第130期 东曹色谱产品在病毒关联药物和疫苗研发中的应用
病毒是一种个体微小，结构简单，只含一种核酸（DNA或RNA），必须在活细胞内寄生并以复制方式增殖的非细胞型生物。
病毒是一种非细胞生命形态，它由一个核酸长链和蛋白质外壳构成，病毒没有自己的代谢机构，没有酶系统。因此病毒离开了宿主细胞，就成了没有任何生命活动、也不能独立自我繁殖的化学物质。它的复制、转录和转译的能力都是在宿主细胞中进行，当它进入宿主细胞后，它就可以利用细胞中的物质和能量完成生命活动，按照它自己的核酸所包含的遗传信息产生和它一样的新一代病毒。
一、病毒和疫苗的特性
※疫苗
·蛋白质作为表面抗原
·天然蛋白、多糖或重组蛋白
·类似于蛋白质分离的纯化策略
·纯化用到的分离模式SEC、IEC、HIC和AFC
※病毒
·巨大的生物分子复合粒子
·流感病毒：100-120nm；腺病毒：80nm；天花病毒：30nm
·不同纯化策略对蛋白质的分离有不同的影响
二、疫苗用色谱填料
※尺寸排除色谱法（SEC）
·病毒分离需要大孔径的填料
病毒尺寸：100nm----TOYOPEARL HW-65，Sepharose 6 FF
病毒尺寸：200nm---- TOYOPEARL HW-75，Sepharose 4 FF
·填料的刚性对装柱更为重要
TOYOPEARL比Sepharose基质具有更好的刚性和稳定性。
※离子交换色谱法（IEC）
·大孔径的填料:TOYOPEARL 650系列
· 接枝型的聚甲基丙烯酸甲酯，分离效果会更好，如GigaCap Q-650M，GigaCap S-650M，SuperQ-650M
※疏水层析（HIC）
·不同填料的疏水性有差异
·疏水性较低的填料可能更有效分离病毒
·DNA在HIC填料上的保留较差
※亲和层析（AFC）
·染料：TOYOPEARL AF-Blue-650M
·肝素：TOYOPEARL AF-Heparin-HC-650M
·其他蛋白质固定化AFC树脂
在这些关联药物的研发、评价、纯化精制中，TSKgel液相色谱柱和Toyopearl层析填料产品有着相当多的应用实例。
TSKgel色谱柱分析柱的应用实例
Year | Mode | TSKgel Column | Target,keyword | Title | Citation at web site |
2020 | SEC | TSKgel G5000PWXL | Coronavirus protein/ 冠状病毒蛋白 | Characterization of the SARS-CoV-2 Spike in an Early Prefusion Conformation/早期融合前构象中SARS-CoV-2峰的表征 | https://www.biorxiv.org/content/10.1101/2020.03.16.994152v1.full |
2020 | IEC | TSKgel DNA-STAT | PS-oligonucleotide / PS-寡聚核苷酸 | Condition Study of Phosphorothioate Oli gonucleotides Separation by High- performance Anion Exchange Chromatography /硫代磷酸寡聚核苷的阴离子高效液相色谱分析的条件研究 | Poster presentation in Asia TIDES 2020 |
2019 | SEC | TSKgel UP-SW2000 | Oligonucleotide / 寡聚核苷酸 | Analysis of olignucleotide by SEC /寡聚核苷酸的分子尺寸排阻色谱分析 | |
2019 | RPC IEC | TSKgel ODS-120H DNA-STAT | Oligonucleotide / 寡聚核苷酸 | Examination of analytical conditions for separation of oligonucleotides for LC/MS /液相色谱进行寡聚核苷酸分析条件的检验 | Poster presentation in HPLC Kyoto, 2019 |
2017 | SEC | TSKgel G6000PWXL G5000PWXL | Conjugated vaccine / 结合疫苗 | Determination of free polysaccharide in Vi glycoconjugate vaccineagainst typhoid fever /伤寒疫苗中游离多糖的测定 | https://www.sciencedirect.com/science/article/pii/S0731708516312511?via%3Dihub |
2017 | SEC | TSKgel G5000PWXL | Virus-like particle / 类病毒颗粒（VLP） | Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysis / 通过尺寸排除色谱和纳米颗粒跟踪定性和定量分析类病毒颗粒 | https://www.sciencedirect.com/science/article/pii/S0021967316317514 |
2016 | SEC | TSKgel G3000PWXL | Vaccine/疫苗 | Immunogenicity and protective efficacy of rotavirus VP8 fused to cholera toxin B subunit in a mouse model/轮状病毒VP8与霍乱毒素B亚单位融合在小鼠模型中的免疫原性和保护作用 | |
2016 | SEC | TSKgel G6000PWXL | Flu virus(VP)/流感病毒 | Size distribution analysis of influenza virus particles using size exclusion chromatography /流感病毒颗粒大小分布的分子尺寸排阻色谱分析 | https://www.sciencedirect.com/science/article/pii/S0021967316311335 |
2015 | SEC | TSKgel G5000PWXL G4000PWXL | Conjugated vaccine/结合疫苗 | Development and characterization of Haemophilus influenzae type b conjugate vaccine prepared using different polysaccharide chain lengths /不同多糖链长流感嗜血杆菌B型偶联疫苗的研制与表征 | https://www.sciencedirect.com/science/article/pii/S0264410X15004958?via%3Dihub |
