CN116355035B - 一种qs21免疫佐剂的制备方法 - Google Patents
一种qs21免疫佐剂的制备方法 Download PDFInfo
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- CN116355035B CN116355035B CN202111627088.6A CN202111627088A CN116355035B CN 116355035 B CN116355035 B CN 116355035B CN 202111627088 A CN202111627088 A CN 202111627088A CN 116355035 B CN116355035 B CN 116355035B
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- saponin
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Abstract
本发明涉及一种QS21免疫佐剂的制备方法,所述方法包括如下步骤:(1)皂素粗品的溶解及过滤,(2)反相粗纯,(3)反相精纯,(4)析出重溶步骤,和(5)超滤置换步骤。使用本发明技术路线获得的QS21佐剂,纯化收率不低于50%,最终获得的QS21的纯度不低于94%,在刺激免疫功能、毒性以及稳定性等多个方面,都不弱于现有相关产品。
Description
技术领域
本发明属于医药生物技术领域,具体的,涉及一种QS21免疫佐剂的制备方法。
背景技术
2017年美国疾病控制和预防中心(CDC)免疫实践顾问委员会(ACIP)发布意见,推荐葛兰素史克重组亚单位疫苗取代默沙东减毒活疫苗/>用于50岁及以上老年人群的免疫接种。该疫苗中,使用了皂树皂苷21(QS21)作为免疫佐剂。
皂树皂苷21(QS21)结构式如下,分子式:C92H148O46,分子量:1990.14,一般性状为无色透明液体。
在癌症患者群体中,QS-21剂量在100~200μg范围内具有最佳活性和良好耐受性。在这个剂量范围内的毒性是大多数患者注射部位出现2~10厘米的红斑和硬结,以及偶尔出现轻微的低度流感样症状。此时,超过1000名患者接种了含有100μg剂量的QS-21疫苗,没有剂量限制毒性被报道。
在癌症患者群体中100或150μg的剂量的QS-21耐受性相对较好,并且已被证明比其他免疫佐剂更有效地增强对各种抗原的免疫应答。然而,QS-21剂量与免疫效力的相关性表明,如果高剂量可以安全使用,免疫原性可以进一步提高。对于大多数患者群体而言,QS-21相关的副作用限制剂量约为50μg,癌症除外。
QS-21浓度低至7~9μg/ml可引起50%引起绵羊红细胞(SRBC)溶血,表明QS-21限制剂量相关的副作用约50μg,癌症患者(如黑色素瘤、乳腺癌和前列腺癌)除外。
QS-21增强了牛对油佐剂口蹄疫疫苗的早期抗体应答,QS-21剂量750μg/牛。
使用相对高剂量的ALFQ免疫调节剂的两个动物模型(恒河猴和兔)获得的安全性数据表明使用特定的脂质和胆固醇比例的ALFQ可以减少QS-21介导的细胞溶解和其他毒性不良反应(ALFD包含100μg/ml QS-21)。
综上,QS-21是一种安全性高,耐受性良好的新型药用佐剂。
QS-21的主要作用可以概括为:1)通过作用于抗原提呈细胞(APC)和T细胞,在体内刺激Th2体液和Th1细胞介导的免疫应答,导致Th1细胞因子的释放,参与清除细胞内病原体。2)激活小鼠APC中的NLRP3型炎症小体,随后释放Caspase-1依赖的细胞因子IL-1β和IL-18,它们对Th1型应答起重要作用。3)单磷酰脂A(MPLA)与脂质体(AS01)中的QS-21在早期IFN-γ应答中的独特协同作用机制,促进疫苗免疫原性。
以QS-21作为佐剂成份的两种疫苗已经上市:1)带状疱疹疫苗(HZ/SU)于2017年获得美国食品和药物管理局的许可,并于2018年在欧盟获得营销授权;2)RTS,S/AS01疟疾疫苗/>该疫苗于2015年获得欧洲药品管理局批准,可在撒哈拉以南国家进一步实施,供常规使用。
但是,目前商品化QS-21供应被少数国际厂商垄断,为了确保疫苗规模化生产时,原辅料供应得到保障。
基于此,提出本发明,涉及QS21免疫佐剂的制备方法。
发明内容
本发明首先涉及一种以半纯化的商品级或食品添加剂级的半纯化皂素为原料,制备皂树皂苷21(QS21)免疫佐剂的制备方法,所述方法包括如下步骤:
(1)皂素溶解及过滤,
以稀释液对皂素原料进行溶解,然后滤器过滤不溶物;
(2)反相粗纯,
取步骤(1)得到的皂素过滤样品溶液,按照如下步骤进行反相粗纯:
1)上样:上样于UniPSN 30-300色谱介质;
