Ⅵ APPROACHES TO DEVELOPING AND ASSESSING EVIDENCE TO DEMONSTRATE BIOSIMILARITY
论证生物相似性的方法
FDA recommends that sponsors use a stepwise approach to develop the evidence needed todemonstrate biosimilarity. FDA intends to consider the totality of the evidence provided by asponsor when the Agency evaluates the sponsor’s demonstration of biosimilarity, consistent witha longstanding Agency approach to evaluating scientific evidence.
FDA 建议申请人采用阶梯式方法来收集证据论证生物相似性。FDA 将整体衡量申请者证明生物相似性的所有证据，这和一直以来用以评价科学证据的方法一致。
A.Using a Stepwise Approach to Demonstrate Biosimilarity 阶梯式方法论证生物相似性
The purpose of a biosimilar development program is to support a demonstration of biosimilaritybetween a proposed product and a reference product, including an assessment of the effects ofany observed differences between the products, but not to independently establish the safety andeffectiveness of the proposed product. FDA recommends that sponsors use a stepwise approachto developing the data and information needed to support a demonstration of biosimilarity. Ateach step, the sponsor should evaluate the extent to which there is residual uncertainty about thebiosimilarity of the proposed product and identify next steps to try to address that uncertainty.Where possible, studies conducted should be designed to maximize their contribution todemonstrating biosimilarity. For example, a clinical immunogenicity study may also provideother useful information about the safety profile of the proposed product.
生物相似性研究的目的是论证一个类似药与参比制剂的生物相似性，包括评估两者之间任何可见差异产生的影响，而不是独立地建立该类似药的安全性和有效性。FDA 建议采用阶梯式方法来建立数据和信息，在实施每一步研究时，申请人应评估还有哪些不确定性因素，并确定进一步论证相似性的后续研究。可能的话，试验设计应最大可能地发挥其论证生物相似性的作用。例如，临床免疫性研究也可能提供类似药安全性方面的有用信息。
The stepwise approach should start with extensive structural and functional characterization of both the proposed product and the reference product, which serves as the foundation of a biosimilar development program (sections VII.A and VII.B). The more comprehensive and robust the comparative structural and functional characterization—the extent to which these studies are able to identify (qualitatively or quantitatively) differences in relevant product attributes between the proposed product and the reference product (including the drug substance, excipients, and impurities)—the more useful such characterization will be in determining what additional studies may be needed. For example, rigorous structural and functional comparisons that show minimal or no difference between the proposed product and the reference product will strengthen the scientific justification for a selective and targeted approach to animal and/or clinical testing to support a demonstration of biosimilarity. It may be useful to further quantify the similarity or differences between the two products using a meaningful fingerprint-like analyses algorithm that covers a large number of additional product attributes and their combinations with high sensitivity using orthogonal methods. Such a strategy may further reduce the possibility of undetected structural differences between the products and lead to a more selective and targeted approach to animal and/or clinical testing. A sufficient understanding of the mechanism of action (MOA) of the drug substance and clinical relevance of any observed structural differences, clinical knowledge of the reference product and its class that indicates low overall safety risks, and the availability of a relevant PD measure(s) may provide further scientific justification for a selective and targeted approach to animal and/or clinical studies.
这种阶梯式方法应基于对类似药和参比制剂全面的结构和功能表征信息，这也是相似性研究的基础( VII.A 和VII.B)。这些结构和功能特性的比较越全面彻底，包括药物成分、辅料和杂质是定量或定性比较研究，对确定下一步具体研究越有利。例如，如果有严格的结构和功能比较表明类似药和参比制剂没有或者仅有很小的差异，越能说明为什么采用一个选择性和目标性的动物或临床试验来证明相似性。也可以利用指纹图谱分析方法（利用正交法能覆盖到大部分产品性质及之间的关系），进一步对这相似性或差异进行定量分析。这种方法能进一步降低检测不到产品间差异存在的可能性，从而使下一步动物或临床试验的选择更有目的性。为了证明选择该试验的合理性，还要对活性成分作用机制、结构差异导致的临床反应、参比制剂的临床信息及其产品类别表明的低风险性、动物或临床相关PD研究方法及数据等进行全面的了解。
The sponsor should then consider the role of animal data in assessing toxicity and, in some cases, in providing additional support for demonstrating biosimilarity and in contributing to the immunogenicity assessment (section VII.C). The sponsor should then conduct comparative human PK and PD studies (if there is a relevant PD measure(s)) (section VII.D.1) and compare the clinical immunogenicity of the two products in an appropriate study population (section VII.D.2). If there is residual uncertainty about biosimilarity after conducting structural analyses, functional assays, animal testing, human PK and PD studies, and the clinical immunogenicity assessment, the sponsor should then consider what additional clinical data may be needed to adequately address that uncertainty (section VII.D.3). FDA encourages sponsors to consult extensively with the Agency after completion of comparative structural and functional analyses (before finalizing the clinical program) and throughout development as needed.
