【作者】 王颖；

【导师】 王升启；

【作者基本信息】 安徽医科大学 ， 药物分析， 2012， 硕士

【摘要】 流感泰得（Flutide，序列5’-CCTTGTTTCTACT-3’）是本室筛选出的作用于流感病毒基因组RNA5’非编码区的新结构、新靶点的抗流感病毒硫代修饰反义寡核苷酸，体内外药效学研究证明其具有较好的抗病毒活性。作为临床前研究药物，流感泰得的大规模制备及质量控制是新药研发中必不可少的技术环节。针对目前国内核酸药物制备规模小的问题和进一步提高核酸药物靶向性和膜透过性的目的我们开展了本课题的研究。一、流感泰得质规模化制备及质量控制方法的建立。本实验将寡核苷酸的制备放大到中试规模，采用经典的亚磷酰胺偶联法合成硫代寡核苷酸药物流感泰得，每个合成循环含四步反应：脱4，4-二甲氧基三苯甲基（DMT）、偶联、封闭和硫代。合成粗品经制备色谱纯化，超滤脱盐后冻干分装。合成规模达到30mmol。为了对Flutide进行质量控制，建立了离子对反相高效液相色谱（IP-RP-LC）质控方法，并将其与其他两种常用方法，离子交换高效液相色谱（IEC）和毛细管电泳（CGE）法对Flutide中常见有关物质的分析进行比较。合成了Flutide及其单核苷酸缩短序列5’n-1、3’n-1和单氧代序列5’(P=O)1，将它们作为已知杂质，在IP-RP-LC上进行分离条件的优化，使用的分析柱为XTerra MS C18柱（250mm×4.6mm，3.5μm）；流动相A为142mM HFIP-36mM TEA-10%甲醇（v:v），流动相B为甲醇；梯度洗脱；检测波长为260nm。结果表明，在该条件下，Flutide能同时与这几种杂质进行分离，其中与缩短序列5’n-1和3’n-1可实现基线分离，而与5’(P=O)1之间的分离度也能达到0.94，解决了其他分析方法（IEC和CGE等）无法同时分离单核苷酸缩短杂质与单氧代杂质的问题。该研究采用建立的IP-RP-LC方法已成功应用于Flutide粗品和纯品中各类杂质的分析和鉴定，证明该方法适用于硫代寡核苷酸类化合物的质量控制。二、流感泰得含量测定及方法学考察。以高纯度的Flutide为标准品，采用离子对反相色谱法对Flutide的含量测定。主峰与最近的杂质峰的分离度R=0.94;含量测定重复性实验结果：峰面积RSD=1.59%，三个浓度样品各重复测定三次获得精密度结果，RSD分别为0.67%,1.0%,0.56%。质量标准曲线回归方程：y=26882x-71957r=0.9999。含量测定范围为20~1200μg/ml。三、流感泰得及其有关物质的结构确证。由于Flutide的结构为硫代修饰的寡核苷酸，其分子与小分子化合物性质差别较大，因此一些鉴定小分子结构常用的手段用于寡核苷酸类药物的结构分析时，结果可能仅能提供部分参考，但无法对其所有基团一一进行确认，因此综合考虑实际情况和参考文献资料，采用紫外光谱、红外光谱、各类质谱、核磁共振谱、原子吸收光谱法等，分别对首次中试制备的样品和工艺稳定后制备得到的一批样品及对照品的特征基团、分子量、序列、等进行分析和推测，确认其化学结构与理论结构完全一致。此外，利用IP-RP-HPLC法良好的分离能力，对Flutide粗品和纯品的各杂质分别进行了手动收集，经电喷雾质谱（ESI-MS）鉴定，对各个杂质做了成分归属。结果表明，除了5’n-1和3’n-1序列，其他一些微量杂质（如5’(P=O)1）也能够得到一定分离。四、金刚乙胺修饰流感泰得化合物(Flutide-5’JG)的纯化方法建立Flutide-5’JG粗品中主要包括修饰失败全长序列，缩短修饰序列，以及未修饰缩合不完全段片段及硫代不完全产物。利用混合物的疏水性不同，采用反相色谱法纯化Flutide-5’JG。由于离子对反相色谱法能同时分开n-1和(P=O)1杂质，故采用此方法对纯化收集样品进行纯度控制。纯化收集样品经电喷雾质谱进行分子量鉴定与理论值相符，证明该方法可行。本实验初步建立了Flutide-5’JG的纯化工艺，纯化规模达到了2.6克/批。优化了其纯化条件，确立了最佳的洗脱梯度。进行了连续三批小试纯化研究并检测了产品纯度。获得的Flutide-5’JG纯度95%以上，而且批次间稳定性高。五、小结本论文建立了Flutide的质量控制方法，在此基础上开展了Flutide及其有关物质检查及结构确证，进一步开展了Flutide-5’JG纯化方法的建立,为Flutide的新药申报奠定了基础。更多还原