2)洗脱:以2.5%B到21.25±5.00%B线性梯度洗脱目的峰,并分段收集高纯度的样品,合并粗纯皂素溶液;
(3)反相精纯,
取步骤(2)得到的粗纯皂素溶液,按照如下步骤进行反相精纯:
1)上样:用水稀释样品溶液至乙腈浓度为22%~28%,上样于UniPS 10-300色谱介质;
2)洗脱:10%B到25±5%B线性梯度洗脱目的峰,分段收集高纯度样品并合并;
3)合并后的样品重复上述1)~2)步骤1~3次,优选的,重复2次;
(4)析出重溶步骤,
1)取步骤(3)制备得到的纯化皂素样本溶液,加入2倍体积的水混匀,室温放置后离心收集沉淀;
2)向沉淀中加入溶解液,搅拌溶解后,去除有机溶剂样品,优选的,溶解液的加入量与步骤(3)制备得到的纯化皂素样本溶液的量相同;
(5)超滤置换步骤,
1)取步骤(4)析出重溶后获得的皂素样品溶液,用超滤置换液进行超滤浓缩获得浓缩后的QS21佐剂溶液;优选的,超滤浓缩至原体积的30%~50%;
上述步骤(1)~(5)中,
稀释液为含30%乙腈、5mM柠檬酸,pH5.0的水溶液;
B液为含99%乙腈0.1%TFA的水溶液;
溶解液为含5mM组氨酸pH6.0的水溶液;
超滤置换液为含5mM组氨酸pH5.0的水溶液。
进一步的,所述方法还包括:
(6)分装,对步骤(5)超滤置换后的QS21溶液进行除菌过滤,分装后-70℃以下温度保存。
进一步的,本发明所述的皂素原料中,QS21含量不低于8%;水分不高于10%,灰分不高于3%,重金属含量和农药残留符合中国药典标准,使用本发明所述方法纯化QS21的收率不低于50%,最终获得的QS21的纯度,不低于94%。
更进一步的,
步骤(1)中,按照1:25(W/V)对皂素原料进行溶解,然后以1.0+0.45μm滤器过滤;
步骤(2)中,上样载量30~60mg/ml,线性流速90~360cm/h,上样结束后,以A1液冲洗,以2.5%B液冲洗杂质;梯度洗脱时,收集纯度高于30%的样品进行合并;
步骤(3)中,上样载量10~50mg/ml,线性流速:100~140cm/h;上样结束后以A2液冲洗,以10%B冲洗杂质,第一次反相精纯收集纯度高于60%的样品,第二次反相精纯收集纯度高于80%的样品,第三次反相精纯收集纯度高于90%的样品,第四次反相精纯收集纯度高于95%的样品;
步骤(4)中,室温放置不少于30min,离心参数为:9000~11500g,2~8℃,10min;
步骤(5)中,超滤参数为:使用5kD膜包,跨膜压0.2~0.4bar,置换次数10次,以超滤置换液洗滤不低于8倍体积;
其中,
A1液为含30%乙腈0.1%TFA的水溶液;
A2液为含20%乙腈0.1%TFA的水溶液。
本发明还涉及所述的QS21免疫佐剂的制备方法在制备疫苗佐剂中的应用。
本发明还涉及所述的QS21免疫佐剂的制备方法在制备药品或疫苗中的应用。
本发明技术路线的关键技术参数和技术效果在于如下几点:
(1)皂素干粉作为原料进行QS-21生产,鉴于QS-21在碱性条件时稳定性较差,选择以pH5.0柠檬酸-柠檬酸钠作为缓冲体系。为了满足后期反相色谱纯化,用含有乙腈的缓冲溶液进行溶解,因此我们选择以30%乙腈5mM柠檬酸pH5.0作为稀释液对皂素进行溶解。
(2)色谱柱柱效(理论塔板数),经优化,UniPSN 30-300装柱柱效不低于1000N/m,UniPS 10-300装柱柱效不低于20000N/m,满足最终QS-21产品的质量标准。
(3)有机溶剂残留,通过对≥95%的QS-21的反相高效液相色谱图(RP-HPLC)进行分析,确定每个杂质峰的去除效果,有机溶剂在超滤置换步骤(连续洗滤不低于8倍体积)最终去除,超滤使用5Kd膜包,最终收率不低于70%。
(4)多步反相纯化中皂素溶液中乙腈浓度发生一定变化,因此,最后进行析出沉淀时,选择以水稀释至3倍体积,控制乙腈浓度在18%以下,QS-21充分析出,离心收集沉淀。
(5)使用本发明技术路线获得的QS21佐剂,收率不低于50%,最终获得的QS21的纯度不低于94%,在刺激免疫功能、毒性以及稳定性等多个方面,都不弱于GSK的QS21佐剂。
附图说明
图1、QS21佐剂的制备工艺流程图。
图2、反相精纯第三步后(四步纯化后)的QS21样品的HPLC色谱图。
图3、各试验组疫苗二次免疫后14天血清抗体型IgG滴度分析。
图4、各试验组疫苗二次免疫后14天细胞免疫分析(CD4+T细胞反应)。
具体实施方式
主要原料
皂素(来源奎拉树皮干燥提取物):购自Desert King公司(名称:VET-SAP,CAS号:8047-15-2)
经高效液相色谱检测,该原料中,QS21含量不低于8%;水分不高于10%,灰分不高于3%,重金属含量和农药残留符合中国药典标准。
UniPSN 30-300色谱介质:苏州纳微科技股份有限公司
UniPS 10-300色谱介质:苏州纳微科技股份有限公司
主要设备
主要缓冲液