申请人要考虑动物试验数据在毒理学研究和某些情况下证明生物相似性的和免疫原性研究当中的重要性（详见VII.C部分），然后在合适的试验人群中进行类似药和参比制剂的人体PK/PD的可比性研究(VII.D.1部分)和临床免疫性的比较研究( VII.D.2部分)。若在结构分析、功能测定、动物试验、人体PK/PD试验后还不能完全确定其生物相似性，则需提供更充分的临床安全数据(VII.D.3部分)。FDA 建议，结构和功能比较分析完成之后（临床试验完成前）或研究过程如有需要，申请者应当和审批机构进行广泛的沟通讨论。
FDA recognizes that some of the aforementioned investigations could be performed in parallel; however, the Agency recommends that sponsors use a stepwise approach to better address residual uncertainty about biosimilarity that might remain at each step and incorporate FDA’s advice provided after FDA review of data and information collected at certain milestones.
FDA要求上述提到的部分研究应当进行平行试验，但是审批机构也建议申请人采用阶梯式方法可以更好的证明每一步生物相似性研究当中的可能存在的不确定性因素；也建议在FDA收到关键的数据信息后，申请人应当和FDA沟通讨论。
B.Using a Totality-of-the-Evidence Approach to Assess a Demonstration of Biosimilarity 采用证据整体法评价生物相似性论证过程
In evaluating a sponsor’s demonstration of biosimilarity, FDA will consider the totality of the data and information submitted in the application, including structural and functional characterization, nonclinical evaluation, human PK and PD data, clinical immunogenicity data, and comparative clinical study(ies) data. FDA intends to use a risk-based approach to evaluate all available data and information submitted in support of the biosimilarity of the proposed product.
评价生物相似性论证时，FDA会全面考察申请人提交的所有的数据信息：包括结构功能特性、非临床研究、人体PK/PD数据、临床免疫性数据和临床研究比较数据。FDA倾向于采用一种基于风险的方法来评估生物相似性论证过程中所有可获得的数据。
Thus, a sponsor may be able to demonstrate biosimilarity even though there are formulation or minor structural differences, provided that the sponsor provides sufficient data and information demonstrating that the differences are not clinically meaningful and the proposed product otherwise meets the statutory criteria for biosimilarity. For example, differences in certain posttranslational modifications or differences in certain excipients (e.g., human serum albumin) might not preclude a finding of biosimilarity if data and information provided by the sponsor show that the proposed product is highly similar to the reference product notwithstanding minor differences in clinically inactive components and that there are no clinically meaningful differences between the products in terms of safety, purity, and potency. Clinically meaningful differences could include a difference in the expected range of safety, purity, or potency of the proposed product and the reference product. By contrast, slight differences in rates of occurrence of certain adverse events between the two products ordinarily would not be considered clinically meaningful differences.
因此，尽管存在处方或者微小的结构差异，只要能够提供有效的数据信息说明这些差异没有临床意义且该类似药符合生物相似性的法规要求，申请人仍然可以证明其生物相似性。例如，如果申请人提供的数据信息可以证明类似药和参比制剂高度相似，且非活性成分差异无论是在安全性、纯度和有效性方面均没有临床有意义差异，那么某些翻译后修饰或某些特定辅料（如人血清蛋白）的不同可能就不会对其生物相似性结果产生影响。有临床意义的差异包括类似药和参比制剂在可控范围内安全性、纯度和效力的差异。相比之下，某些不良反应发生率方面的微小差异通常被认为无临床有意义差异。
ⅦDEMONSTRATING BIOSIMILARITY生物相似性的论证
This section discusses scientific considerations in the stepwise approach to developing data and information needed to support a demonstration of biosimilarity. To demonstrate biosimilarity, a sponsor must provide sufficient data and information to show that the proposed product and the reference product are highly similar notwithstanding minor differences in clinically inactive components and that there are no clinically meaningful differences between the two products in terms of safety, purity, and potency. The type and amount of analyses and testing that will be sufficient to demonstrate biosimilarity will be determined on a product-specific basis.
