药学学报

Characteristics	In vivo method	In vitro method
Sensitivity	High, capable of reflecting the overall immune response in living organisms.	High, capable of detecting subtle changes in specific antigens or antibodies, but limited in simulating complex immune responses.
Specificity	Lower, influenced by individual animal differences and environmental factors.	Higher, capable of precise detection of specific antigens or antibodies with minimal external interference.
Reproducibility	Lower, results are affected by individual animal differences and experimental conditions, leading to higher variability.	Higher, experimental conditions are controllable, results are more stable, and variability is lower.
Experimental duration	Longer, involving animal breeding, vaccination, observation, and other time-consuming steps.	Shorter, simplified experimental procedures, and faster results.
Cost	Higher, involving costs related to animal breeding, experimental operations, and ethical reviews.	Lower, relatively lower costs for materials and equipment, and no expenses related to animal experiments.
Ethical considerations	Involves animal experiments, raising ethical concerns and not aligning with the 3R principles (replacement, reduction, refinement).	No animal experiments required, aligns with the 3R principles, and has fewer ethical concerns.
Applicable scenarios	Suitable for evaluating the overall immune response of vaccines, especially for simulating complex immune reactions.	Suitable for monitoring production consistency, detecting antigen content, and assessing specific immune responses.
Limitations	Results are significantly affected by individual animal differences, making it difficult to precisely control experimental conditions.	Limited ability to simulate complex immune responses, and potential interference in detecting certain vaccines (e.g., adjuvanted vaccines).
Regulatory acceptance	Traditional methods, widely accepted but gradually being replaced by in vitro methods.	Increasingly accepted by regulatory agencies but requires rigorous validation to ensure correlation with in vivo methods.

Characteristics	In vivo method	In vitro method
Sensitivity	High, capable of reflecting the overall immune response in living organisms.	High, capable of detecting subtle changes in specific antigens or antibodies, but limited in simulating complex immune responses.
Specificity	Lower, influenced by individual animal differences and environmental factors.	Higher, capable of precise detection of specific antigens or antibodies with minimal external interference.
Reproducibility	Lower, results are affected by individual animal differences and experimental conditions, leading to higher variability.	Higher, experimental conditions are controllable, results are more stable, and variability is lower.
Experimental duration	Longer, involving animal breeding, vaccination, observation, and other time-consuming steps.	Shorter, simplified experimental procedures, and faster results.
Cost	Higher, involving costs related to animal breeding, experimental operations, and ethical reviews.	Lower, relatively lower costs for materials and equipment, and no expenses related to animal experiments.
Ethical considerations	Involves animal experiments, raising ethical concerns and not aligning with the 3R principles (replacement, reduction, refinement).	No animal experiments required, aligns with the 3R principles, and has fewer ethical concerns.
Applicable scenarios	Suitable for evaluating the overall immune response of vaccines, especially for simulating complex immune reactions.	Suitable for monitoring production consistency, detecting antigen content, and assessing specific immune responses.
Limitations	Results are significantly affected by individual animal differences, making it difficult to precisely control experimental conditions.	Limited ability to simulate complex immune responses, and potential interference in detecting certain vaccines (e.g., adjuvanted vaccines).
Regulatory acceptance	Traditional methods, widely accepted but gradually being replaced by in vitro methods.	Increasingly accepted by regulatory agencies but requires rigorous validation to ensure correlation with in vivo methods.

疫苗类产品效价测定体外方法替代体内方法的有关考量

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张媛 ¹ , 陈钢 ² , 谭德讲 ¹^,^*

药学学报 | 综述 2025,60(9): 2724-2729

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药学学报 |综述 2025 , 60 (9) : 2724 -2729

疫苗类产品效价测定体外方法替代体内方法的有关考量

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张媛¹, 陈钢², 谭德讲¹^,^*

作者信息

¹中国食品药品检定研究院, 北京 102629

²上海市食品药品检验研究院, 上海 201203

通讯作者:

谭德讲, Tel: 86-10-53851589, E-mail: tandj@nifdc.org.cn

作者简介:

作者贡献:

张媛负责文章撰写与文献检索; 陈钢负责文章提纲框架的拟定; 谭德讲对文章进行了修改及审核。

Considerations of in vitro methods to alternate in vivo methods for vaccine potency determination