缓冲液名称及成份 |
清洗液1(0.5M NaOH) |
保存液1(100mM NaOH) |
稀释液(30%乙腈~5mM柠檬酸pH5.0) |
A1液(30%乙腈0.1%TFA) |
A2液(20%乙腈0.1%TFA) |
B液(99%乙腈0.1%TFA) |
溶解液(5mM组氨酸pH6.0) |
超滤置换液(5mM组氨酸pH5.0) |
实施例1、皂素的溶解过滤及粗纯
1、皂素溶解及过滤
投料量:皂素200g,稀释液5L;
皂素以稀释液(30%乙腈~5mM柠檬酸pH5.0)按照1:25(W/V)进行溶解,以1.0+0.45μm滤器过滤,过滤后得到皂素过滤样品5.11~5.15L。
2、皂素溶液的反相粗纯
(1)前处理:连接好层析柱与液相色谱系统,分别控制A1液进入A泵口,B液进入B泵口,系统线性流速100~140cm/h,经过100%B冲洗后100%A平衡。
(2)上样:取步骤1得到的皂素过滤样品(约5L)上样于UniPSN 30-300色谱介质,载量30~60mg/ml,线性流速90~360cm/h,上样结束后以A1液(4.5L)冲洗,再依次以2.5%B冲洗杂质;
(3)洗脱:2.5%B到21.25±5.00%B线性梯度洗脱目的峰,并分段收集进行纯度检测,将纯度高于30%的样品合并,获得4.7~5.5L的粗纯皂素溶液,
反相粗纯步骤上样及洗脱的物料用量如下表所示,
实施例2、皂素的精纯
1、皂素溶液反相精纯第一步
(1)前处理:连接好层析柱与液相色谱系统,分别控制A2液进入A泵口,B液进入B泵口,系统线性流速100~140cm/h,经过100%B冲洗后100%A平衡;
(2)上样:用水稀释粗纯皂素溶液,控制乙腈浓度为22%~28%,上样于UniPS 10-300色谱介质(将稀释后的粗纯皂素溶液全部上样,载量约为10~50mg/ml),线性流速:100~140cm/h;上样结束后以A2液冲洗,再依次以10%B冲洗杂质;
(3)洗脱:10%B到25±5%B线性梯度洗脱目的峰,分段收集并进行纯度检测,将纯度高于60%的样品合并,获得4.94~5.30L二步纯化皂素样本溶液;
反相精纯第一步上样及洗脱的物料用量如下表所示,
2、皂素溶液反相精纯第二步
(1)前处理:连接好层析柱与液相色谱系统,分别控制A2液进入A泵口,B液进入B泵口,系统线性流速100~140cm/h,经过100%B冲洗后100%A平衡;
(2)上样:用水稀释二步纯化的皂素样本溶液,控制乙腈浓度为22%~28%,上样于UniPS 10-300色谱介质(将稀释完毕的样本全部上样,载量约为10~50mg/ml),线性流速:100~140cm/h;上样结束后以A2液冲洗,再依次以10%B冲洗杂质;
(3)洗脱:10%B到25±5%B线性梯度洗脱目的峰,并分段收集进行纯度检测。将纯度高于80%的样品合并,获得1.76~2.30L,作为三步纯化皂素样本溶液。
反相精纯第二步上样及洗脱的物料用量如下表所示,
3、皂素溶液反相精纯第三步
(1)前处理:连接好层析柱与液相色谱系统,分别控制A2液进入A泵口,B液进入B泵口,系统线性流速100~140cm/h,经过100%B冲洗后100%A平衡。
(2)上样:用水稀释三步纯化的皂素样品溶液,控制乙腈浓度为22%~28%,上样于UniPS 10-300色谱介质(将稀释完毕的样本全部上样,载量约为10~50mg/ml),线性流速:100~140cm/h;上样结束后以A2液冲洗,再依次以10%B冲洗杂质;
(3)洗脱:10%B到25±5%B线性梯度洗脱目的峰,并分段收集进行纯度检测。将纯度高于95%的样品合并,获得1.41~1.81L,即为四步纯化皂素样本溶液。
反相精纯第三步上样及洗脱的物料用量如下表所示,
实施例3、皂素的析出重溶、超滤置换及分装
1、析出重溶步骤
(1)取实施例2制备得到的四步纯化皂素样本溶液(1.4~1.8L),加入2倍体积的水混匀,室温放置不少于30min,再以9000~11500g,2~8℃离心10min,收集沉淀,
(2)向沉淀中加入溶解液1.5~3.0L,搅拌溶解后,获得1.5~3.0L去除有机溶剂样品。
2、超滤置换步骤
(1)以超滤置换液(5mM组氨酸pH5.0)润洗超滤膜;取本实施例步骤1析出重溶后获得的去除有机溶剂的皂素样品(约1.5~3.0L)超滤浓缩至0.91~0.93L;5kD膜包,跨膜压:0.2~0.4bar,置换次数10次。
(2)以超滤置换液洗滤不低于8倍体积,获得0.91~0.93L皂素超滤样品。
使用5kD膜包后,收率不低于70%,相比与常规的10kD超滤置换膜包,有显著提升。
3、分装
以0.45+0.2μm滤器过滤除菌超滤置换后的皂素超滤样品,即得本申请所述QS21佐剂溶液,分装后-70℃以下温度保存。
实施例4、关键工艺质量检测
1、检测方法
