔玻璃层析法及醇分级分离法纯化干扰素粗品。
人白细胞干扰素(下文简称为IFN),是一类具有抗病毒活性及某些其他生物学效应的蛋白质。干扰素抑制细胞增殖、激活天然杀伤细胞并可影响免疫系统的功能。基于这些性质,人白细胞干扰素适于有效的治疗应用。
治疗上有用的IFN以及制备IFN的适宜方法,必须符合这样几个特殊的要求:即IFN不应含有细菌内毒素;制造过程应保证没有病毒污染,并能产生最广的生物学活性亚型谱以及具有满意的产率。
有许多方法已用于制备有适宜纯度的IFN,如使用交联葡聚糖凝胶(Sephadex)G-100或G-150〔G.Bodo:Methods Enzymol.78,69(1981)〕、Ultragel AcA54〔J.E.Whitmann等;J.Interferon Res.1,305(1981)〕、磺丙基葡聚糖凝胶〔P.J.Bridgen等,J.Biol.Chem.252,6585(1977)〕或苯基琼脂糖凝胶〔J.E.Whitmann等,J.Interferon Res.1,305(1981)〕的层析法;或使用铜螯合剂〔K.Berg等,J.Immunol.11,489(1980)〕;或使用CPG层析法〔K.C.Chanda:J.Interferon Res.2,229(1082)〕。
虽然联合使用上述方法能够制得均质IFN产品,但是产率却很低,而且有些方法并不适于大规模工业生产。
迄今所发展的用于大量制备治疗上有用之IFN的方法如下所述。
Cantell等人曾详细描述了一种纯化方法〔Methods Enzymol.78,499(1981)〕,其包括一系列控制PH的沉淀步骤,可产生一种具有比活性为2×106IU/mg的蛋白质产品,其产率为50%。PH3.5时用异硫氰酸钾沉淀白细胞IFN粗品,然后溶解于含酸乙醇中,逐渐增加PH到5.3,然后再增加到5.8而选择性地沉淀出杂质。经将乙醇溶液的PH值增加到8.0,沉淀出IFN,然后重新溶解于含有0.5M异硫氰酸钾的0.1M磷酸盐缓冲液中。再将PH降至5.2而使污染物沉淀下来。溶解的IFN则于PH3.0时沉淀。然后,将该沉淀物溶解于0.1M磷酸盐缓冲液(PH8.0)中并对PBS(磷酸盐缓冲盐水)透析。
Wellcome研究实验室研究了另一种纯化由Namalva细胞产生之IFN的方法〔S.Rouveny等,Ann.Virol.13,191(1982)〕,由此可得到平均比活性为6×107IU/mg的蛋白质。根据该方法,用0.5%三氯乙酸溶液沉淀类淋巴母细胞IFN粗品,然后在PH3.5时用94%乙醇提取沉淀物。再经逐步提高PH值而纯化乙醇提取物。可使用多克隆抗体-亲和层析法进一步纯化如此制得的干扰素。
纽约血液中心的B.Horowitz等人建立了一种制备高纯度IFN的两步骤层析法〔Methods Enzymol.119,39(1986)〕。即将IFN粗品吸附在CPG-10775柱上并用含50%乙二醇的缓冲液洗脱。使用单克隆抗体亲和层析法(NKZ-Sepharose柱,Celltech)进一步纯化洗脱物,得到比活性为3.6×108IU/mg蛋白的产品,其产率为50%。
使用本发明的方法,能够得到一种可在很大程度上满足上述要求的产品。所得产品的纯度高,其内毒素含量很低。由于采取了某些工艺步骤,使得最终进入产品中的病毒都是被失活了的。所获产品含有很宽的IFN亚型谱,包含一高比例的酸不稳定性组分。产率很高,所以生产的经济效宜很好。
本发明涉及制备一种具有高滴度的IFN粗产品,以及结合使用可控制的微孔玻璃层析法及醇分级分离和膜分离技术以纯化IFN。方法的各个步骤一起确保了产品的有利性质。
本发明的方法中以氯化铵诱导溶血作用破坏红细胞从中纯化出人血白细胞。根据痉⒚鞣椒ǖ挠叛∈凳┓桨福苎饔檬前炊椒ǎ?~5℃下使用缓冲到PH值为7.2~7.4的0.83%氯化铵溶液完成的。第一步中,细胞悬浮液对氯化铵的比例为1∶3,第二步比例为1∶9。以1000~2000×g离心,由溶血介质中沉淀出白细胞。将所得白细胞悬浮液悬浮在合适的营养液(Eagle MSH、RPMI、MSKD等)中,细胞浓度为1~1.3×107细胞/ml。营养液还含有0.5~2.5mg/ml无γ球蛋白人血清。
首先,将细胞培养物与100~200IU/ml人IFN在37℃下共同保温1.5~3小时。然后用100~200HA单位/ml仙台病毒诱导IFN产生。
诱导后1~3小时,适当改变保温条件(PH、盐浓度、温度、添加过氧化氢),继续保温15~25小时,然后经离心由营养液中回收白细胞,得到IFN粗制品。
过滤除去IFN粗品中的浮动杂质和细胞碎片,然后加压通过CPG10/15柱进行层析。先用PBS,然后用含有0.05~0.1MTris-HCl和1.5M氯化钠的溶液(PH8.0)进行梯度洗脱。
PBS(磷酸盐缓冲盐水)的组成如下:
mg/ml
NaCl 8800
KCl 220
Na2HPO4·12H2O 3500
NaH2PO4·H2O 224
pH:7.2~7.4
Tris缓冲液含有三(羟甲基)-氨基甲烷,它不会使IFN失活,但可大大改善洗脱效率。当然也可以使用其他伯、仲或叔胺。