Yuan ZHANG¹, Gang CHEN², De-jiang TAN¹^,^*

Affiliations

¹National Institutes for Food and Drug Control, Beijing 102629, China

²Shanghai Institute for Food and Drug Control, Shanghai 201203, China

出版时间: 2025-09-12 doi: 10.16438/j.0513-4870.2025-0169

文章导航

摘要

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本文讨论了疫苗效价测定的体外方法替代传统体内方法的可行性与挑战及相关考量。疫苗效价体内测定方法长期用于疫苗质量控制, 但存在变异性大、实验效率低、涉及动物伦理等问题。随着3R原则和科技进步, 体外方法逐渐成为替代体内方法的趋势。体外测定方法在评估疫苗生产一致性、监测生产过程中变化方面具有优势, 且可更高效地评估疫苗的稳定性和免疫效果。尽管体外和体内方法的机制不同, 二者的质量控制属性也不完全一致, 体外方法仍然能够有效在保证产品质量特别是效价一致的前提下替代体内效价测定, 尤其是在疫苗开发早期进行质量控制时。常见的体外替代方法包括免疫测定、理化分析和分子生物学测定, 选择合适的方法需根据疫苗类型和作用机制。此外, 文章强调应在疫苗开发初期建立体外方法, 并确保其稳定性、重现性和与临床效果的相关性。尽管体外替代方法的推广面临挑战, 但随着技术进步, 未来有望进一步替代动物实验, 提高疫苗质量控制的效率和伦理性。

关键词

疫苗 / 效价 / 方法替代 / 体内方法 / 体外方法 / 关键质量属性

Abstract

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This article focuses on the methods for determining vaccine potency, especially the feasibility and challenges of replacing traditional in vivo methods with in vitro methods, as well as related considerations. The in vivo method for determining vaccine potency has been used for a long time for vaccine quality control, but it has problems such as high variability, low experimental efficiency, and animal ethics issues. With the 3R principle and technological progress, in vitro methods have gradually become a trend to replace in vivo methods. In vitro testing methods have advantages in evaluating the consistency of vaccine production and monitoring changes during the production process, and can more effectively evaluate the stability and immune effects of vaccines. Although the mechanisms of in vitro and in vivo methods are different, and the quality control attributes of the two methods are not completely consistent, in vitro methods can still effectively replace in vivo potency assays, especially for quality control during the early stages of vaccine development. Common in vitro alternative methods include immunoassay, physicochemical analysis, and molecular biological assay. The selection of appropriate methods depends on the type of vaccine and its mechanism of action. In addition, the article emphasizes that the establishment of in vitro methods should be included in the vaccine development process as early as possible, and ensure its stability, reproducibility, and correlation with clinical efficacy. Although the promotion of in vitro alternative methods faces challenges, with technological progress, it is expected to further replace animal experiments in the future, improving the efficiency and ethicality of vaccine quality control.

Key words

vaccine / potency / method replacement / in vivo method / in vitro method / critical quality attribute

引用本文

张媛, 陈钢, 谭德讲. 疫苗类产品效价测定体外方法替代体内方法的有关考量. 药学学报, 2025 , 60 (9) : 2724 -2729 . DOI: 10.16438/j.0513-4870.2025-0169

Yuan ZHANG, Gang CHEN, De-jiang TAN. Considerations of in vitro methods to alternate in vivo methods for vaccine potency determination[J]. Acta Pharmaceutica Sinica, 2025 , 60 (9) : 2724 -2729 . DOI: 10.16438/j.0513-4870.2025-0169

正文

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疫苗的效价是指疫苗产生免疫反应以预防指定疾病的能力, 是反映疫苗有效性的关键质量属性(critical quality attributes, CQAs)。疫苗效价测定方法则是证明疫苗具有实现其预期结果能力的一种检测手段^[1]。疫苗效价测定是疫苗研发及其监管中的核心环节, 直接关系到疫苗临床应用的有效性乃至安全性。效价测定用于评估疫苗诱导免疫反应的能力, 确保其能够提供预期的保护效果。疫苗效价测定的应用可贯穿包括研发(疫苗候选株的筛选、工艺优化等)、生产(批放行检验、生产过程监控等) 以及监管(注册、上市后监测等) 在内的疫苗产品的整个生命周期。