取三批次中试生产的各个步骤的中间样品,进行RP-HPLC检测,对比分析每个层析步骤杂质峰去除效果。色谱条件如下表:
2、主要杂质去除效率
反相粗纯、反相精纯第一步得到样品的HPLC杂质峰较多,其中QS-21主峰保留时间在22.622min,保留时间在20.376min(1号杂质)、20.734min(2号杂质)、23.109min(3号杂质)的三个峰为主要杂质峰。
反相精纯第三步后(四步纯化后)的QS21样品的检测色谱图如图2所示;
由于反相粗纯及反相精纯第一步得到样品杂质峰较多,在此仅对反相精纯第二步(三步纯化后)和反相精纯第三步四步纯化后的结果计算层析杂质去除效果,四步纯化相比与三步纯化的主要杂质去除效果如下表所示,
去除率=层析收获样品的杂质峰总量/层析上样样品的杂质峰总量,其中,
杂质峰总量=(RP-HPLC检测紫外峰峰面积/进样体积)*层析步骤样品体积
3、QS21纯度及收率检测
采用同样的RP-HPLC对QS21的纯度进行检测,结果如下表所示,
采用同样的RP-HPLC对QS21的收率进行检测,结果如下表所示,
层析步骤 | MA001-20200810 | MA001-20200815 | MA001-20200819 |
反相粗纯 | 76.77% | 75.37% | 80.60% |
反相精纯第一步 | 82.64% | 83.21% | 71.87% |
反相精纯第二步 | 37.87% | 51.13% | 60.10% |
反相精纯第三步 | 80.46% | 53.78% | 63.17% |
实施例5、QS-21功能性验证
试验样品:商品化QS-21配制的gE/复合佐剂疫苗,自制QS-21配制的gE/复合佐剂疫苗,gE/缓冲液疫苗,具体信息见下表:
备注:
gE为SEQ ID NO.1所示的gE蛋白抗原(带状疱疹疫苗),混合脂质体为阳离子脂质体和中性脂质体混合物,gE抗原及混合脂质体的制备方法见本申请人同日申请的发明专利:一种带状疱疹疫苗制剂及其制备方法。
SEQ ID NO.1:
SVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRY
本实验将本发明自制的QS-21与外购QS-21分别配制成gE/复合佐剂候选疫苗,比较两者免疫原性差异。
试验方法:在研究开始时,首先进行预免疫,再分为3组,试验组每组10只,gE/缓冲液对照组5只;
预免疫方法:经皮下给予5~6周龄雌性C57BL/6小鼠104-5TCID50个VZV致弱病毒;
在预免疫5周后,于0天、28天分别对小鼠肌肉注射,每次0.05ml/0.1剂/只;
在第二次免疫后14天对小鼠采血,并使用ELISA法检测个体血清中的结合抗体。
同时分离脾脏,通过细胞内细胞因子染色(流式细胞术法)对产生细胞因子(IL-2/IFN-γ)的CD4+T细胞频数进行评估。
实验结果显示:采用两种不同来源QS-21配制的gE/复合佐剂候选疫苗免疫后结合抗体滴度无明显差异(图3);两种疫苗制剂均能够诱导刺激产生(IL-2/IFN-γ)CD4+T细胞反应(图4);可见,采用本发明的方法流程制备的QS-21和外购QS-21配制的gE/复合佐剂候选疫苗在诱导激活免疫方面无明显差异。
最后需要说明的是,以上实施例仅用于帮助本领域技术人员理解本发明的实质,不用于限定本发明的保护范围。
序列表
<110> 成都迈科康生物科技有限公司
<120> 一种QS21免疫佐剂的制备方法
<130> TBD
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 508
<212> PRT
<213> Varicella zoster
<400> 1
Ser Val Leu Arg Tyr Asp Asp Phe His Thr Asp Glu Asp Lys Leu Asp
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Ser Trp Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn
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Ser Pro Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn
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Ala His Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser
65 70 75 80
Gly Glu Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu
