FDA关于破坏实验的一些最新看法和要求(中文版本)

FDA关于ANDA强制降解试验的观点：强制降解试验为方法学验证中的重要内容，为了解国外对强制降解试验的要求，根据Pharmaceutical Technology第36卷5期中“FDAPerspectives: Scientific Considerations of Forced DegradationStudies in ANDA Submissions”一文（发布时间为2012年5月2日，作者为Ragine Maheswaran），对FDA关于强制降解试验的相关要求进行了翻译整理，具体内容如下：一、强制降解试验简介强制降解试验也称破坏性试验，其试验目的明确。

强制降解试验可预测原料药的稳定性或影响制剂的纯度、有效性和安全性的因素。

了解不同破坏条件下药物的降解产物和降解途径是非常必要的。

强制降解试验可以为分析方法的建立、说明书的制定和处方设计的确定等提供有益的参考。

样品破坏的程度取决于药物本身的性质和产品的剂型。

ICHQ1B为光稳定性试验提出了一些建议，在ICH稳定性指导原则和验证指南中，没有可以参考的关于其他降解条件的建议，对于氧化和水解降解研究也仅有有限的信息。

原料药与辅料分析方面的药物专著可以为不同原料药的各降解条件提供参考。

二、仿制药强制降解试验研究存在的问题仿制药申请时提供的强制降解试验研究数据不完整是申报的一大缺陷。

美国仿制药申报常见缺陷解读（CMC部分）已经出版，常见的一些例子说明，强制降解试验的缺陷包括以下几个方面：原料药在各破坏条件下均不产生降解。

请重复破坏试验以获得足够的降解产物,若没有产生降解,请提供依据。

破坏条件过于剧烈，导致大部分药物均被降解。

请用温和的破坏条件或减少样品暴露时间以产生相关的降解产物。

请注意即使你已经用含量测定的方法对破坏的样品进行了检测，为了验证有关物质的检测方法具有稳定性指示功能，破坏的样品也应用有关物质的方法进行测定。

请提供所做的验证试验数据，以证明用以检测未破坏样品和破坏样品的方法能够检测出所有的降解杂质。

1II. 背景 (2)III. 分析方法的类型 (3)A. 法定分析方法 (3)B. 可选择分析方法 (3)3 C. 稳定性指示分析 (3)IV. 对照品……………………………………………………………………………4A. 对照品的类型 (4)B. 分析报告单 (4)C. 对照品的界定 (4)V. IND 中的分析方法验证 (6)VI. NDA, ANDA, BLA 和PLA 中分析方法验证的内容和格式 (6)A. 原则 (6)B. 取样 (7)C. 仪器和仪器参数 (7)D. 试剂 (7)E. 系统适应性实验 (7)F. 对照品的制备 (7)G. 样品的制备 (8)H. 分析方法 (8)L. 计算 (8)J. 结果报告 (8)VII. NDA，ANDA,BLA 和PLA 中的分析方法验证 (9)A．非法定分析方法 (9)1．验证项目 (9)2. 其它分析方法验证信息 (10)a. 耐用性 (11)b. 强降解实验 (11)c. 仪器输出/原始资料 (11)3．各类检测的建议验证项目 (13)B．法定分析方法 (15)VIII. 统计分析…………………………………………………………………………15A. 总则 (15)C. 统计 (16)IX. 再验证 (16)X. 分析方法验证技术包：内容和过程……………………………………………17A. 分析方法验证技术包 (17)B. 样品的选择和运输 (18)C. 各方责任 (19)XI. 方法………………………………………………………………………………20A. 高效液相色谱(HPLC) (20)B. 气相色谱(GC) (22)C. 分光光度法，光谱学，光谱法和相关的物理方法 (23)D. 毛细管电泳 (23)E. 旋光度 (24)F. 粒径相关的分析方法 (25)G. 溶出度 (26)H. 其它仪器分析方法 (27)附件A：NDA，ANDA，BLA 和PLA 申请的内容 (28)附件B：分析方法验证的问题和延误 (29)参考文献……………………………………………………………………………………30术语表………………………………………………………………………………………32This guidance provides recommendations to applicants on submitting analytical procedures, validation data, and samples to support the documentation of the identity, strength, quality, purity, and potency of drug substances and drug products.1. 绪论本指南旨在为申请者提供建议，以帮助其提交分析方法，方法验证资料和样品用于支持原料药和制剂的认定，剂量，质量，纯度和效力方面的文件。