疫苗效价测定方法包括体内测定或体外测定两种, 有时也可以结合使用。目前, 收载于中国药典的疫苗各论中包括大量体内效价测定方法, 虽然这些方法在保障疫苗质量方面一直发挥着核心作用, 但由于此类方法变异度较大、实验效率较低且涉及动物伦理, 因此, 与其相比, 设计合理的体外方法显然更适用于监测疫苗产品的一致性(或生产稳定性) 以及评估由生产变更带来的潜在影响^[2]。近年来, 随着生物学等学科技术的飞速发展, 使得新型检测技术日新月异、部分疫苗的作用机制也日益明朗化, 加之3R (替代、减少、优化) 原则以及质量源于设计(quality by design, QbD) 理念的推广和认可, 世界卫生组织(WHO)、各国监管机构和疫苗生产商等均致力于疫苗体内方法的替代。此外, WHO等国际组织同时也致力于制定和推广疫苗效价测定的国际标准, 通过国际标准的协调统一, 全球疫苗质量控制水平得到提升, 疫苗的质量和效力得到更好的保障。方法替代是分析方法全生命周期的一部分, 方法的改变可能涉及疫苗产品CQAs的重新定义及质量标准的变更。尽管疫苗效价测定方法的替代面临诸多挑战, 但整体趋势仍然向体外方法发展。

本文主要针对体外替代方法与原有体内方法检测原理、机制完全不同, 且两类方法控制的产品质量属性间没有必然或完全因果关系的情况^[3], 对疫苗效价测定体内外方法替代提出相关的考虑要点, 供从事疫苗相关工作的研究者作为参考。此外, 还需强调的一点是疫苗效价体外测定方法最好在产品开发过程中即开始考虑建立^[2]。本文的研究对象虽为人用疫苗, 但其他生物制品、生化药品等产品的质控方法替代亦可参考。

1　方法替代的考虑要点

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疫苗产品的常规质控方法旨在监控产品的一致性, 并确保上市批次与最初在临床研究中被证实安全、有效的批次间具有可比性。方法替代的前提是所涉及的疫苗具有良好的安全性和有效性, 且其生产工艺具有较好的一致性。

疫苗的体内效价检测方法通常已被证明适用于其使用目的, 并已证明在保证疫苗有效性方面具有至关重要的作用。但由于体内方法的固有变异性, 使得其在与稳健性更好的体外方法进行典型的一对一比较研究时面临巨大挑战。早先开发的部分疫苗效价体内测定方法缺乏完整规范的验证要求, 正是这一原因导致一对一体内、外方法比较研究难度较大, 甚至难以评估两种方法间的差异。由于药典方法通常被认为是经过验证的, 现在对这些方法进行回顾性验证不仅不切实际且成本过高, 也不符合伦理要求, 因而很难对药典收录的体内方法进行再验证^[2]。但即使无法证明体外和体内方法间的一致性, 也不意味着体外方法不适用于疫苗效价的检测。在临床疫苗开发过程中, 可以通过测试不同生产过程的样品以及稳定性研究, 评估体内外效价测定方法的相关性^[4]。当二者相关性难以评价或不相关时, 还可以使用临床研究或常规使用证实安全有效的库存样本来建立体外测定结果与临床疫苗性能的联系。但需要强调一点, 与在注册产品上市阶段直接用体外检测相比, 这种方法可能会更具挑战性。例如: 乙型肝炎疫苗的效价测定由早期的小鼠法转换到后来的酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA) 方法, 即使用的上述策略^[4]。后续建立的ELISA方法能够准确反映疫苗的免疫原性, 该方法已被WHO和多个国家的监管机构认可, 并广泛应用于乙型肝炎疫苗的质量控制。除此之外, 体外测定方法还应能够确证疫苗是否达到其质量标准的要求, 并与体内方法结论一致, 如灭活脊髓灰质炎疫苗^[5], 随着对脊髓灰质炎病毒D抗原(病毒衣壳蛋白) 的研究深入, 体外D抗原检测方法被开发出来。该方法通过检测疫苗中D抗原的含量来评估疫苗的效力, 具有更高的精确性和可重复性。而人乳头瘤病毒(human papillomavirus, HPV) 疫苗也随着对HPV病毒结构和免疫机制的理解加深, 成功地由原有的小鼠法替代为体外中和抗体检测法, 该方法通过检测疫苗诱导的中和抗体与病毒颗粒的结合能力来评估疫苗的效力。在多数情况下, 体外方法可检测出疫苗生产工艺中无法通过体内方法检测到的变化。因此, 体外方法在监测疫苗生产一致性方面是优于体内方法的, 可更有效地评估工艺变化对产品带来的影响^[2]。

