理想的实验室操作下.ppt

理想的實驗室操作(下),Analytical Method 分析方法 Transfer, Verification, and Validation 转移、确认和验证,Method Transfer 方法转移,The process to establish documented evidence that the analytical method works as well in the receiving laboratory as in the originators laboratory, or the transferring laboratory, is called Analytical method transfer. 用文件化的证明分析方法在接受实验室和原始实验 室或者转移实验室一致的程序叫做方法转移,Method Transfer 方法转移,Regulatory testing requires that a transfer of analytical methodology from research development (RD) to quality control (QC), contract (e.g. CRO), or process laboratory is performed to ensure and certify that other laboratories are qualified to execute the method. 法规要求从研发到QC，合约实验室，或者中控实验室的分 析方法转移是为了确保和证明这些实验室有资质执行这个分 析方法,Method Transfer 方法转移, Original laboratory provides a method validation report to receiving laboratory. 原始实验室提供给接收实验室一份方法验证报告。 After reading the report, the receiving lab writes a “Report Summary” and a “Method Transfer Protocol”. 收到报告后，接收实验室写一份“报告总结”和“方法转移方案” Then execute the transferring work. 然后执行方法转移工作。 Finally, document a “Method Transfer Report”. 最后，形成一份“方法转移报告”文件。,Method Transfer 方法转移,Review the validation report from originating laboratory 回顾原始实验室提供的验证报告 Assay conditions 含量条件 Calculations 计算 System suitability 系统适用性 Receiving laboratory should perform the method 接收实验室应按照分析方法执行。 To determine whether there are differences and gaps in documentation 确认文件中是否存在差异和差别。,Method Transfer 方法转移, Assess a subset of validation parameters 评估验证参数 Equivalence Testing 等价性测试 Precision and Accuracy 精密度和准确度 Key Attributes 关键的属性 Precision and Accuracy 精密度和准确度 LOD/LOQ 检测限和定量限 Identity 鉴别 Linearity 线性,Method Transfer 方法转移,Typical Transfer should include: 典型的方法转移应该包括： More than one lot of testing materials 超过一批产品的检测 Reference Standard(s) 工作标准品 Samples 样品 Forced Degradation (Stress) Samples 强制降解样品 Typically the originating lab will prepare initial sample to be tested at both laboratories 通常原始实验室会提供初始的样品供两个实验室检测 One analyst at the originating lab and multiple analysts in the receiving lab. 原始实验室的一个分析员和接收实验室的多个分析员,Method Transfer 方法转移,Training of Analysts 分析员的培训 Training at originating laboratory 在原始实验室的培训 Review of relevant SOPs 回顾相关的SOPs Observation of test procedures 观察测试程序 Performing test procedures 执行测试程序 Evaluate 评估 Reference Standard 工作标准品 Chemicals, Reagents 化学品，试剂 Solvents 溶剂,Points to consider 需要考虑的要点,Equipment 仪器设备 Available? 是否可用？ Same Manufacturer? 是否同一生产商？ Calibrated or Qualified prior to use? 使用前是否校验或确认 Gradient HPLC 梯度HPLC Dwell volume correction may have to be performed 需对死体积进行校正,Method Transfer Protocol 方法转移方案,Test Method 测试方法 Parameters being assessed 需要评估的参数 Sample/Reagent preparations 样品和试剂准备 Instrument/Equipment/Apparatus 仪器设备 Performance Parameters 性能参数 Different analyst on different days 不同天不同分析员 Predefined Acceptance Criteria 预先定好的可接受标准 Statistical Analysis 数据分析,Points to consider 考虑的重点,Some minimal revalidation should be performed by the receiving laboratory: 接收实验室最少应该重新验证的内容： Linearity 线性 LOD 检测限 LOQ 定量限 Recovery 回收率,Method Transfer Report 方法转移报告,Include representative and/or full data sets 包括有代表性的或者全套数据： Chromatograms 色谱图 Standard Curves 标准曲线 Tabular Data 数据列表 Deviations 偏差 Statistical Analysis 数据分析 Conclusion 结论,Method Transfer Report 方法转移报告,Follow the regulation requirements: 遵循下列要求： Retain