【Abstract】 This research mainly focuses on synthesis and quality control of a phosphorothioateantisense oligonucleotide flutide and the purification of its modifications. First, anion-pair reversed phased LC method (IP-RPLC) was developed for the analysis ofrelated substances of a phosphorothioate antisense oligonucleotide flutide and routinequality control. The analytical result of IP-RPLC was compared with ion exchangechromatography(IEC) and capillary gel electrophoresis(CGE). The synthesized flutideand its analogs of monphodiester5’(P=O)1as well as deletion sequences5’ n-1and3’n-1were analyzed. The optimized conditions of IP-RPLC were as follows: XTerra MSC18(250mm X4.6mm,3.5μm) column; the mobile phase A was142mol/L1,1,1,3,3,3-hexafluoroisopropanol (HFIP)-36mmol/L triethylamine acetate (TEA)-10%methanol(V/V), the mobile phase B was methanol; The result showed that under theseconditions, flutide, deletion sequences5’ n-1and3’ n-1could be completelly separatedfrom each other and the resolution R of flutide and5’(P=O)1could reach to0.94. Theproposed IP-RPLC method has been applied for the related substances analysis andidentification of crude and purified flutide samples, indicating that this method can beused for the quality control of phoshorothioate antisense oligonucleotide.Second, IP-RPLC method was used as quantitation method of phosphorothioateoligonecleotides flutide. The separation degree of the nearest peak was0.94;theconcentration samples were all reapted three times and precision RSD were0.67%,1.0%,0.56%separately. There was good linear relationship among amount of flutidesand its function was:=26882x-71957, linearity correlation coefficient was0.9999. Thecontent determination range is from20to1200μg/ml.Third, the structure of flutide and its related substances were identified here.Because flutide belonging to oligonucleotides, its nature was different to small molecules. Thecommon identification methods of small molecules could only provide partlyinfomation of oligonucleotides. Cosidering the actural situration, we using ultravioletspectrum, infrared spectrum, mass spectrometry, nuclear magnetic resonancespectrosocopy, atomic absorption spectra and so on together for the first time toidentificate the structure and molecular of flutide and its related substances. In addition,separated by IP-RPLC, the crude and pure flutide samples were collected manually.Then it was identified by ESI-MS. The ESI-MS result showed that both flutide and itsrelated substances5’ n-1,3’ n-1and5’(P=O)1were match to theorical molecular.Then the development of purification method of modified flutide was as following:the crude product of solid-phase synthesis of modified oligonucleotide comprises thefull-length modified phosphorothioate oligonucleotide (n-mer), unmodified full lengthsequence, nucleotide deletion sequences (n-x) and unfully sulfed sequences etc.According to the hydrophobic diferences among sequences, reverse phasechromatography was used to purify modified flutide. Due to good separation ability of5’ n-1,3’ n-1and5’(P=O)1impurities, IP-RPLC method was used to evaluate the purityof collected samples of modified flutide after purification. Then, the purified sampleswere identified by ESI-MS, which showed that the molecular was match to theory. Thusproving that the reverse phase chromatography purification method of modified flutidewas feasible.This experiment developed the purification process of modified flutide.The purification scale reached2.6/time. We also optimized its purification conditionsand established the best elution gradient. Three small scale crude products were purified,the average purity was>95%and method showed high stability among different batchs.更多还原