Trace level detection of microbiological contaminants in pharmaceutical environment using Rapid Evaporative Ionisation Mass Spectrometry

快速蒸发电离质谱（REIMS）产生不同细菌和霉菌的高度特异性磷脂谱，可用于区分不同微生物。REIMS为开发基于脂质、无需样品制备的微生物鉴定系统提供了新的机会。该技术将比目前的方法快得多，并且与其他技术（例如MALDI）相比具有灵敏度和耐用性。该项目的重点是开发技术，以适当的检测限检测和分类片剂制剂和液体中存在的原料药、辅料中的不同微生物。该项目的另一个重点是开发一种技术，用于从环境监测板中识别分离株，这将有助于未来的真实的时间释放（作为更广泛的控制策略的一部分）。制药用水分析、一系列药物剂量规格所需的特定微生物的存在/不存在条款是本项目关注的另外两个可能的REIMS应用。与制药公司现有的方法和程序相比，REIMS技术可能是一种为企业节省大量资金的技术（例如，节省时间和金钱，提高可持续性、完整性和稳健性，减少批次和库存放行的延迟）。我已经把前2年的项目计划放在一起，并介绍了时间表，以显示计划的哪一部分已经开始或将在在接下来的几个月里。该项目的大部分是环境监测。为了开发一种能够识别平板的技术，需要构建药物环境分离物数据库。数百个样本（细菌和霉菌）每周从阿斯利康送到帝国，其中将样品保存在样品数据库中以供进一步使用和通过MALDI或16 S测序鉴定（大约80%的样品必须进行测序以获得真正的鉴定，这对于进一步的技术开发至关重要，因为AZ使用的鉴定技术和Imperial MALDI的结果不匹配）。第二年工作计划：1。建立环境REIMS识别数据库a。环境样本与临床样本的比较。REIMS识别数据库的构建和验证。与AZ（阿斯利康）内部技术的比较d。使用REIMS进行样品分析，参考模型建立和验证。研究样品储存条件对REIMS 2分析的影响。在REIMS上进行AZ水样分析（纯化水、注射用水、钻孔水）a.开发浓缩和丰富分析样品的技术b。确定法规要求的细菌检测下限/CFU c。鉴定大肠杆菌和大肠菌群检测方法，如短时间孵育、同位素标记、磁性分离或生物标志物分析。活菌检测的鉴定方法（预测方法的参考模型构建）3.真菌鉴定a. REIMS识别数据库的构建和验证b.真菌菌落在REIMSc上生长7天期间代谢变化的调查。使用REIMS进行样品分析，参考模型建立和验证d。研究样品储存条件对REIMS分析的影响4.原料药或药物本身的免培养方法a）研究适合溶解原料药的溶剂（如需要）b）开发浓缩样品进行分析的技术（过滤方法）c）法规要求的细菌下限检测/CFU d）确定活细胞检测方法，如短时间孵育、同位素标记、磁性分离或生物标志物分析。仪器开发a。调查

Rapid evaporative ionization mass spectrometry (REIMS) yields highly specific phospholipid profiles of different bacteria and moulds which can be used to distinguish between different microorganisms. REIMS offers a new opportunity for the development of a lipid based, sample-preparation-free microbial identification system. This technique would be considerably faster than current methods and have the sensitivity and robustness compared to other techniques (e.g. MALDI).This project is focusing on developing technique to detect and categorise different microorganisms in the presence of drug substances, excipients in a tablet formulation and in liquids at a suitable detection limit. Another focus of the project is to develop a technique which would be used for identifying isolates from environmental monitoring plates which would be an enabler for future real time release (as part of a wider control strategy). Pharmaceutical water analysis, absence/presence of specified organisms clauses which are required for a range of pharmaceutical dosage presentations are another two possible REIMS applications which this project is focusing. Comparing with existing methods and procedures in pharmaceutical companies, REIMS technology could potentially be a technique with great savings for the business (e.g. saving time and money, increasing sustainability, integrity and robustness, reducing delays for batch and stock release).Below, I have put together project plan for the first 2 years and introduced timelines to show which part of the plan have been started or will be started in the upcoming months.Big part of the project is environmental monitoring. In order to develop a technique which would be able to identify the plates, construction of pharmaceutical environmental isolates data base is required. Hundreds of samples (bacteria and moulds) are send to Imperial everyweek from AstraZeneca, where samples are saved in to the sample data base for further use and identification by MALDI or 16S sequencing (about 80% of samples have to be sequenced to obtain true identification which is crucial for further technique development due to the not matching results from AZ used techniques for identification and Imperial MALDI).1st -2nd Year Work Plan:1. Construction of environmental REIMS identification databasea. Comparison between environmental and clinical samplesb. Construction and validation of REIMS identification database c. Comparison to AZ (AstraZeneca) in-house techniquesd. Samples analysis using REIMS, reference model building and validatione. Investigation of samples storage conditions effect for analysis on REIMS2. AZ water samples analysis on REIMS (purified water, water for injections, borehole water)a. Develop technique to concentrate and enrich samples for analysisb. Identify lower limit detection/CFU of bacteria required by regulationsc. Identify method for E.coli and Coliform detection such as short incubation, isotopic labelling, magnetic separation or biomarkers analysis.d. Identify method for viable bacteria detection (reference model building forprediction approach)3. Fungi Identificationa. Construction and validation of REIMS identification databaseb. Investigation of fungi colonies metabolic changes during 7 day growth onREIMSc. Samples analysis using REIMS, reference model building and validation d. Investigation of samples storage conditions effect for analysis on REIMS4. Culturing free approach for drug substances or drug itselfa) Investigate solvents suitable for dissolving drug substances if requiredb) Develop technique to concentrate samples for analysis (filtering approach)c) Lower limit detection/CFU of bacteria required by regulationsd) Identify method for viable cell detection such as short incubation, isotopic labelling, magnetic separation or biomarkers analysis.5. Instrument developmenta. Investiga