美国FDA分析方法验证指南中英文对照2012-03-05 10:37I. INTRODUCTIONThis guidance provides recommendations to applicants on submitting analytical procedures, validation data, and samples to support the documentation of the identity, strength, quality, purity, and potency of drug substances and drug products.1. 绪论本指南旨在为申请者提供建议，以帮助其提交分析方法，方法验证资料和样品用于支持原料药和制剂的认定，剂量，质量，纯度和效力方面的文件。

This guidance is intended to assist applicants in assembling information, submitting samples, and presenting data to support analytical methodologies. The recommendations apply to drug substances and drug products covered in new drug applications (NDAs), abbreviated new drug applications (ANDAs), biologics license applications (BLAs), product license applications (PLAs), and supplements to these applications.本指南旨在帮助申请者收集资料，递交样品并资料以支持分析方法。

这些建议适用于NDA，ANDA，BLA，PLA及其它们的补充中所涉及的原料药和制剂。

药品及生物制品的分析方法和方法验证指导原则目录1.介绍 (1)2.背景 (2)3.分析方法开发 (3)4.分析程序内容 (3)A.原则/范围 (4)B.仪器/设备 (4)C.操作参数 (4)D.试剂/标准 (4)E.样品制备 (4)F. .................................................................................................................... 标准对照品溶液的制备 (5)G.步骤 (5)H.系统适应性 (5)I.计算 (5)J.数据报告 (5)5.参考标准和教材 (6)6 分析方法验证用于新药，仿制药，生物制品和DMF (6)A.非药典分析方法 (6)B.验证特征 (7)C.药典分析方法 (8)7.统计分析和模型 (8)A.统计 (8)B.模型 (8)8.生命周期管理分析程序 (9)A.重新验证 (9)B.分析方法的可比性研究 (10)1.另一种分析方法 (10)2.分析方法转移的研究 (11)C.报告上市后变更已批准的新药，仿制药，或生物制品 (11)9.美国FDA 方法验证 (12)10.参考文献前言本指导原则草案，定稿后，将代表美国食品和药物管理局（FDA）目前关于这个话题目前的想法。

它不会创造或赋予或任何人的任何权利，不约束FDA 或公众。

您可以使用另一种方法，如果该方法符合适用的法律和法规的要求。

如果你想讨论一个替代方法，请与FDA 工作人员负责实施本指南。

如果你不能确定适当的FDA 工作人员，请拨打本指南的标题页上所列的电话号码。

介绍：该修订指南草案将取代行业2000 年的指导分析方法和方法验证草案，并最终确定后，也将取代1987 年美国FDA 行业指南《提交的样品和分析数据的方法验证》。

该草案提供了有关申请人如何提交分析程序和方法验证数据来支持说明原料药和制剂具有强度、质量、纯度和效用的文件。

FDA关于ANDA强制降解试验的观点强制降解试验为方法学验证中的重要内容，为了解国外对强制降解试验的要求，本公司根据Pharmaceutical Technology 第36卷5期中“FDA Perspectives: Scientific Considerations of Forced Degradation Studies in ANDA Submissions”一文（发布时间为2012年5月2日，作者为Ragine Maheswaran），对FDA关于强制降解试验的相关要求进行了翻译整理，具体内容如下：一、强制降解试验简介强制降解试验也称破坏性试验，其试验目的明确。