2014 | SEC | TSKgel G5000PWXL-CP | Bacteriophage particle/噬菌体颗粒 | Size exclusion-based purification and PCR-based quantitation of MS2 bacteriophage particles for environmental applications/用于环境应用的MS2噬菌体颗粒的储存排阻纯化与PCR定量 | https://www.sciencedirect.com/science/article/pii/S0166093414004704?via%3Dihub |
2013 | SEC | TSKgel G5000PWXL-CP | Virus particle/病毒颗粒 | A Gel Filtration -Based Method for the Purification of Infectious Rotavirus Particles for Environmental Research Applications /一种用于环境研究应用的感染性轮状病毒的凝胶过滤纯化方法 | |
2013 | HIC | TSKgel Butyl-NPR | DNA vaccine/DNA 疫苗 | Antibiotic-free production of a herpes simplex virus 2 DNA vaccine in a high yield cGMP process /无抗生素生产单纯疱疹病毒2型DNA疫苗的高产cGMP过程 | |
2008 | SEC | TSKgel G4000PWXL | Flu vaccine/流感疫苗 | Development of physicochemical methods for analysis of pandemic influenza vaccine/大流行性流感疫苗理化分析方法的发展 | https://journal.lib.uoguelph.ca/index.php/surg/article/view/420 |
2007 | IEC | TSKgel DNA-NPR | Plasmid DNA/质粒DNA | Separation of topological forms plasmid DNA by anion -exchange HPLC: Shifts in elution order of linear DNA/阴离子交换高效液相色谱法分离质粒DNA的拓扑结构 | https://www.sciencedirect.com/science/article/pii/S1570023207002747 |
2007 | SEC | TSKgel G5000PWXL | Conjugated vaccine /结合疫苗 | Quality and Immunogenicity of MenAfriVac, a Conjugate Vaccine Against Meningococcal Group ADisease/脑膜炎球菌 a群疾病的结合疫苗MenAfriVac的质量和免疫原性 | https://www.meningvax.org/files/PharmaPhase2PosterSept07.pdf |
2007 | SEC | TSKgel G5000PWXL | Flu vaccine /流感疫苗 | A Retrospective Study on the Quality of Haemophilus influenzae type b Vaccines Used in the UK Between 1996 and 2004 / 对1996年至 2004 年英国使用的 B型流感嗜血杆菌疫苗的质量进行回顾性研究 | |
2005 | SEC | TSKgel G-DNA-PW G6000PWXL | Plasmid DNA/质粒DNA | High-speed chromatograph purification of plasmid DNA with a customized biporous hydrophobic adsorbent/用定制的双孔疏水吸附剂对质粒DNA进行高速色谱纯化 | https://www.sciencedirect.com/science/article/abs/pii/S1369703X05002044 |
2003 | SEC | TSKgel G5000PWXL G4000PWXL | Liposome/脂质体 | Characterization of loaded liposomes by size exclusion chromatography/已包载脂质体的分 子尺寸排阻色谱分析 | https://www.sciencedirect.com/science/article/pii/S0165022X03000599?via%3Dihub |
Toyopearl层析填料的分离纯化应用实例
Year | Mode | TSKgel Column | Target,keyword | Title | Citation at web site |
2019 | HIC | PPG-600M | Flu virus/流感病毒 | Hydrophobic-interaction chromatography for purification of influenza A and B virus / 疏水层析纯化甲型和乙型流感病毒 | https://www.sciencedirect.com/science/article/pii/S1570023219301655?via%3Dihub |
2019 | IEC | TSKgel SuperQ-5PW | Nucleotides/核苷酸 | Generic anion -exchange chromatography method for analytical and preparative separation of nucleotides in the development andmanufacture of drug substances /通用阴离子交换色谱法用于核酸药物的研发、生产中的分析 | https://www.sciencedirect.com/science/article/pii/S0021967318315255?via%3Dihub |
2018 | IEC | TSKgel SuperQ-5PW | siRNA | Comprehensive evaluation of canonical versus Dicericericer-substrate siRNA in vitro and in vivo | |
