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

快速蒸发电离质谱法（REIMS）可获得不同细菌和霉菌的高特异性磷脂谱，可用于区分不同的微生物。REIMS为开发一种基于脂质，无需样品制备的微生物鉴定系统提供了新的机会。该技术将比现有方法快得多，并且与其他技术（例如MALDI）相比具有灵敏度和鲁棒性。该项目的重点是开发一种技术，以在适当的检测限内检测和分类药片制剂中存在的原料药、赋形剂和液体中的不同微生物。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物，这将有助于将来实时释放（作为更广泛的控制战略的一部分）。制药水分析、特定生物的缺失/存在条款是该项目关注的另外两个可能的REIMS应用。这些条款是一系列药物剂量展示所必需的。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成本的技术（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）。下面，我列出了前两年的项目计划，并介绍了时间表，以显示计划的哪些部分已经开始或将在未来几个月内开始。这个项目的很大一部分是环境监测。为了开发一种能够对药物环境分离物进行鉴别的技术，需要建立药物环境分离物数据库。阿斯利康每周将数百个样品（细菌和霉菌）送到帝国理工学院，样品被保存到样品数据库中，以便通过MALDI或16S测序进一步使用和鉴定（大约80%的样品必须进行测序才能获得真正的鉴定，这对于进一步的技术开发至关重要，因为阿斯利康使用的鉴定技术和帝国MALDI的结果不匹配）。1 -2年工作计划：环境rems识别数据库的构建。环境样本与临床样本的比较。REIMS鉴定数据库的构建和验证c.与AZ（阿斯利康）内部技术的比较。使用REIMS进行样品分析，参考模型的建立和验证。样品贮存条件对REIMS2分析的影响。AZ水样rems（纯净水、注射用水、钻孔水）分析发展技术，以浓缩和丰富样品进行分析。确定法规要求的细菌下限检测/CFU。大肠杆菌和大肠菌群检测的鉴定方法，如短孵育、同位素标记、磁分离或生物标志物分析。鉴定活菌检测方法（建立预测方法的参考模型）真菌Identificationa。REIMS鉴定数据库的构建与验证。真菌菌落在reimsc上生长7 d代谢变化的研究。使用REIMS进行样品分析，建立参考模型并验证d.研究样品储存条件对REIMS分析的影响。原料药或药物本身的无培养法a)如果需要，研究适合溶解原料药的溶剂b)开发浓缩样品分析的技术（过滤法）c)法规要求的细菌下限检测/CFU d)确定活细胞检测方法，如短孵育，同位素标记，磁分离或生物标志物分析。开发工具。室内外

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