强制降解试验可预测原料药的稳定性或影响制剂的纯度、有效性和安全性的因素。

了解不同破坏条件下药物的降解产物和降解途径是非常必要的。

强制降解试验可以为分析方法的建立、说明书的制定和处方设计的确定等提供有益的参考。

样品破坏的程度取决于药物本身的性质和产品的剂型。

ICH Q1B为光稳定性试验提出了一些建议，在ICH稳定性指导原则和验证指南中，没有可以参考的关于其他降解条件的建议，对于氧化和水解降解研究也仅有有限的信息。

原料药与辅料分析方面的药物专著可以为不同原料药的各降解条件提供参考。

二、仿制药强制降解试验研究存在的问题仿制药申请时提供的强制降解试验研究数据不完整是申报的一大缺陷。

美国仿制药申报常见缺陷解读（CMC部分）已经出版，常见的一些例子说明，强制降解试验的缺陷包括以下几个方面：原料药在各破坏条件下均不产生降解。

请重复破坏试验以获得足够的降解产物,若没有产生降解,请提供依据。

破坏条件过于剧烈，导致大部分药物均被降解。

请用温和的破坏条件或减少样品暴露时间以产生相关的降解产物。

请注意即使你已经用含量测定的方法对破坏的样品进行了检测，为了验证有关物质的检测方法具有稳定性指示功能，破坏的样品也应用有关物质的方法进行测定。

请提供所做的验证试验数据，以证明用以检测未破坏样品和破坏样品的方法能够检测出所有的降解杂质。

药品及生物制品的分析方法和方法验证指导原则目录1.介绍...................... (1)2.背景..................... .. (2)3.分析方法开发. ..................... . (3)4.分析程序内容.............................................. ......... ..................................... .. 3A.原则/范围 (4)B.仪器/设备............................................. . (4)C.操作参数.............................................. .. (4)D.试剂/标准............................................. . (4)E.样品制备.............................................. .. (4)F.标准对照品溶液的制备............................................ .. (5)G.步骤......... ....................................... (5)H.系统适应性..... (5)I.计算 (5)J.数据报告 (5)5.参考标准和教材............................................ (6)6分析方法验证用于新药，仿制药，生物制品和DMF (6)A.非药典分析方法............................................. (6)B.验证特征 (7)C.药典分析方法............................................. .. (8)7.统计分析和模型 (8)A.统计 (8)B.模型 (8)8.生命周期管理分析程序 (9)A.重新验证 (9)B.分析方法的可比性研究............................................ . (10)1.另一种分析方法............................................... .. (10)2.分析方法转移的研究 (11)C.报告上市后变更已批准的新药，仿制药，或生物制品 (11)9.美国FDA方法验证............................................... . (12)10.参考文献前言本指导原则草案，定稿后，将代表美国食品和药物管理局（FDA）目前关于这个话题目前的想法。

FDA关于ANDA强制降解试验的观点2016-02-15HPC药闻药事风险管理谢大侠强制降解试验为方法学验证中的重要内容，为了解国外对强制降解试验的要求，根据Pharmaceutical Technology 第36卷5期中“FDA Perspectives: Scientific Considerations of Forced Degradation Studies in ANDA Submissions”一文（发布时间为2012年5月2日，作者为Ragine Maheswaran），对FDA关于强制降解试验的相关要求进行了翻译整理，具体内容如下：一、强制降解试验简介强制降解试验也称破坏性试验，其试验目的明确。