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Gly Asp Asp Thr Gly Ile His Val Ile Pro Thr Leu Asn Gly Asp Asp
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Arg His Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe
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Lys Gly Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val
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Ser Val Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg
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Ile Tyr Gly Val Arg Tyr Thr Glu Thr Trp Ser Phe Leu Pro Ser Leu
165 170 175
Thr Cys Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys
180 185 190
His Thr Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu
195 200 205
Asn Thr Lys Glu Asp Gln Leu Ala Glu Ile Ser Tyr Arg Phe Gln Gly
210 215 220
Lys Lys Glu Ala Asp Gln Pro Trp Ile Val Val Asn Thr Ser Thr Leu
225 230 235 240
Phe Asp Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu
245 250 255
Lys Val Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn
260 265 270
Met Arg Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr
275 280 285
Trp Lys Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro
290 295 300
Gln Pro Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val
305 310 315 320
Phe Ser Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys
325 330 335
Ile His Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro
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Ile Asp Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr
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His Pro Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr
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Phe Thr Ser Pro His Leu Ala Gln Arg Val Ala Ser Thr Val Tyr Gln
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Asn Cys Glu His Ala Asp Asn Tyr Thr Ala Tyr Cys Leu Gly Ile Ser
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His Met Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr
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Leu Lys Phe Val Asp Thr Pro Glu Ser Leu Ser Gly Leu Tyr Val Phe