2018 | SEC | HW-65F | Virus-like particle (VLP) / 类病毒颗粒 | Dengue virus-like particles mimic the antigenic properties of the infectious dengue virus envelope /等革类病毒颗粒模拟感染性登革病毒包膜的抗原特性 | |
2017 | HIC | Butyl-650M | BBC vaccine/BBC疫苗 | Multi-subunit BCG booster vaccine GamTBvac: Assessment of immunogenicity and protective efficacy in murine and guinea pig TB models/多亚单位卡介苗增强疫苗GamTBvac：小鼠和豚鼠结核病模型免疫原性和保护效果的评估 | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409163/ |
2016 | HIC | Butyl-650M | Malaria vaccine/疟疾疫苗 | Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation/由三种高度相似的PfAMA1蛋白分子组成（克服抗原变异）的疟疾疫苗的生产、质量控制、稳定性和药物毒性 | https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164053 |
2016 | IEC | NH2-750F GigaCap Q-650M | Flu virus/流感病毒 | Mono- and polyprotic buffer systems in anion- exchange chromatography of influenza virus particles /流感病毒颗粒阴离子交换层析中单、多质子缓冲体系的研究 | https://www.sciencedirect.com/science/article/pii/S0021967316304708?via%3Dihub |
2015 | AFC | AF-Heparin 650M | Pertussis vaccine /百日咳疫苗 | Single Amino Acid Polymorphism s of Pertussis Toxin Subunit S2 (PtxB) Affect Protein Function /百日咳毒素亚基S2(PtxB)单氨基酸多态性影响蛋白功能 | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573519/ |
2013 | IEC | DEAE-650M | HBV-VLP/HBV 类病毒颗粒 | Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties /从重组毕赤酵母中纯化乙型肝炎表面抗原样病毒颗粒，并在体内分析其免疫原性 | https://www.sciencedirect.com/science/article/pii/S1570023213005126?via%3Dihub |
2012 | HIC | Phenyl-600M | HBV vaccine/乙肝疫苗 | Novel membrane extraction procedure for the purification of hepatitis B surface antigen from Pichia pastoris/新型膜提取法从毕赤酵母中分离纯化乙型肝炎表面抗原 | https://www.sciencedirect.com/science/article/pii/S1570023212002115?via%3Dihub |
2012 | IEC | DEAE-650M | Anti-viral component/抗病毒成分 | Isolation of antiviral components against Influenza from food in Yamagata (report Japanese) /从山形县食物中分离抗流感病毒成分 | http://www.eiken.yamagata.yamagata.jp/pdfshohou/shohou45.pdf |
2009 | HIC (HAC) | Phenyl-650M (Ca++Pure-HA) | Botlinum vaccine/肉毒疫苗 | Bivalent recombinant vaccine for botlinum neurotoxin types A and B based on a polypeptide comprising their effector and translocation domains that is protective against the predominant A and B subtypes /基于包含其效应域和易位域的多肽肉毒杆菌神经毒素A型和B型的双价重组疫苗 | https://www.ncbi.nlm.nih.gov/pubmed/19398544 |
2006 | IEC | DEAE-650M | HBV vaccine/乙肝疫苗 | Expression in and purification of Hepatitis B surface antigen (S-protein) from methylotrophic yeast Pichia pastoris/乙型肝炎表面抗原（S-蛋白）的表达及纯化 | https://www.sciencedirect.com/science/article/pii/S107599640600045X?via%3Dihub |
2004 | IEC | DEAE-650M | Adenovirus/腺病毒 | Development and optimization of an adenovirus production process/腺病毒生产工艺的开发和优化 | https://onlinelibrary.wiley.com/doi/10.1002/jgm.503 |
2002 | IEC SEC | SP-650M,HW-65S | Botlinum vaccine/肉毒疫苗 | Mucosal immunisation with Clostridium botulinum type C 16 S toxoid and its non-toxic component / C16 S-型肉毒杆菌及其无毒成分的粘膜免疫 | https://www.microbiologyresearch.org/content/journal/jmm/10.1099/0022-1317-51-10-813 |
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