本部分主要讨论采用阶梯式方法收集生物相似性的数据信息时应注意的科学问题。为了论证生物相似性，申请者必须提供有效的数据信息来说明尽管临床非活性成分存在微小差异，类似药和参比制剂仍然高度相似，且两种产品间的安全性、纯度和效能无临床有意义差别。应根据每个产品的特性，分别采用不同的分析试验类型和数量充分证明其生物相似性。
A.Structural Analyses结构分析
The PHS Act requires that a 351(k) application include information demonstrating biosimilarity based on data derived from, among other things, analytical studies that demonstrate that the biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components, unless FDA determines that an element is unnecessary in a 351(k) application. FDA expects that first, a sponsor will extensively characterize the proposed product and the reference product with state-of-the-art technology, because extensive characterization of both products serves as the foundation for a demonstration of biosimilarity. It is expected that the expression construct for a proposed product will encode the same primary amino acid sequence as its reference product. However, minor modifications such as N- or Cterminal truncations that are not expected to change the product performance may be justified and should be explained by the sponsor. Additionally, sponsors should consider all relevant characteristics of the protein product (e.g., the primary, secondary, tertiary, and quaternary structure; posttranslational modifications; and biological activities) to demonstrate that the proposed product is highly similar to the reference product notwithstanding minor differences in clinically inactive components. The more comprehensive and robust the comparative structural and functional characterization is, the stronger the scientific justification for a selective and targeted approach to animal and/or clinical testing.
除非FDA认为不需要，PHS Act要求 351(k) 申请提交的证明生物相似性性的信息，除了其他必需事项外，还应包括能够证明即使临床非活性成分存在微小差异，类似药和参比制剂仍然高度相似的分析试验研究。首先，FDA 希望申请者采用最先进的技术对类似药和参比制剂进行广泛研究，因为这些研究结果是论证生物相似性的基础。通常情况下，FDA 希望类似药和参比制剂的氨基酸序列表达构造相同，但是，像N-或C-端断裂等不会影响安全有效性的微小差异，申请者只要给出科学合理性的解释即可。另外，申请人还必须考虑到蛋白制品所有相关性质（包括一、二、三、四级结构、翻译后修饰、生物活性），以证明即使在临床非活性成分存在微小差异的情况下，其与参比制剂仍具有高度生物相似性。对结构功能比较得越全面，动物和/或临床试验的选择和针对性越科学合理。
Sponsors should use appropriate analytical methodologies with adequate sensitivity and specificity for structural characterization of the proteins. Generally, such tests include the following comparisons of the proposed product and the reference product:
• Primary structures, such as amino acid sequence
• Higher order structures, including secondary, tertiary, and quaternary structure (including aggregation)
• Enzymatic posttranslational modifications, such as glycosylation and phosphorylation
• Other potential variations, such as protein deamidation and oxidation
• Intentional chemical modifications, such as PEGylation sites and characteristics
申请人需要使用灵敏度高、专属性强的分析方法分析蛋白质的结构特性。一般而言，分析实验通常需要比较以下几个方面：
（1）一级结构，如氨基酸序列；
（2）高级结构，包括二级、三级和四级结构（包括聚合）；
（3）酶转录后的修饰，如糖基化和磷酸化；
（4）其他可能的变化，如蛋白脱酰基化反应和氧化反应；
（5）意向化学修饰，如聚乙二醇化结合位点及其特征。
Sponsors should conduct extensive structural characterization of both the proposed product and the reference product in multiple representative lots to understand the lot-to-lot variability of both products in the manufacturing processes. Lots used for the analyses should support the biosimilarity of both the clinical material used in the clinical study(ies) intended to support a demonstration of biosimilarity, and the to-be-marketed proposed product, to the reference product. Characterization of lots manufactured during process development for the proposed product may also be useful. Sponsors should justify the selection of the representative lots, including the number of lots.
申请人应该对类似药和参比制剂多个有代表性批次进行广泛的结构确认，以了解生产过程中不同批次间的变化。用于进行生物相似性分析的批次应能够代表临床试验、待上市的产品和参比制剂。类似药产品工艺开发期间批次的性质也是有用信息，申请者应能够说明批次及批数选择的合理性。
In addition, FDA recommends that sponsors analyze the finished dosage form of multiple lots of the proposed product and the reference product, assessing excipients and any formulation effect on purity, product- and process-related impurities, and stability. Differences in formulation between the proposed product and the reference product are among the factors that may affect the extent and nature of subsequent animal or clinical testing. A sponsor considering manufacturing changes after completing the initial analytical similarity assessment or after completing clinical testing intended to support a 351(k) application should perform an additional analytical similarity assessment with lots manufactured by the new process and the reference product and establish comparability of the proposed product manufactured by the old and new manufacturing processes. The nature and extent of the changes may determine the extent of the analytical similarity and comparability studies and any necessary additional studies.
