1.1 该测试方法描述了一种测量程序，用于可重复地分离脂质体药物制剂中存在的成分大小群体，并表征其相关的大小和大小分布。该方法还可以产生关于脂质体的形状和物理稳定性的信息，并且适用于在存在血清蛋白的情况下的测量。可以使用本试验方法中未规定的各种技术收集馏分进行离线分析。 1.2 本试验方法适用于设计用于药物递送并分散在天然水溶液中的单层和多层脂质体。该方法通常适用于约10的颗粒尺寸范围（半径） nm至250 nm，并且对于注射的脂质质量为20 µg至200 µg。 1.3 此测试方法基于- 检测器非对称流场流动分馏（MD-AF4）技术，配置在典型的商业仪器平台上，具有在线检测器，如多角度（静态）光散射（MALS）、动态光散射（DLS）、紫外-可见光（UV-Vis）吸收率和差折射率（dRI） ( 1. ) . 2. 1.4 该方法不涉及脂质体组成。参考测试方法 E3297 , E3323 或 E3324 用于脂质定量。 1.5 单位-- 以国际单位制表示的数值应视为标准。在适当的情况下，除了SI外，还提供了cgs单位。 1.6 本标准并不旨在解决与其使用相关的所有安全问题（如有）。本标准的使用者有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 1.7 本国际标准是根据世界贸易组织技术性贸易壁垒委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认的标准化原则制定的。 ====意义和用途====== 5.1 用于治疗癌症和其他疾病的脂质体制剂是目前提交市场批准和临床应用的最常见的纳米技术药物产品。对其物理化学性质的准确表征对于支持此类产品的开发和评估至关重要 ( 2. ) 特别地，尺寸、尺寸分布、形状和物理稳定性是已被广泛确定为脂质体药物产品的关键质量属性（CQA）的关键特性（以及其他特性）。 5.1.1 非对称流场流动分馏（AF4）是一种类似色谱的技术，它使用流体动力根据大小和扩散率将分析物轻轻地分离成其组分群体 ( 3和 4. ) 然后，分馏的样品通过专门为应用要求选择的一个或多个在线检测器。低剪切分离、对复杂基质的耐受性和异常宽的尺寸范围使AF4成为应用于纳米技术药物产品（如脂质体）的首选技术 ( 1. 3. , 5. 9 ) . 5.1.2 多探测器AF4适用于研发、制造质量控制、产品稳定性/保质期测试和监管评估。 5.1.3 MD-AF4的应用有多种固有的假设，包括用于解释在线光散射数据的模型的适当性，以及分析物和基质与形成分馏通道中积聚壁表面的膜的兼容性。 其他假设是检测器或分析物特定的。 5.2 流动相的化学成分不得诱导脂质体聚集或以其他方式显著改变其物理性质。 5.3 在解释不同散射探测器使用不同散射模型和分析模式获得的尺寸数据时，应使用离散度。 5.4 现行成品药良好生产规范（见《美国联邦法规汇编》第21卷第211.194（a）（2）节）和ICH分析程序验证协调三方指南Q2（R1） ( 10 ) ，说明所有试验方法的适用性应在实际使用条件下进行验证。 5.5 MD-AF4可以与间歇模式动态光散射（DLS）进行比较，以测定脂质体的平均尺寸和尺寸分布，其中MD-AF4提供复杂混合物的去卷积，从而对存在的群体进行更准确的评估。 批处理模式DLS（例如，参见测试方法 E3247 )提供了一种可用于在MD-AF4分析之前筛选材料的快速、低成本的方法。后者需要更多的分析师时间和精力，以及适当的培训和专业知识。定性地说，MD-AF4通过与多个检测器相结合的分馏过程，对样品的物理复杂性提供了更深入的了解。

1.1 This test method describes a measurement procedure to reproducibly separate component size populations present within liposomal drug formulations and to characterize their associated size and size distribution. The method can also yield information on the shape and physical stability of the liposomes and is applicable to measurements in the presence of serum proteins. Fractions can be collected for off-line analysis using various techniques not specified in this test method. 1.2 This test method applies to uni-lamellar and multi-lamellar liposomes that are designed for drug delivery and which are dispersed in a native solution that is aqueous in nature. The method is generally applicable over a particle size range (radius) of approximately 10 nm to 250 nm, and for injected lipid mass from 20 µg to 200 µg. 1.3 This test method is based on the multi-detector asymmetrical-flow field-flow fractionation (MD-AF4) technique as configured on a typical commercial instrument platform with online detectors such as multi-angle (static) light scattering (MALS), dynamic light scattering (DLS), ultraviolet-visible (UV-Vis) absorbance, and differential refractive index (dRI) ( 1 ) . 2 1.4 This method does not address liposome composition. Refer to Test Methods E3297 , E3323 , or E3324 for lipid quantification. 1.5 Units— The values stated in SI units are to be regarded as standard. Where appropriate, cgs units are given in addition to SI. 1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. 1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee. ====== Significance And Use ====== 5.1 Liposomal formulations for the treatment of cancer and other diseases are the most common form of nanotechnology-enabled drug products submitted for market approval and in clinical application at the present time. The accurate characterization of their physical-chemical properties is critical to support the development and assessment of such products ( 2 ) . In particular, size, size distribution, shape, and physical stability are key properties (among others) that have been widely identified as critical quality attributes (CQAs) for liposomal drug products. 5.1.1 Asymmetrical-flow field-flow fractionation (AF4) is a chromatographic-like technique that uses hydrodynamic forces to gently separate analytes into their component populations according to size and diffusivity ( 3 and 4 ) . The fractionated sample then passes through one or more online detectors chosen specifically for the application requirements. The combination of low-shear separation, tolerance for complex matrices, and exceptionally broad size range make AF4 a technique of choice for application to nanotechnology-enabled drug products such as liposomes ( 1 3 , 5- 9 ) . 5.1.2 Multi-detector AF4 is suitable for research and development, manufacturing quality control, product stability/shelf-life testing and regulatory assessments. 5.1.3 There are multiple assumptions inherent in the application of MD-AF4, including the appropriateness of models used to interpret online light scattering data and the compatibility of the analyte and matrix with the membrane that forms the surface of the accumulation wall in the fractionation channel. Other assumptions are detector or analyte specific. 5.2 Chemical components of the mobile phase must not induce agglomeration of liposomes or otherwise significantly alter their physical properties. 5.3 Discretion should be used in the interpretation of size data obtained by different scattering detectors using different scattering models and modes of analysis. 5.4 The Current Good Manufacturing Practice for Finished Pharmaceuticals (see 21 CFR 211.194(a)(2)) and the ICH Harmonized Tripartite Guideline on Validation of Analytical Procedures Q2(R1) ( 10 ) , state that the suitability of all test methods shall be verified under actual conditions of use. 5.5 MD-AF4 can be compared with batch mode dynamic light scattering (DLS) for the determination of liposome mean size and size distribution, where MD-AF4 provides deconvolution of complex mixtures yielding a more accurate assessment of the populations present. Batch mode DLS (see, for instance, Test Method E3247 ) provides a rapid, low-cost approach that can be used to screen materials prior to analysis by MD-AF4. The latter requires substantially more analyst time and effort along with appropriate training and expertise. Qualitatively, MD-AF4 offers greater insight into the physical complexity of a sample via the fractionation process in combination with multiple detectors.