用55~75%乙醇于-20℃下处理含IFN的洗脱物。用离心法除去沉淀的蛋白质。用超滤膜将含有IFN的乙醇上清液浓缩10至20倍,然后用适当缓冲液通过透滤或透析置换乙醇。或者可再次吸附存在于醇上清液中的IFN。可将如此得到的高纯度IFN溶液直接冷冻干燥。
一方面进行层析,另一方面用55~75%乙醇,可确保没有病毒。因为内毒素可被乙醇沉淀,所以用乙醇处理即可除去任何最后存在的内毒素。
对酸不稳定的IFN亚型不会丢失其活性,因为本发明的方法中没有包括任何于低PH条件完成的步骤。
下列非限定性实施例旨在进一步阐明本发明的方法。
实施例1
得自人血的血沉棕黄层(富含白细胞的血液部分),在冷却(置于0至4℃冰上)条件下与3倍体积的0.85%氯化铵溶液一同搅拌10分钟。10分钟后,以1500xg,4℃离心而由混合物中沉淀出白细胞。将白细胞悬浮于PBS中,并用9倍体积的0.83%氯化铵溶液再次处理之。10分钟后,依上述相似方法离心沉淀细胞并以1.0×107细胞/ml的浓度悬浮于Eagle MEC营养液中。营养液中还含有1mg/ml无γ球蛋白人血清。向细胞培养物内加入150IU/ml人IFN,然后在37℃恒温槽中搅拌2小时。继之加入100HA单位/ml仙台病毒并于37℃保温15至20小时以诱导IFN产生。所得营养溶液即为IFN粗品,其比活性为200,000IU/mg蛋白质。
IFN粗品以100ml/mlCPG的比例压入装有CPG10/75的、预先用PBS平衡的柱内。用PBS洗柱直到其中没有蛋白质,然后用Tris-缓冲的1.5M氯化钠溶液洗脱IFN。洗脱物的比活性为10,000,000IU/mg蛋白质。洗脱物于-20℃下与保持-20℃的乙醇混合,使乙醇终浓度达到75%。醇混合物于-20℃放置24小时,然后离心。
倾去沉淀物后,上清于-20℃通过超滤膜而浓缩。经透滤法除去乙醇,同时用适当缓冲溶液置换之。将所得产品分装到安瓶内并冷冻干燥。
实施例2
按实施例1中所述的方法制备IFN粗品并进行继后的CPG层析。
亦可用沉淀法从含有IFN的75%乙醇溶液中分离出干扰素。含IFN的乙醇溶液于-10℃下对含30%乙醇的0.05M柠檬酸盐缓冲液(PH5)透析。离心分离沉淀的蛋白质并重新溶解于相应量的PBS中。所得产品可直接冻干。
实施例3
与实施例2所述方法相似,不同的是使用超滤膜浓缩IFN的75%乙醇溶液,然后对含有20%乙醇的0.05M柠檬酸盐缓冲液(PH5)透析。接下来则按实施例2中所述方法处理沉淀的蛋白质。
Claims (8)
1、一种制备人干扰素的方法,其包括分离血液中的白细胞、除去红细胞、将白细胞悬浮于营养溶液中、用干扰素预处理、用诱导剂诱导、将含有干扰素的液体与细胞分离开及纯化干扰素粗制品等步骤,特征在于使用氯化铵溶液除去红细胞、营养溶液中添加人血清、将白细胞以0.5×107至1.5×107细胞/ml的浓度悬浮于营养溶液中进行诱导,并联合使用可控制的微孔玻璃层析法及乙醇分级分离法进行纯化。
2、根据权利要求1的方法,其包括使用可控制的微孔玻璃10/75柱进行纯化。
3、根据权利要求1或2的方法,其中包括每1ml柱体积加50至200ml干扰素粗品。
4、根据权利要求1至3中任一项的方法,其包括用0.1至1.5M伯、仲、叔胺或季铵溶液进行洗脱。
5、根据权利要求1至4中任一项的方法,其包括用终浓度为55至75%的乙醇处理洗脱液。
6、根据权利要求1至5中任一项的方法,其包括于0°~40℃下用乙醇处理。
7、根据权利要求1至6中任一项的方法,其包括用已缓冲到PH6至7.5的0.83%氯化铵溶液除去红细胞。
8、根据权利要求1至7中任一项的方法,其包括在营养溶液中添加0.5至2.5mg/ml用可控制的微孔玻璃柱层析法纯化的无γ球蛋白人血清。
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HU881050A HU201100B (en) | 1988-03-04 | 1988-03-04 | Process for large-scale production of high purity human leukocyte alpha interferon with reduced endotoxin content and with improved therapeutic effect |
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CN89102018.7A Pending CN1036961A (zh) | 1988-03-04 | 1989-03-04 | 高纯度治疗上有用的人类白细胞干扰素的工业规模制备方法 |
Country Status (7)
Country | Link |
---|---|
JP (1) | JPH01281097A (zh) |
CN (1) | CN1036961A (zh) |
AT (1) | AT391482B (zh) |
DE (1) | DE3906871A1 (zh) |
