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1.重组均质纯净固态及非免疫原型干扰素之生产方法,该干扰素含有低于5%之甲硫胺酸干扰素,不具减数型及干扰素之碎屑,含有低于0.2%之寡聚物及低于2%之二体物/三体物/四体物,且其中高于90%之单体含量组成天然单体干扰素,此方法之特性为:含干扰素基因之E.coli在正常情况下培养,细胞在正常观察期于pH2时在匀浆器中杀死,及收成,其中形成不超过20%,较佳不超过5%,最佳不超过1%之甲硫胺酸干扰素,实验干扰素以正常方式分离,细胞碎屑在稍微呈 性之介质中分离,干扰素浓缩并以衔接色谱法纯化之,该衔接色谱由己填充DE-52纤维素之纤维素前柱及己填充与载体结合之单源抗体EBI 1之亲和性色谱组成,其中欲纯化物质以TRIS-NaCl缓冲剂pH7.5在两柱上清筅,干扰素在25%含0.1M柠檬酸之乙二醇中从亲和栓上洗提,调节洗提剂至pH値为4.5以便移去杂质,最后藉色谱法在MONO-S,HR 10/10型阳离子交换柱纯化干扰素,以线性浓度梯度自0.1至1.0M,较佳为0.1至0.5M,之乙酸铵缓冲剂在pH4.0-5.0,较佳为pH4.5,为洗提剂,及尔后冻乾之。2.根据申请专利范围第1.项之方法,特性为寄主生物含胺基酸顺序如下之人体干扰素编码基因:Cys Aap Lau Pro Gln Thr His Ser Leu Gly Ser Arq Qrq Thr LeuMet LeuLeu Ala Gln Met Arq Arq Ile Ser Leu Phe Ser Cys LeuLys Asp Arq Arq Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly AsnGln Phe Gln Lys Ala Glu ThrIle Pro Val Leu His Glu Met IleGln Gln Ile Phe Asn Leu Pho Ser Thr Lys Asp Ser Ser Ala AlaTrp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr GluLeu Tyr GlnGln Lou Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly ValThr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Lou Ala Val ArqLys Tyr Phe Gln Arq Ile Thr Lou Tyr Lou Lyo Glu Lys Lys TyrSer Pro Cys Ala Trp Glu Val Val Arq Ala Glu Ile Met Arq SerPhe Ser Leu Set Thr Aan Leu Gln Glu Ser Lou Arq SeL Lys Glu3.根据申请专利范围第1.或2.项之方法,生产重组,均质固态及非免疫原型人体干扰素具如下胺基酸顺序Cys Aap Lau Pro Gln Thr His Ser Leu Gly Ser ArqQrq Thr LeuMet Leu Leu Ala Gln Met Arq Arq Ile Ser Leu Phe Ser Cys LeuLys Asp Arq Arq Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly AsnGln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met IleGln Gln Ile Phe Asn Leu Pho Ser Thr Lys Asp Ser Ser Ala AlaTrp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr GlnGln LouAsn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly ValThr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Lou Ala Val ArqLys Tyr Phe Gln Arq Ile ThrLou Tyr Lou Lyo Glu Lys Lys TyrSer Pro Cys Ala Trp Glu Val Val Arq Ala Glu Ile Met Arq SerPhe Ser Leu Set Thr Aan Leu Gln Glu Ser Lou ArqSeL Lys Glu及含少于5%之甲硫胺酸干扰素,不含减数型及干扰素之碎屑,所含寡聚物低于0.2%,二体物/三体物/四体物少于2%,其中天然单体物干扰素在位置1及98及29及138半胱氮酸间均含二硫键。4.干扰素,其可以申请专利范围第1.项之方法生产者。5.重组干扰素,其特性为呈均质,纯净型态,含甲硫胺酸干扰素低于20%,较佳是低于5%,最佳是低于1%。6.根据申请专利范围第5.项之干扰素,其特性为呈均质,纯净型态,不含减数及碎屑,所含之寡聚体低于0.2%,二体物/三体物/四体物低于2%。