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Val Val Tyr Phe Asn Gly His Val Glu Ala Val Ala Tyr Thr Val Val
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Ser Thr Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro
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Pro Thr Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr
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Pro Val Asn Pro Gly Thr Ser Pro Leu Leu Arg Tyr
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Claims (10)
1.一种以半纯化皂素为原料,制备皂树皂苷21免疫佐剂的制备方法,其特征在于,所述方法包括如下步骤:
(1)皂素溶解及过滤,以稀释液对皂素原料进行溶解,然后滤器过滤不溶物;所述皂素中,皂树皂苷21含量不低于8%,水分不高于10%,灰分不高于3%,重金属含量和农药残留符合中国药典标准。
(2)反相粗纯,取步骤(1)得到的皂素过滤样品溶液,按照如下步骤进行反相粗纯:
1)上样:上样于UniPSN 30-300色谱介质;
2)洗脱:以2.5%B到21.25±5.00%B线性梯度洗脱目的峰,并分段收集高纯度的样品,合并粗纯皂素溶液;
(3)反相精纯,取步骤(2)得到的粗纯皂素溶液,按照如下步骤进行反相精纯:
1)上样:用水稀释样品溶液至乙腈浓度为22%~28%,上样于UniPS10-300色谱介质;
2)洗脱:10%B到25±5%B线性梯度洗脱目的峰,分段收集高纯度样品并合并;
3)合并后的样品重复上述1)~2)步骤1~3次;
(4)析出重溶步骤,取步骤(3)制备得到的纯化皂素样本溶液,加入2倍体积的水混匀,室温放置后离心收集沉淀;然后向沉淀中加入溶解液,搅拌溶解后,去除有机溶剂样品;
(5)超滤置换步骤,取步骤(4)析出重溶后获得的皂素样品溶液,超滤置换液进行超滤浓缩获得浓缩后的皂树皂苷21佐剂溶液;
其中,
稀释液为含30%乙腈、5mM柠檬酸,pH5.0的水溶液;
B液为含99%乙腈0.1%TFA的水溶液;
溶解液为含5mM组氨酸pH6.0的水溶液;
超滤置换液为含5mM组氨酸pH5.0的水溶液。
2.根据权利要求1所述的方法,其特征在于,
反相精纯步骤中,3)合并后的样品重复上述1)~2)步骤2次;
析出重溶步骤中,溶解液的加入量与步骤(3)制备得到的纯化皂素样本溶液的量相同;
超滤置换步骤中,超滤浓缩至原体积的30%~50%。
3.根据权利要求1所述的方法,其特征在于,所述方法还包括:
(6)分装,对步骤(5)超滤置换后的皂树皂苷21溶液进行除菌过滤,分装后-70℃以下温度保存。
4.根据权利要求1-3任一所述的方法,其特征在于,
步骤(1)中,按照1:25对皂素原料进行溶解,然后以1.0+0.45μm滤器过滤。
5.根据权利要求1-3任一所述的方法,其特征在于,
步骤(2)中,上样载量30~60mg/ml,线性流速90~360cm/h,上样结束后,以A1液冲洗,以2.5%B液冲洗杂质;梯度洗脱时,收集纯度高于30%的样品进行合并;A1液为含30%乙腈0.1%TFA的水溶液。
6.根据权利要求1-3任一所述的方法,其特征在于,
步骤(3)中,上样载量10~50mg/ml,上样结束后以A2液冲洗,以10%B冲洗杂质,第一次反相精纯收集纯度高于60%的样品,第二次反相精纯收集纯度高于80%的样品,第三次反相精纯收集纯度高于90%的样品,第四次反相精纯收集纯度高于95%的样品;A2液为含20%乙腈0.1%TFA的水溶液。
7.根据权利要求1-3任一所述的方法,其特征在于,
步骤(4)中,室温放置不少于30min,离心参数为:9000~11500g,2~8℃,10min。
8.根据权利要求1-3任一所述的方法,其特征在于,
步骤(5)中,超滤参数为:使用5kD膜包,跨膜压0.2~0.4bar,置换次数10次,以超滤置换液洗滤不低于8倍体积。
9.权利要求1-8任一所述的方法在制备疫苗佐剂中的应用。
10.权利要求1-8任一所述的方法在制备疫苗中的应用。
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