the testing articles 保留测试记录 Retain the testing raw data 保留测试原始数据 Verify the results by second person 第二个人对数据进行复核 Review and Sign by laboratory management 实验室管理人员审核并且签名,Validation or Verification 验证和确认,Analytical Methods described in 21 CFR 194(a)(2) “current version of USPNF, AOAC International Books of Methods, and methods detailed in an approved regulatory applications (NDA or ANDA) ” are not required to validate the accuracy and reliability of these methods, but need to verify their suitability under actual conditions of use. 在21CFR 194（a）（2）描述：如果是USP现行版本的方 法，或者是分析化学师协会（AOAC） 国际卷的方法，或是 在已经批准过的（NDA或者ANDA）申请中的方法不要求确 认这些方法的准确度和可靠性，但是需要确认在实际测试条 件下的系统适用性。,Method Verification 方法确认,The process of demonstrating that analytical procedures are suitable for their intended use (FDA) 这个过程证明分析程序适合于使用要求。 The process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for the intended Analytical applications (USP) 通过实验室研究和建立的，具有一定性能参数的程序要符合 分析应用的要求。,Method Verification 方法确认,Verification of an analytical procedure is the demonstration that a laboratory is capable of replicating with an Acceptable level of performance a standard method. 分析方法确认的目的是证明该实验室有能力重复出 分析方法可接受的精度。 Ensuring that an analytical method is suitable under actual conditions of use. 确保分析方法在实际应用的条件下是适用的。,Method Verification 方法确认,Not required for basic compendial test procedures: 一些简要的测试程序是不要求验证的 Loss on drying 干燥失重 Residue on ignition 炽灼残渣 Wet chemical procedures, e.g.: acid value 化学过程，例如：酸度。 Simple instrumental methods, e.g.: pH measurements 简单的仪器方法，例如：pH测量。,Method Verification Approach 方法确认步骤,Verify whether the system suitability can be met by carrying out the methods as written for test samples of interests. 按照写下来的检测样品的方法执行，确认系统适用性能否符 合。 If possible, use the particular brand of column specified in the monograph. One can use a different brand of the same type of column. 如果有可能，使用专论中提到的品牌的色谱柱。可以使用不 同品牌但是是同一类型的色谱柱。,Method Verification Approach 方法确认步骤,If system suitability cant be met by changing the brand of column, method conditions may be adjusted within the ranges defined by USP Chromatography. 如果更换了不同品牌的色谱柱导致系统适用性不合格，可以在（621）色谱学专论中提到的范围内调整方法。,Method Verification Approach 方法确认步骤,pH of Mobile Phase (HPLC): within 0.2 units of the value or range specified 流动相（HPLC）的pH值：可以在0.2单位 或者是 指定的范围内调整 Conc. of Salts in Buffer (HPLC): within10%, provided the permitted pH variation is met 缓冲盐（HPLC）的浓度：倘若pH值改变在允许范围内，缓冲盐浓度可以有10%的改变。,Method Verification Approach 方法确认步骤,Ratio of Components in Mobile Phase (HPLC): 流动相的比例： Minor components of the mobile phase 流动相最小比例的组成 Can be adjusted by 30% relative 可以调整相对 30%的比例 But the change cant exceed 10% absolute 但是比例改变不能超过绝对值的10% Only to one minor component in a ternary mixture 对于三种组分的流动相只能调整最小比例的那种成分,Method Verification Approach 方法确认步骤,Wavelength of UV/Vis Detector (HPLC): 紫外可见检测器的波长 Deviations from the wavelengths specified in the method are not permitted 背离方法中的波长是不允许的 At most 3 nm on another validated procedures 超过3 nm 就需要另一个确认程序 Column Length (GC, HPLC): As much as70% 柱子长度（GC，HPLC）：最大可以有70%的改变,Method Verification Approach 方法确认步骤, Column Inner Diameter (GC, HPLC): 25% for HPLC and 50% for GC 柱子内径：对于HPLC可以有25% ，对于GC可以有 50% 的改变 Particle Size (HPLC): Can be reduced as much as 50% 粒径（HPLC）：可以减少至50% Flow Rate (GC, HPLC): 50% 流速（GC，HPLC）： 