第130期 東曹色譜產品在病毒關聯藥物和疫苗研發中的應用
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病毒是一種個體微小，結構簡單，只含一種核酸（DNA或RNA），必須在活細胞內寄生并以復制方式增殖的非細胞型生物。
病毒是一種非細胞生命形態，它由一個核酸長鏈和蛋白質外殼構成，病毒沒有自己的代謝機構，沒有酶系統。因此病毒離開了宿主細胞，就成了沒有任何生命活動、也不能獨立自我繁殖的化學物質。它的復制、轉錄和轉譯的能力都是在宿主細胞中進行，當它進入宿主細胞后，它就可以利用細胞中的物質和能量完成生命活動，按照它自己的核酸所包含的遺傳信息產生和它一樣的新一代病毒。
一、病毒和疫苗的特性
※疫苗
·蛋白質作為表面抗原
·天然蛋白、多糖或重組蛋白
·類似于蛋白質分離的純化策略
·純化用到的分離模式SEC、IEC、HIC和AFC
※病毒
·巨大的生物分子復合粒子
·流感病毒：100-120nm；腺病毒：80nm；天花病毒：30nm
·不同純化策略對蛋白質的分離有不同的影響
二、疫苗用色譜填料
※尺寸排除色譜法（SEC）
·病毒分離需要大孔徑的填料
病毒尺寸：100nm----TOYOPEARL HW-65，Sepharose 6 FF
病毒尺寸：200nm---- TOYOPEARL HW-75，Sepharose 4 FF
·填料的剛性對裝柱更為重要
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TOYOPEARL比Sepharose基質具有更好的剛性和穩定性。
※離子交換色譜法（IEC）
·大孔徑的填料:TOYOPEARL 650系列
·? 接枝型的聚甲基丙烯酸甲酯，分離效果會更好，如GigaCap Q-650M，GigaCap S-650M，SuperQ-650M
※疏水層析（HIC）
·不同填料的疏水性有差異
·疏水性較低的填料可能更有效分離病毒
·DNA在HIC填料上的保留較差
※親和層析（AFC）
·染料：TOYOPEARL AF-Blue-650M
·肝素：TOYOPEARL AF-Heparin-HC-650M
·其他蛋白質固定化AFC樹脂
?? 在這些關聯藥物的研發、評價、純化精制中，TSKgel液相色譜柱和Toyopearl層析填料產品有著相當多的應用實例。
TSKgel色譜柱分析柱的應用實例
Year | Mode | TSKgel Column | Target,keyword | Title | Citation at web site |
2020 | SEC | TSKgel G5000PWXL | Coronavirus protein/ 冠狀病毒蛋白 | Characterization of the SARS-CoV-2 Spike in an Early Prefusion ? Conformation/早期融合前構象中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陰離子高效液相色譜分析的條件研究 | 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譜分析 | |
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 /液相色譜進行寡聚核苷酸分析條件的檢驗 | Poster presentation in HPLC Kyoto, 2019 |
2017 | SEC | TSKgel G6000PWXL G5000PWXL | Conjugated vaccine / 結合疫苗 | Determination of free polysaccharide in Vi glycoconjugate vaccineagainst typhoid fever /傷寒疫苗中游離多糖的測定 | https://www.sciencedirect.com/science/article/pii/S0731708516312511?via%3Dihub |
2017 | SEC | TSKgel G5000PWXL | Virus-like particle / 類病毒顆粒（VLP） | Quantification and characterization of virus-like particles by ? size-exclusion chromatography and nanoparticle tracking analysis / 通過尺寸排除色譜和納米顆粒跟蹤定性和定量分析類病毒顆粒 | 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/輪狀病毒VP8與霍亂毒素B亞單位融合在小鼠模型中的免疫原性和保護作用 | |
2016 | SEC | TSKgel G6000PWXL | Flu virus(VP)/流感病毒 | Size distribution analysis of influenza virus particles using size ? exclusion chromatography /流感病毒顆粒大小分布的分子尺寸排阻色譜分析 | https://www.sciencedirect.com/science/article/pii/S0021967316311335 |
2015 | SEC | TSKgel G5000PWXL G4000PWXL | Conjugated vaccine/結合疫苗 | Development and characterization of Haemophilus influenzae type b conjugate ? vaccine prepared using different polysaccharide chain lengths /不同多糖鏈長流感嗜血桿菌B型偶聯疫苗的研制與表征 | https://www.sciencedirect.com/science/article/pii/S0264410X15004958?via%3Dihub |
2014 | SEC | TSKgel G5000PWXL-CP | Bacteriophage particle/噬菌體顆粒 | Size exclusion-based purification and PCR-based quantitation of MS2 ? bacteriophage particles for environmental applications/用于環境應用的MS2噬菌體顆粒的儲存排阻純化與PCR定量 | https://www.sciencedirect.com/science/article/pii/S0166093414004704?via%3Dihub |
2013 | SEC | TSKgel G5000PWXL-CP | Virus particle/病毒顆粒 | A Gel Filtration -Based Method for the Purification of Infectious ? Rotavirus Particles for Environmental Research Applications /一種用于環境研究應用的感染性輪狀病毒的凝膠過濾純化方法 | |