强制降解试验可预测原料药的稳定性或影响制剂的纯度、有效性和安全性的因素。

了解不同破坏条件下药物的降解产物和降解途径是非常必要的。

强制降解试验可以为分析方法的建立、说明书的制定和处方设计的确定等提供有益的参考。

样品破坏的程度取决于药物本身的性质和产品的剂型。

ICH Q1B为光稳定性试验提出了一些建议，在ICH稳定性指导原则和验证指南中，没有可以参考的关于其他降解条件的建议，对于氧化和水解降解研究也仅有有限的信息。

原料药与辅料分析方面的药物专著可以为不同原料药的各降解条件提供参考。

二、仿制药强制降解试验研究存在的问题仿制药申请时提供的强制降解试验研究数据不完整是申报的一大缺陷。

美国仿制药申报常见缺陷解读（CMC部分）已经出版，常见的一些例子说明，强制降解试验的缺陷包括以下几个方面：1.原料药在各破坏条件下均不产生降解。

请重复破坏试验以获得足够的降解产物,若没有产生降解,请提供依据。

2.破坏条件过于剧烈，导致大部分药物均被降解。

请用温和的破坏条件或减少样品暴露时间以产生相关的降解产物。

3.请注意即使你已经用含量测定的方法对破坏的样品进行了检测，为了验证有关物质的检测方法具有稳定性指示功能，破坏的样品也应用有关物质的方法进行测定。

药品及生物制品的分析方法和方法验证指导原则目录1.介绍...................... (1)2.背景..................... .. (2)3.分析方法开发. ..................... . (3)4.分析程序内容.............................................. ......... ..................................... .. 3A.原则/范围 (4)B.仪器/设备............................................. . (4)C.操作参数.............................................. .. (4)D.试剂/标准............................................. . (4)E.样品制备.............................................. .. (4)F.标准对照品溶液的制备............................................ .. (5)G.步骤......... ....................................... (5)H.系统适应性..... (5)I.计算 (5)J.数据报告 (5)5.参考标准和教材............................................ (6)6分析方法验证用于新药，仿制药，生物制品和DMF (6)A.非药典分析方法............................................. (6)B.验证特征 (7)C.药典分析方法............................................. .. (8)7.统计分析和模型 (8)A.统计 (8)B.模型 (8)8.生命周期管理分析程序 (9)A.重新验证 (9)B.分析方法的可比性研究............................................ . (10)1.另一种分析方法............................................... .. (10)2.分析方法转移的研究 (11)C.报告上市后变更已批准的新药，仿制药，或生物制品 (11)9.美国FDA方法验证............................................... . (12)10.参考文献前言本指导原则草案，定稿后，将代表美国食品和药物管理局（FDA）目前关于这个话题目前的想法。