50% Injection Volume (GC, HPLC): Can be reduced as far as is consistent with accepted precision and detection limits 进样体积：只要精密度和检测限可以达到要求，可以一直减少,Method Verification Approach 方法确认步骤, Column Temperature (HPLC): 10C 柱温（HPLC）： 10C Oven Temperature (GC): As much as10% 柱温（GC）： 最大可以10% Oven Temperature Program (GC): 柱温程序(GC): Temperature within the range stated above 温度在上述规定的范围之内 Time: up to 20% 时间：最大20%,Method Verification 方法确认,The system suitability still cant be met by playing those adjustments, then what? 通过上面的这些调整，系统适用性仍然不合格， 然后怎么办？,Method Verification 方法确认,Repeatability: Establish precision by testing the product of interest under the method condition as stated. Single analyte should evaluate a minimum of six replicate. 可重复性：通过按照规定方法测试感兴趣样品确定精密度， 同一个样品应该至少测试六次 Specificity: Verify the test matrix does not introduce interference that would inhibit quantitation for the peak of interest by analyzing blank. Verify the peak purity of each peak of interest. 专属性：通过分析空白确认分析不会引入可能影响感兴趣峰的含量干扰，确认每个感兴趣峰的纯度。,Method Verification 方法确认,Filter Compatibility: If filters are used, demonstrate that the filtration process does not impact the Analyte final concentration in the solution. 过滤：如果用到了过滤，要证明过滤过程没有影响 到样品在溶液中的最终浓度,Method Verification 方法确认,Document the final method, incorporating all the instrument conditions, system suitability test, sample/solutions preparation details and reporting requirements, if method verified. 方法确认结束，要把最后的方法，连同仪器条件、系统适用 性测试、样品/溶液准备的细节一同形成文件，并且报告要求,Method Verification 方法确认,If above attempts have been tried and acceptable results still cant be obtained, then what? 如果上面的尝试都已经做过，但是依然没有得到可 接受结果，怎么办呢？,Method Validation 方法验证,The laboratory shall validate nonstandard methods, laboratory designed/developed methods, standard methods used outside their intended scope to confirm that the methods are fit for their intended purpose. 实验室应该验证非标准化的方法，实验室自己开发 的方法，超出了适用范围的标准化方法以确认这个 方法达到他们的目的要求,Method Validation 方法验证,Typical Analytical Characteristics used in Method Validation: 典型的分析方法验证应该验证的内容： Accuracy 准确度 Precision 精密度 Specificity 专属性 Limit of Detection (LOD) 检测限 Limit of Quantitation (LOQ) 定量限 Linearity 线性 Range 范围 Robustness 耐用性,Considerations Prior to Method Validation 方法验证优先考虑的方面,Suitability of Instrument 仪器是否合适 Status of Qualification and Calibration 仪器确认和校验的状态 Suitability of Materials 材料是否合适 Reference Standard, Reagents, Blank Matrix 工作标准品，试剂，空白 Suitability of Analyst 分析员是否合适 Training and Qualification 培训和资质 Suitability of Documentation 文件适用性 SOP, Protocol with preestablished criteria SOP,预先建立可接受标准的方案,Validation Steps 验证步骤,Define the application, purpose, and scope of the Method 先详细说明应用，目的和方法的适用范围 Analyte(s)? Concentration? Sample Matrices? 分析的样品？浓度？样品空白？ Develop an analytical method 建立一个分析方法 Develop a validation protocol 建立一个验证方案 Qualification/Calibration of instrument 确认/校验仪器 Analyst(s) training/qualification 分析员培训/资质确认,Accuracy 准确度,The accuracy of an analytical procedure is the closeness of test results obtained by the procedure to the true value. The accuracy of an analytical procedure should be established across its range. 分析方法的准确度是测量值和真实值的接近程度， 分析方法在整个范围内都应该是准确的。,Accuracy (drug substance) 准确度（原料药）,The application of the analytical procedure to an analyte of know purity, e.g. Reference Standard. 