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 /無抗生素生產單純皰疹病毒2型DNA疫苗的高產cGMP過程 | |
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發展 | https://journal.lib.uoguelph.ca/index.php/surg/article/view/420 |
2007 | IEC | TSKgel DNA-NPR | Plasmid DNA/質粒DNA | Separation of topological forms plasmid DNA by anion -exchange HPLC: Shifts ? in elution order of linear DNA/陰離子交換高效液相色譜法分離質粒DNA的拓撲結構 | https://www.sciencedirect.com/science/article/pii/S1570023207002747 |
2007 | SEC | TSKgel G5000PWXL | Conjugated vaccine /結合疫苗 | Quality and Immunogenicity of MenAfriVac, a Conjugate Vaccine Against ? Meningococcal Group ADisease/腦膜炎球菌 a群疾病的結合疫苗MenAfriVac的質量和免疫原性 | 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 / 對1996年至 2004 年英國使用的 B型流感嗜血桿菌疫苗的質量進行回顧性研究 | |
2005 | SEC | TSKgel G-DNA-PW G6000PWXL | Plasmid DNA/質粒DNA | High-speed chromatograph purification of plasmid DNA with a customized biporous ? hydrophobic adsorbent/用定制的雙孔疏水吸附劑對質粒DNA進行高速色譜純化 | https://www.sciencedirect.com/science/article/abs/pii/S1369703X05002044 |
2003 | SEC | TSKgel G5000PWXL G4000PWXL | Liposome/脂質體 | Characterization of loaded liposomes by size exclusion chromatography/已包載脂質體的分 子尺寸排阻色譜分析 | https://www.sciencedirect.com/science/article/pii/S0165022X03000599?via%3Dihub |
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Toyopearl層析填料的分離純化應用實例
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 / 疏水層析純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 /通用陰離子交換色譜法用于核酸藥物的研發、生產中的分析 | 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) / 類病毒顆粒 | Dengue virus-like particles mimic the antigenic properties of the ? infectious dengue virus envelope /等革類病毒顆粒模擬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/多亞單位卡介苗增強疫苗GamTBvac：小鼠和豚鼠結核病模型免疫原性和保護效果的評估 | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409163/ |
2016 | HIC | Butyl-650M | Malaria vaccine/瘧疾疫苗 | Production, Quality Control, Stability and Pharmacotoxicity of a ? Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome ? Antigenic Variation/由三種高度相似的PfAMA1蛋白分子組成（克服抗原變異）的瘧疾疫苗的生產、質量控制、穩定性和藥物毒性 | 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 /流感病毒顆粒陰離子交換層析中單、多質子緩沖體系的研究 | 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 /百日咳毒素亞基S2(PtxB)單氨基酸多態性影響蛋白功能 | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4573519/ |
2013 | IEC | DEAE-650M | HBV-VLP/HBV 類病毒顆粒 | Purification of hepatitis B surface antigen virus-like particles from ? recombinant Pichia pastoris and in vivo analysis of their immunogenic ? properties /從重組畢赤酵母中純化乙型肝炎表面抗原樣病毒顆粒，并在體內分析其免疫原性 | 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/新型膜提取法從畢赤酵母中分離純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) /從山形縣食物中分離抗流感病毒成分 | 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 /基于包含其效應域和易位域的多肽肉毒桿菌神經毒素A型和B型的雙價重組疫苗 | 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）的表達及純化 | 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/腺病毒生產工藝的開發和優化 | 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-型肉毒桿菌及其無毒成分的粘膜免疫 | https://www.microbiologyresearch.org/content/journal/jmm/10.1099/0022-1317-51-10-813 |
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