FDA关于破坏实验的一些最新看法和要求FDA Perspectives: Scientific Considerations of Forced Degradation Studies in ANDA SubmissionsThe author outlines the scientific aspects of forced degradation studies that should be considered in relation to ANDA submissions.May 2, 2012By:Ragine MaheswaranPharmaceutical TechnologyVolume 36, Issue 5, pp. 73-80Forced degradation is synonymous with stress testing and purposeful degradation. Purposeful degradation can be a useful tool to predict the stability of a drug substance or a drug product with effects on purity, potency, and safety. It is imperative to know the impurity profile and behavior of a drug substance under various stress conditions. Forced degradation also plays an important role in the development of analytical methods, setting specifications, and design of formulations under the quality-by-design (QbD) paradigm. The nature of the stress testing depends on the individual drug substance and the type of drug product (e.g., solid oral dosage, lyophilized powders, and liquid formulations) involved (1).The International Conference on Harmonization (ICH) Q1B guideline provides guidance for performing photostability stress testing; however, there are no additional stress study recommendations in the ICH stability or validation guidelines (2). There is also limited information on the details about the study of oxidation and hydrolysis. The drug substance monographs of Analytical Profiles of Drug Substances and Excipients provide some information with respect to different stress conditions of various drug substances (3).The forced degradation information provided in the abbreviated new drug application (ANDA) submissions is often incomplete and in those cases deficiencies are cited. An overview of common deficiencies cited throughout the chemistry, manufacturing, and controls (CMC) section of the ANDAs has been published (4–6). Some examples of commonly cited deficiencies related to forced degradation studies include the following:Your drug substance does not show any degradation under any of the stress conditions. Please repeat stress studies to obtain adequate degradation. If degradation is not achievable, please provide your rationale.Please note that the conditions employed for stress study are too harsh and that most of your drug substance has degraded. Please repeat your stress studies using milder conditions or shorter exposure time to generate relevant degradation products.It is noted that you have analyzed your stressed samples as per the assay method conditions. For the related substances method to be stability indicating, the stressed samples should be analyzed using related substances method conditions.Please state the attempts you have made to ensure that all the impurities including the degradation products of the unstressed and the stressed samples are captured by your analytical method. Please provide a list summarizing the amount of degradation products (known and unknown) in your stressed samples.Please verify the peak height requirement of your software for the peak purity determination. Please explain the mass imbalance of the stressed samples.Please identify the degradation products that are formed due to drug-excipient interactions.Your photostability study shows that the drug product is very sensitive to light. Please explain how this is reflected in the analytical method, manufacturing process, product handling, etc.In an attempt to minimize deficiencies in the ANDA submissions, some general recommendations to conduct forced degradation studies, to report relevant information in the submission, and to utilize the knowledge of forced degradation in developing stability indicating analytical methods, manufacturing process, product handling, and storage are provided in this article.Stress conditionsTypical stress tests include four main degradation mechanisms: heat, hydrolytic, oxidative, and photolytic degradation. Selecting suitable reagents such as the concentration of acid, base, or oxidizing agent and varying the conditions (e.g., temperature) and length of exposure can achieve the preferred level of degradation. Over-stressing a sample may lead to the formation of secondary degradants that would not be seen in formal shelf-life stability studies and under-stressing may not serve the purpose of stress testing. Therefore, it is necessary to control the degradation to a desired level. A generic approach for stress testing has been proposed to achieve purposeful degradation that is predictive of long-term and accelerated storage conditions (7). The generally recommended degradation varies between 5-20% degradation (7–10). This range covers the generally permissible 10% degradation for small molecule pharmaceutical drug products, for which the stability limit is 90%-110% of the label claim. Although there are references in the literature that mention a wider recommended range (e.g., 10-30%), the more extreme stress conditions often provide data that are confounded with secondary degradation products.Photostability.Photostability testing should be an integral part of stress testing, especially for photo-labile compounds. Some recommended conditions for photostability testing are described in ICH Q1B Photostability Testing of New Drug Substances and Products (2). Samples of drug substance, and solid/liquid drug product, should be exposed to a minimum of 1.2 million lux hours and 200 watt hours per square meter light. The same samples should be exposed to both white and UV light. To minimize the effect of temperature changes during exposure, temperature control may be necessary. The light-exposed samples should be analyzed for any changes in physical properties such as appearance, clarity, color of solution, and for assay and degradants. The decision tree outlined in the ICH Q1B can be used to determine the photo stability testing conditions for drug products. The product labeling should reflect the appropriate storage conditions. It is also important to note that the labeling for generic drug products should be concordant with that of the reference listed drug (RLD) and with United States Pharmacopeia (USP) monograph recommendations, as applicable.Heat.Thermal stress testing (e.g., dry heat and wet heat) should be more strenuous than recommended ICH Q1A accelerated testing conditions. Samples of solid-state drug substances and drug products should be exposed to dry and wet heat, whereas liquid drug products can be exposed to dry heat. It is recommended that the effect of temperature be studied in 10 °C increments above that for routine accelerated testing, and humidity at 75% relative humidity or greater (1). Studies may be conducted at higher temperatures for a shorter period (10). Testing at multiple time points could provide information on the rate of degradation and primary and secondary degradation products. In the event that the stress conditions produce little or no degradation due to the stability of a drug molecule, one should ensure that the stress applied is in excess of the energy applied byaccelerated conditions (40 °C for 6 months) before terminating the stress study.Acid and base hydrolysis.Acid