在考虑从体内到体外质量控制方法过渡时, 了解体内方法能或不能提供什么最为关键^[2]。首先需考虑的关键因素是体外替代方法应能反映疫苗类的免疫有效性, 并具有较低的变异和较高的灵敏度。虽然疫苗的体内效力检测方法可检测与有效性相关的免疫功能反应, 但不能预测其在目标群体中的实际免疫反应。而体外方法通常具有较低的变异性和较高的灵敏度, 能够检测与免疫反应相关的特定指标。另一需考虑的关键因素是当体外方法替代体内方法时, 疫苗的CQAs是否会以不同方式进行评估, 例如以检测抗原含量或功能反应(病毒或毒素中和) 的体外方法替代体内效力检测方法等。总之, 体外方法必须能控制相关产品的CQA, 并监测生产一致性从而确保产品的有效性。

用于疫苗质量控制的体外方法一般需具有检测疫苗抗原含量和功能的能力。因此, 确定可识别包括抗原功能、稳定性和/或其他产生有效免疫反应重要特征在内的关键表位的相关抗体是此类体外检测方法开发的关键^[6]。如果使用单一方法, 最好通过靶向测定与疫苗保护相关的表位来检测抗原的含量和完整性。例如, 选择与作为产生中和抗体主要靶标的表位(epitope) 或表位簇相对应的单克隆抗体进行测定。此外, 为使得检测方法更具稳定性, 应优选具有构象的表位或表位簇。如单一体外方法无法充分反映疫苗的抗原含量和功能, 可考虑使用多个体外方法替代, 如对结合多糖疫苗的效价检测方法的替代, 可以通过测定分子大小、结合物完整性、总体及游离多糖含量等检测方法替代原有的动物体内试验^[2]。

许多疫苗制剂含有佐剂, 其作用是能够非特异性地改变或增强机体对抗原的特异性免疫应答, 因此佐剂的存在对疫苗的功效至关重要。但与体内检测方法不同, 佐剂的存在会给体外效力检测带来更多的挑战, 如铝盐等佐剂的存在可能会干扰靶抗原的检测。因此, 在这些情况下, 效价测定方法必须能够在不破坏功能性表位的前提下, 使得抗原可以与佐剂分离(例如, 使用适当的缓冲处理)^[1]。也可在测定前将抗原从佐剂中解吸附, 以规避佐剂对测定的影响^[7-9]。

虽然本文的研究重点是使用疫苗效价体外检测方法替代已批准产品的现有体内方法, 但仍需强调的是在疫苗产品开发过程中即开始考虑采用体外方法进行质量控制是更值得推广的, 而采用体内方法测定疫苗效价也并非强制性的^{[2, 10]}。对于很多已经上市的疫苗, 在进行方法替代时, 由于原来对体内方法的验证不足, 积累的数据不规范, 建议初始阶段可实行两类方法并行的方式, 待积累到足够评价数据后, 经专业的统计比较分析, 再确定是否删除原体内方法。疫苗效价体内、外测定方法特性比较详见表1。

由表1可看出, 体外方法在重现性、成本、伦理考虑等方面具有明显优势, 而体内方法在评估复杂免疫反应方面仍有其独特价值。随着技术进步, 体外方法有望在更多领域替代体内方法, 但两者在疫苗研发和质量控制中仍将长期共存。

2　体外替代方法的分类^[4]