此外，FDA 建议申请人分析多个批次的成品来评价辅料和处方对纯度、产品和工艺相关杂质和稳定性的影响。类似药和参比制剂处方不同可能是后续动物或临床试验的影响因素之一。在完成初始相似性评估或临床试验后，申请人为支持其351（K）申请考虑到生产工艺变化，应对新工艺和参比制剂生产的批次进行额外的相似性分析评估，并对由新旧生产工艺生产的产品进行相似性分析。生产工艺的变化可能决定相似性分析的程度，是否需要对比及其他附加试验。
If the reference product or the proposed product cannot be adequately characterized with stateof-the-art technology, the application for the proposed product may not be appropriate for submission under section 351(k) of the PHS Act; and the sponsor should consult FDA for guidance on the appropriate submission pathway.
如果利用最先进的技术仍不能全面描述类似药和参比制剂的特征，则PHS Act351(k)可能不适用于该产品的申请，申请人应咨询FDA，请FDA指导PHS Act 351(k)是否适用于这种蛋白产品的申请。
B.Functional Assays功能性实验
The pharmacologic activity of protein products should be evaluated by in vitro and/or in vivo functional assays. In vitro assays may include, but are not limited to, biological assays, binding assays, and enzyme kinetics. In vivo assays may include the use of animal models of disease (e.g., models that exhibit a disease state or symptom) to evaluate functional effects on pharmacodynamic markers or efficacy measures. A functional evaluation comparing a proposed product to the reference product using these types of assays is also an important part of the foundation that supports a demonstration of biosimilarity and may be used to scientifically justify a selective and targeted approach to animal and/or clinical testing.
蛋白产品的药理学活性应当通过体内和/或体外功能性实验评估。体外分析包括但不仅限于生物分析、结合分析和酶动力学。体内分析包括应用动物病理模型（例如表现出疾病状态或病症的模型）来评价对药效学标志物或疗效测定的影响。功能性试验不仅是论证生物相似性的基础，还可用于合理和有针对性的选择动物和/或临床试验。
Sponsors can use functional assays to provide additional evidence that the biologic activity and potency of the proposed product are highly similar to those of the reference product and/or to support a conclusion that there are no clinically meaningful differences between the proposed product and the reference product. Such assays also may be used to provide additional evidence that the MOA of the two products is the same to the extent the MOA of the reference product is known. Functional assays can be used to provide additional data to support results from structural analyses, investigate the consequences of observed structural differences, and explore structure-activity relationships. These assays are expected to be comparative so they can provide evidence of similarity or reveal differences in the performance of the proposed product compared to the reference product, especially differences resulting from variations in structure that cannot be detected using current analytical methods. FDA also recommends that sponsors discuss limitations of the assays they used when interpreting results in their submissions to FDA. Such discussions would be useful for the evaluation of analytical data and may guide whether additional analytical testing would be necessary to support a demonstration of biosimilarity.
申请人可以利用功能性实验进一步说明类似药的生物活性和效力与参比制剂高度相似，证明两者没有临床有意义差异；亦可用于证明两者作用机制相同。功能性试验也可以用于结构分析的结果分析，研究结构差异的影响和结构-活性之间关系。功能性试验应该是比较性的，从而能够证明类似药和参比制剂之间的相似性或揭示其差异，尤其是在现有分析方法不能检测到结构变化导致的结果差异的情况下。此外，FDA 建议申请人在提交的结果中应当讨论功能性实验的限制条件。此类讨论将有助于评估分析数据，并可以用来判断是否需要进行额外的分析测试以支持其生物相似性。
Functional assays can also provide information that complements the animal and clinical data in assessing the potential clinical effects of minor differences in structure between the proposed product and the reference product. For example, cell-based bioactivity assays may be used to detect the potential for inducing cytokine release syndrome in vivo. The available information about these assays, including sensitivity, specificity, and extent of validation, can affect the amount and type of additional animal or clinical data that may be needed to establish biosimilarity. As is the case for the structural evaluation, sponsors should justify the selection of the representative lots, including the number of lots.
功能性实验也可以为用于评价类似药和参比制剂存在微小结构差异时可能会有的临床反应的动物、临床试验提供补充数据。例如，基于细胞的生物活性测定实验可用于检测诱发体内细胞因子释放综合能力的潜能。这些功能性实验分析信息，包括灵敏性、特异性和有效范围，均能够影响下一步所需的动物或临床数据的数量和类型。对于结构评价，申请人应选择有代表性的批次和批次数量。