and base hydrolytic stress testing can be carried out for drug substances and drug products in solution at ambient temperature or at elevated temperatures. The selection of the type and concentrations of an acid or a base depends on the stability of the drug substance. A strategy for generating relevant stressed samples for hydrolysis is stated as subjecting the drug substance solution to various pHs (e.g., 2, 7, 10–12) at room temperature for two weeks or up to a maximum of 15% degradation (7). Hydrochloric acid or sulfuric acid (0.1 M to 1 M) for acid hydrolysis and sodium hydroxide or potassium hydroxide (0.1 M to 1 M) for base hydrolysis are suggested as suitable reagents for hydrolysis (10). For lipophilic drugs, inert co-solvents may be used to solubilize the drug substance. Attention should be given to the functional groups present in the drug molecule when selecting a co-solvent. Prior knowledge of a compound can be useful in selecting the stress conditions. For instance, if a compound contains ester functionality and is very labile to base hydrolysis, low concentrations of a base can be used. Analysis of samples at various intervals can provide information on the progress of degradation and help to distinguish primary degradants from secondary degradants.Oxidation.Oxidative degradation can be complex. Although hydrogen peroxide is used predominantly because it mimics possible presence of peroxides in excipients, other oxidizing agents such as metal ions, oxygen, and radical initiators (e.g., azobisisobutyronitrile, AIBN) can also be used. Selection of an oxidizing agent, its concentration, and conditions depends on the drug substance. Solutions of drug substances and solid/liquid drug products can be subjected to oxidative degradation. It is reported that subjecting the solutions to 0.1%-3% hydrogen peroxide at neutral pH and room temperature for seven days or up to a maximum 20% degradation could potentially generate relevant degradation products (10). Samples can be analyzed at different time intervals to determine the desired level of degradation.Different stress conditions may generate the same or different degradants. The type and extent of degradation depend on the functional groups of the drug molecule and the stress conditions. Analysis methodThe preferred method of analysis for a stability indicating assay is reverse-phase high-performance liquid chromatography (HPLC). Reverse-phase HPLC is preferred for several reasons, such as its compatibility with aqueous and organic solutions, high precision, sensitivity, and ability to detect polar compounds. Separation of peaks can be carried out by selecting appropriate column type, column temperature, and making adjustment to mobile phase pH. Poorly-retained, highly polar impurities should be resolved from the solvent front. As part of method development, a gradient elution method with varying mobile phase composition (very low organic composition to high organic composition) may be carried out to capture early eluting highly polar compounds and highly retained nonpolar compounds. Stressed samples can also be screened with the gradient method to assess potential elution pattern. Sample solvent and mobile phase should be selected to afford compatibility with the drug substance, potential impurities, and degradants. Stress sample preparation should mimic the sample preparation outlined in the analytical procedure as closely as possible. Neutralization or dilution of samples may be necessary for acid and base hydrolyzed samples. Chromatographic profiles of stressed samples should be compared to those of relevant blanks (containing no active) and unstressed samples to determinethe origin of peaks. The blank peaks should be excluded from calculations. The amount of impurities (known and unknown) obtained under each stress condition should be provided along with the chromatograms (full scale and expanded scale showing all the peaks) of blanks, unstressed, and stressed samples. Additionally, chiral drugs should be analyzed with chiral methods to establish stereochemical purity and stability (11, 12).The analytical method of choice should be sensitive enough to detect impurities at low levels (i.e., 0.05% of the analyte of interest or lower), and the peak responses should fall within the range of detector's linearity. The analytical method should be capable of capturing all the impurities formed during a formal stability study at or below ICH threshold limits (13, 14). Degradation product identification and characterization are to be performed based on formal stability results in accordance with ICH requirements. Conventional methods (e.g., column chromatography) or hyphenated techniques (e.g., LC–MS, LC–NMR) can be used in the identification and characterization of the degradation products. Use of these techniques can provide better insight into the structure of the impurities that could add to the knowledge space of potential structural alerts for genotoxicity and the control of such impurities with tighter limits (12–17). It should be noted that structural characterization of degradation products is necessary for those impurities that are formed during formal shelf-life stability studies and are above the qualification threshold limit (13).Various detection types can be used to analyze stressed samples such as UV and mass spectroscopy. The detector should contain 3D data capabilities such as diode array detectors or mass spectrometers to be able to detect spectral non-homogeneity. Diode array detection also offers the possibility of checking peak profile for multiple wavelengths. The limitation of diode array arises when the UV profiles are similar for analyte peak and impurity or degradant peak and the noise level of the system is high to mask the co-eluting impurities or degradants. Compounds of similar molecular weights and functional groups such as diastereoisomers may exhibit similar UV profiles. In such cases, attempts must be made to modify the chromatographic parameters to achieve necessary separation. An optimal wavelength should be selected to detect and quantitate all the potential impurities and degradants. Use of more than one wavelength may be necessary, if there is no overlap in the UV profile of an analyte and impurity or degradant peaks. A valuable tool in method development is the overlay of separation signals at different wavelengths to discover dissimilarities in peak profiles.Peak purity analysis.Peak purity is used as an aid in stability indicating method development. The spectral uniqueness of a compound is used to establish peak purity when co-eluting compounds are present.Peak purity or peak homogeneity of the peaks of interest of unstressed and stressed samples should be established using spectral information from a diode array detector. When instrument software is used for the determination of spectral purity of a peak, relevant parameters should be set up in accordance with the manufacturer's guidance. Attention should be given to the peak height requirement for establishing spectral purity. UV detection becomes non linear at higher absorbance values. Thresholds should