HU (1) | HU201100B (zh) |
IN (1) | IN169468B (zh) |
IT (2) | IT1228560B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104622777A (zh) * | 2015-02-15 | 2015-05-20 | 广州赛莱拉干细胞科技股份有限公司 | 一种白细胞提取物及其制备方法与应用 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2888443B2 (ja) * | 1989-12-07 | 1999-05-10 | 日本ケミカルリサーチ株式会社 | ヒト白血球インターフェロン亜種の抗体の製造法及び測定法 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NL7907791A (nl) * | 1979-10-23 | 1981-04-27 | Stichting Rega V Z W | Werkwijze voor het zuiveren van interferon. |
US4382027A (en) * | 1981-08-18 | 1983-05-03 | Meloy Laboratories, Inc. | Purification of human immune interferon |
JPS5889196A (ja) * | 1981-11-24 | 1983-05-27 | Fujisawa Pharmaceut Co Ltd | インタ−フエロンの精製法 |
HU184972B (en) * | 1981-12-01 | 1984-11-28 | Egyt Gyogyszervegyeszeti Gyar | Process for preparing human gamma interferone |
HU192254B (en) * | 1983-12-13 | 1987-05-28 | Egyt Gyogyszervegyeszeti Gyar | Process for producing human leucocite and human gamma interferons in consecutive steps |
-
1988
- 1988-03-04 HU HU881050A patent/HU201100B/hu not_active IP Right Cessation
-
1989
- 1989-03-03 IN IN179/MAS/89A patent/IN169468B/en unknown
- 1989-03-03 JP JP1050210A patent/JPH01281097A/ja active Pending
- 1989-03-03 IT IT8919634A patent/IT1228560B/it active
- 1989-03-03 DE DE3906871A patent/DE3906871A1/de active Granted
- 1989-03-03 AT AT0047989A patent/AT391482B/de not_active IP Right Cessation
- 1989-03-03 IT IT8919633A patent/IT1228559B/it active
- 1989-03-04 CN CN89102018.7A patent/CN1036961A/zh active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104622777A (zh) * | 2015-02-15 | 2015-05-20 | 广州赛莱拉干细胞科技股份有限公司 | 一种白细胞提取物及其制备方法与应用 |
Also Published As
Publication number | Publication date |
---|---|
IT1228559B (it) | 1991-06-21 |
HUT49894A (en) | 1989-11-28 |
DE3906871C2 (zh) | 1991-08-14 |
IN169468B (zh) | 1991-10-19 |
DE3906871A1 (de) | 1989-09-21 |
JPH01281097A (ja) | 1989-11-13 |
AT391482B (de) | 1990-10-10 |
IT8919634A0 (it) | 1989-03-03 |
IT8919633A0 (it) | 1989-03-03 |
HU201100B (en) | 1990-09-28 |
ATA47989A (de) | 1990-04-15 |
IT1228560B (it) | 1991-06-21 |
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