采用分析方法分析一个已知纯度的分析样品，例如工作标 准品 Comparison of the results of the procedure with those of a second, wellcharacterized procedure, the accuracy of which has been stated or defined. 和另一个，确认好的，准确度已经确认过的分析方法比较分 析出的结果。,Accuracy (drug product) 准确度（制剂）,For Assay Calculate the percentage of recovery of the known added amount of analyte in the sample. 对于百分含量计算，加入已知量的被分析物，计算回收率 For Impurities Assess on samples (of drug substance or drug product) spiked with know amounts of impurities. 对于样品中杂质，在样品（制剂或原料药）加入已知量的杂质计算。,Accuracy 准确度,Accuracy should be assessed using a minimum of nine determinations over a minimum of three concentration levels, covering the specified range. 准确度评估至少应该有三个不同的浓度，九个不同的点，三个浓度应该涵盖整个指标范围。 Assay: 80%, 100%, and 120% 含量：80%，100%和120% Dissolution: 70%, 100%, and 130% 溶出：70%，100%和130%,Nine Determinations 九个点,Can I prepare three spiked solutions with concentration of 80%, 100%, and120%; then three injections for each solution? 我可以配80%，100%，120%三个不同浓度的溶液，然后每 个溶液各进三针吗？ Or should I prepare nine spiked solutions with three in 80%, three in 100%, and three in 120%. Then one injection for each solution? 或者是我配80%，100%，120%三个不同浓度的九个溶液， 然后每个溶液各进一针？,Precision 精密度,Precision is the agreement between a set of replicate measurements without assumption of knowledge of the true value. 精密度是一组测量值（不一定需要知道真实值）的重复性 Precision is usually defined in terms of standard deviation or relative standard deviation (%RSD). 精密度通常用标准偏差或相对标准偏差来判定。,Precision 精密度,Reproducibility the use of the analytical procedure in different labs. 重现性分析程序在不同实验室中使用。 Intermediate precision (ruggedness) within lab variations, as on different days or with different analysts or equipment within the same lab. 中间精密度在同一实验室内变更条件，例如不同天数，或者是不同分析员，或者是同一实验室内的不同仪器。 Repeatability the use of the analytical procedure within a lab over a short period of time using the same analyst and equipment 重复性分析程序在实验室内同样的分析员和仪器在一小段时间内的使用。,Reproducibility Study 重现性研究,Precision 精密度,Repeatability should be assessed using a minimum of nine determinations covering the specified range for the procedure. 可重复性应该通过最少九个点涵盖整个指标范围来评估。 Three conc. and three replicates of each conc. 三个浓度，每个浓度各重复三次。 A minimum of six determinations at 100% of the test Concentration 100%浓度的测试液最少六个点。,Specificity 专属性,The ability to assess unequivocally the analyte in the presence of components which may be expected to be present, such as impurities, degradation products, and matrix components. 评定被分析物与其他组分。例如杂质，降解产物，和 空白基 质 的区分的能力,Specificity 专属性,For qualitative analysis (ID test) the ability to select between compounds of closely related structure that are likely to be present should be demonstrated 对于定性分析区分可能出现的相近结构的的组分的能力应 该被证明。 For quantitative analysis (Assay) Demonstrate that the procedure is unaffected by the presence of impurities or excipients. This may be done by spiking with impurities. 对于定量分析（含量）证明分析程序不受可能出现的杂质和赋形剂的影响，这个可以通过加入杂质来证明。,Specificity 专属性,Positive results the retention time of the analyte matches with that of reference standard. 阳性结果被分析物的保留时间和对照标准品一致。 Negative results Reagent blank and/or method blank has no peak at the retention time of analyte. 阴性结果试剂空白或者方法空白在被分析物保留 时间未出峰,Specificity (Peak Purity) 专属性（峰纯度）,Peak purity test may be useful to show that the analyte chromatographic peak is not attributable to more than one component. 