be set such that co-eluting peaks can be detected. Optimum location of reference spectra should also be selected. The ability of the software to automatically correct spectra for continuously changing solvent background in gradient separations should be ascertained.Establishing peak purity is not an absolute proof that the peak is pure and that there is noco-elution with the peak of interest. Limitations to peak purity arise when co-eluting peaks are spectrally similar, or below the detection limit, or a peak has no chromophore, or when they are not resolved at all.Mass balance.Mass balance establishes adequacy of a stability indicating method though it is not achievable in all circumstances. It is performed by adding the assay value and the amounts of impurities and degradants to evaluate the closeness to 100% of the initial value (unstressed assay value) with due consideration of the margin of analytical error (1).Some attempt should be made to establish a mass balance for all stressed samples. Mass imbalance should be explored and an explanation should be provided. Varying responses of analyte and impurity peaks due to differences in UV absorption should also be examined by the use of external standards. Potential loss of volatile impurities, formation of non-UV absorbing compounds, formation of early eluants, and potential retention of compounds in the column should be explored. Alternate detection techniques such as RI LC/MS may be employed to account for non-UV absorbing degradants.Termination of studyStress testing could be terminated after ensuring adequate exposure to stress conditions. Typical activation energy of drug substance molecules varies from 12–24 kcal/mol (18). A compound may not necessarily degrade under every single stress condition, and general guideline on exposure limit is cited in a review article (10). In circumstances where some stable drugs do not show any degradation under any of the stress conditions, specificity of an analytical method can be established by spiking the drug substance or placebo with known impurities and establishing adequate separation.Other considerationsStress testing may not be necessary for drug substances and drug products that have pharmacopeial methods and are used within the limitations outlined in USP <621>. In the case where a generic drug product uses a different polymorphic form from the RLD, the drug substance should be subjected to stress testing to evaluate the physiochemical changes of the polymorphic form because different polymorphic forms may exhibit different stability characteristics.Forced degradation in QbD paradigmA systematic process of manufacturing quality drug products that meet the predefined targets for the critical quality attributes (CQA) necessitates the use of knowledge obtained in forced degradation studies.A well-designed, forced degradation study is indispensable for analytical method development in a QbD paradigm. It helps to establish the specificity of a stability indicating method and to predict potential degradation products that could form during formal stability studies. Incorporating all potential impurities in the analytical method and establishing the peak purity of the peaks of interest helps to avoid unnecessary method re-development and revalidation.Knowledge of chemical behavior of drug substances under various stress conditions can also provide useful information regarding the selection of excipients for formulation development. Excipient compatibility is an integral part of understanding potential formulation interactions during product development and is a key part of product understanding. Degradation products due to drug-excipient interaction or drug-drug interaction in combination products can be examined by stressing samples of drug substance, drug product, and placebo separately and comparing theimpurity profiles. Information obtained regarding drug-related peaks and non-drug-related peaks can be used in the selection and development of more stable formulations. For instance, if a drug substance is labile to oxidation, addition of an antioxidant may be considered for the formulation. For drug substances that are labile to acid or undergo stereochemical conversion in acidic medium, delayed-release formulations may be necessary. Acid/base hydrolysis testing can also provide useful insight in the formulation of drug products that are liquids or suspensions.Knowledge gained in forced degradation studies can facilitate improvements in the manufacturing process. If a photostability study shows a drug substance to be photolabile, caution should be taken during the manufacturing process of the drug product. Useful information regarding process development (e.g., wet versus dry processing, temperature selection) can be obtained from thermal stress testing of drug substance and drug product.Additionally, increased scientific understanding of degradation products and mechanisms may help to determine the factors that could contribute to stability failures such as ambient temperature, humidity, and light. Appropriate selection of packaging materials can be made to protect against such factors.ConclusionAn appropriately-designed stress study meshes well with the QbD approaches currently being promoted in the pharmaceutical industry. A well-designed stress study can provide insight in choosing the appropriate formulation for a proposed product prior to intensive formulation development studies. A thorough knowledge of degradation, including mechanistic understanding of potential degradation pathways, is the basis of a QbD approach for analytical method development and is crucial in setting acceptance criteria for shelf-life monitoring. Stress testing can provide useful insight into the selection of physical form, stereo-chemical stability of a drug substance, packaging, and storage conditions. It is important to perform stress testing for generic drugs due to allowable qualitative and quantitative differences in formulation with respect to the RLD, selection of manufacturing process, processing parameters, and packaging materials. AcknowledgmentsThe author would like to thank Bob Iser, Naiqi Ya, Dave Skanchy, Bing Wu, and Ashley Jung for their scientific input and support.Ragine Maheswaran, PhD, is a CMC reviewer at the Office of Generic Drugs within the Office of Pharmaceutical Science, under the US Food and Drug Administration's Center for Drug Evaluation and Research, Ragine.Maheswaran@Disclaimer: The views and opinions in this article are only those of the author and do not necessarily reflect the views or policies of the US Food and Drug Administration.References1. ICH, Q1A(R2) Stability Testing of New Drug Substances and Products (Geneva, Feb. 2003).2. ICH, Q1B Stability Testing: Photostability Testing of New Drug Substances and Products (Geneva, Nov. 1996).3. H. Brittain, Analytical Profiles of Drug Substances and Excipients (Academic Press, London, 2002).4. A. Srinivasan and R. Iser, Pharm. Technol. 34 (1), 50–59 (2010).5. A. Srinivasan, R. Iser, and D. Gill, Pharm. Technol. 34(8), 45–51 (2010).6. A. 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