7.根据申请专利范围第5.或6.项之干扰素,无寡聚体及二体物/三体物/四体物。8.根据申请专利范围第5.或6.项之干扰素,其中单体物成份有90%以上为天然单体物干扰素。9.根据申请专利范围第5.或6.项之干扰素无非天然单体物干扰素。10.根据申请专利范围第5.或6.项之干扰素,特性为不刺激产生任何一种抗体。11.根据申请专利范围第5.或6.项之干扰素,特性为呈固态。12.根据申请专利范围第5.或6.项之人体干扰素。13.根据申请专利范围第5.或6.项之干扰素,特性为胺基酸顺序相当于如下所示:Cys Aap Lau Pro Gln Thr His Ser Leu Gly Ser Arq Qrq Thr LeuMet Leu Leu AlaGln Met Arq Arq Ile Ser Leu Phe Ser Cys LeuLys Asp Arq Arq Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly AsnGln Phe Gln Lys Ala Glu Thr Ile ProVal Leu His Glu Met IleGln Gln Ile Phe Asn Leu Pho Ser Thr Lys Asp Ser Ser Ala AlaTrp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu TyrGlnGln Lou Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly ValThr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Lou Ala Val ArqLys Tyr Phe Gln Arq Ile Thr Lou Tyr Lou Lyo Glu Lys Lys TyrSer Pro Cys Ala Trp Glu Val Val Arq Ala Glu Ile Met Arq SerPhe Ser Leu Set Thr Aan Leu Gln Glu Ser Lou Arq SeL Lys Glu14.重组非免疫原型固态人体干扰素,具如下之胺基酸顺序:Cys Aap Lau Pro Gln Thr His Ser Leu Gly Ser Arq QrqThr LeuMet Leu Leu Ala Gln Met Arq Arq Ile Ser Leu Phe Ser Cys LeuLys Asp Arq Arq Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly AsnGln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met IleGln Gln Ile Phe Asn Leu Pho Ser Thr Lys Asp Ser Ser Ala AlaTrp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr GlnGln Lou AsnAsp Leu Glu Ala Cys Val Ile Gln Gly Val Gly ValThr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Lou Ala Val ArqLys Tyr Phe Gln Arq Ile Thr LouTyr Lou Lyo Glu Lys Lys TyrSer Pro Cys Ala Trp Glu Val Val Arq Ala Glu Ile Met Arq SerPhe Ser Leu Set Thr Aan Leu Gln Glu Ser Lou Arq SeLLys Glu呈均质,纯净型式,所含之甲硫胺酸干扰素少于5%,寡聚物少于0.2%,二体物/三体物/四体物少于2%,并无减数型及干扰素之碎屑,其中单体物成份有95%以上由天然单体物干扰素组成,其中在位置1及98,29及138半胱氮酸间均有一个二硫键。15.根据申请专利范围第4.、5.、6.或14.项之干扰素,适用于药理治疗滤过性病毒及肿瘤疾病。16.药理治疗用之制剂,特性为包含根据申请专利范围第4.、5.、6.或14.项之干扰素并含一或多种药理钝性之辅助剂及/或载体成分。图示简单说明:图1:经衔接色谱法后酸性洗提液之逆构HPLC(高压液相层析)之色谱;成份以K1-K7表示。图2:经衔接色谱法后酸性洗提液之凝胶渗透-HPLC之色谱。图3:在pH4.5沈淀后之逆相色谱,成分以K1,K2,K3及K6表示。图4:在pH4.5沈淀后之凝胶渗透-HPLC之色谱图。图5:FPLC在MONO-S后逆相HPLC色谱图。图6:「肩部馏分」逆相HPLC之色谱图。图7:「肩部馏分」凝胶渗透色谱图。图8:「主要馏分」逆相HPLC色谱图。图9:「主要馏分」凝胶渗透-HPLC色谱图。图10:「主要馏分」色聚焦色谱图。图11:经衔接色谱法后及藉逆相HPLC分离成分K1-K7酸性洗提液之凝胶电泳图。图12a-12f:抗干扰素之抗体结果,各项显示研究。图13:抗干扰素之抗体检定图。 |