峰纯度测试表明被分析物色谱峰不是多个组分形成的非常有 用。 Peak purity tests can be evaluated with 峰纯度测试可以用下面来评价： The spectra of photodiode array detector 二极管阵列 Mass Spectrometry 质谱,Limit of Detection 检测限,The detection limit is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated, under the stated experimental conditions 检测限是在指定的实验条件下，被分析物能够检测出来的， 不需要定量的最小量。 Typically acceptable signal to noise ratios is 3 : 1. 可接受标准一般是信噪比为3：1.,Limit of Quantitation 定量限,The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. 定量限是指样品中被分析物满足精密度和准确度要求的最低 量，,Limit of Quantitation 定量限,The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or Degradation products. 定量限是样品中低含量组分定量计算时的一个参数，主要用 来检测样品中的杂质和/或者降解产物。,Limit of Quantitation 定量限,The quantitation limit should be shown to be sufficiently low by the analysis of samples with known concentrations of analyte above and below the quantitation level. 通过分析已知的在定量限上下的样品浓度以确定定 量限，定量限应该尽量低， Typically acceptable signal to noise ratio is 10:1 at which the analyte can reliably be quantitated. 通常被分析物可以定量，可接受信噪比为10：1,Linearity and Range 线性和范围,Determine linearity over a minimum of five concentrations 通过至少五个浓度来确定线性 Assay of drug substance: 80% 120% 原料药含量：80%-120% Determination of an impurity: 50% 120% of the specification 杂质：指标的50% 120% Content uniformity: A minimum of 70% 130% of the test concentration 含量均匀度：测试溶液的70% 130% Dissolution testing: 20% throughout the specified range of the dissolution test. 溶出测试：溶出测试指标范围的20%,Robustness 耐用性,Robustness is the capacity of a method to remain unaffected by small, deliberate variations in method parameters; it is a measurement of the reliability of a method. 耐用性是分析方法参数发生微小的，有意的改变，分析方法 仍然有效的能力，反应了分析方法的可靠性。,System Suitability 系统适用性,System suitability is the checking of a system to ensure system performance before or during the analysis of unknowns. 系统适用性是在分析未知样品前或者中间确保系统性能的系 统检查。 Its parameters are established as a direct result of ruggedness and robustness studies 系统适用性参数通过重现性和耐用性研究结果确定。,System Suitability 系统适用性,Theoretical plate count (N), peak tailing factor (T), resolution (R), and reproducibility (%RSD) are determined from replicate injections of a standard compared with method specifications. 通过测定理论塔板数，峰拖尾因子，分离度，标准品重复进 样重现性和方法标准相比较， If RSD is stated less than 2.0%, five replicates should be used. 如果RSD规定了小于2.0%，重复进样五次。 If RSD is stated greater than 2.0%, six replicates should be used. 如果RSD规定了大于2.0%，重复进样六次。,QC Samples QC 配样,Two separate weight of Reference Standards (RS) and used one of them as an initial concentration verification, e.g.: 称取两个不同重量的工作标准品，用其中一个作为初始浓度 确认，例如： 20 mg 100.0 mL 10 mg 50.0 mL Two separate weight of Samples, e.g.: 两个不同重量的样品 20.2345 mg 100.0 mL 21.8765 mg 100.0 mL Spike solution (equal vol. of RS and Sample) 配样：工作标准品和样品应该等体积,液相序列,Data Recording 数据记录, How is raw data controlled 原始数据怎么控制 How is data maintained / stored 原始数据怎么保持和存储 Bound books / Notebooks 装订成册 Sequentially numbered forms(Worksheets) 数据表格 Logbooks 使用记录 LIMS 实验室信息管理系统 Traceability 可追踪性,Method Validation Report 方法验证报告,A summary report should include: 一个完整的报告应该包括： Instrument/Equipment used 使用的仪器/设备 Reagents, Chemicals, and Reference Standard(s) used 试剂，化学品，工作标准品 Number of samples tested 测试的样品数 Summary of each validation test 每个验证测试的概要 Acceptance Criteria 可接受标准 Revie