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效价测定方法的选择主要取决于以下两个因素, 一是疫苗形式, 如亚单位疫苗、活病毒疫苗、灭活疫苗等; 二是疫苗的作用机制。例如: 对于活病毒疫苗, 常使用感染性测定方法以测量病毒复制; 而对于亚单位疫苗, 理化方法或免疫方法可能是最佳选择^[4]。虽然疫苗效价检测方法各不相同, 但通常均可反映出疫苗的复杂性以及对疫苗作用方式的理解程度。尽管相同类型的疫苗具有共性, 但效价测定方法仍需要逐案验证评估, 每类疫苗通常都有个例。目前, 常用的疫苗体外测定方法分为如下几类。

2.1　免疫测定类

如果疫苗产生保护性抗体的模式适宜, 且表位已知, 则可以开发体外免疫测定方法。通过抗原特异性抗体测量表位的存在及其数量可以推断出潜在的疫苗效力。但需要注意的一点是: 疫苗中的其他成分是否会对测定产生影响, 例如前文所述的佐剂的存在。对于一些疫苗, 效力与抑制病毒进入和感染的中和抗体的产生直接相关。因此, 在体外结合中和抗体可用于免疫测定以评估疫苗的效价, 但获得稳定来源的中和抗体是此类体外测定方法开发的关键因素之一。由于中和表位的确定困难重重, 因此可以制造单克隆抗体用于效力测定。此类免疫测定方法较体内方法而言通常具有更好的重现性。该类方法适于所有类型的疫苗。

对于减毒活疫苗和细菌疫苗, 通常采用下列方法: ①体外感染性实验测定法。常见方法有噬斑测定法, 通过将不同稀释倍数的病毒接种于易感细胞, 病毒复制后细胞溶解, 计数细胞层中的噬斑。同理, 使用指定数量的病毒颗粒感染指定数量的细胞, 从而计算感染细胞与非感染细胞的比例, 以50%的感染细胞为限, 可确定半数组织细胞感染剂量(50% tissue culture infectious dose, TCID₅₀)。②荧光定量法: 即以荧光抗体对感染细胞进行染色, 并通过荧光定量, 从而测量出斑点形成单位(focus-forming units)。但使用此类测定方法的前提是找到合适的易感细胞株以及病毒/细菌具有适宜的裂解能力。③特异性生物标志物法: 体外生物标志物检测可以替代斑点测定实验, 特别是对于非裂解病毒。所选细胞系需具有生物标志物反应, 如感染疫苗后的线粒体呼吸。尽管生物标志物检测增加了细胞培养和感染条件的复杂性, 但其测量精密度较高, 因此最终测定结果的变异度通常远低于斑点测定。

2.2　理化分析类

此类方法适用于细菌多糖疫苗及细菌多糖蛋白结合疫苗。其中细菌多糖疫苗(如脑膜炎球菌、肺炎球菌、伤寒沙门氏菌Vi和b型流感嗜血杆菌疫苗等) 的效力与各自多糖的数量和大小有关。多糖疫苗是第一种基于纯化学和物理化学方法测定效价的疫苗, 其效力可能还包括对多糖某些附加基团(如O-乙酰基) 含量的测量。细菌多糖蛋白结合疫苗的效力同样主要通过化学和物理化学方法进行测定, 但较前者更为复杂。测试参数除了包括多糖含量、游离多糖含量, 还需包括多糖与蛋白质的比例、结合程度以及结合物的尺寸/分子量(和多分散性), 而后者更是决定此类疫苗效力的主要衡量标准^[1]。常用的检测手段包括核磁共振或高效液相色谱等理化方法。核磁共振能够更好地评估分子结构, 但这类方法需要更广泛的专业知识和昂贵的仪器设备, 因此在推广上存在一定局限性。

2.3　分子生物学测定类

脱氧核糖核酸(deoxyribonucleic acid, DNA) 质粒疫苗作为一种新型疫苗, 其效价测定可通过体外方法评估DNA质粒转染宿主细胞的能力, 或检测质粒产物转录和/或翻译为相关蛋白抗原的能力, 如TaqMan RT-PCR、流式细胞术等^[1]。

随着医药科技的飞速发展, 新型疫苗的出现是时代发展的必然趋势, 是人类对抗疾病的重要武器。新型疫苗出现可能对其效价测定方法有着更多的挑战。如信使核糖核酸(messenger ribonucleic acid, mRNA)疫苗作为近年来疫苗领域的一项革命性技术, 其效价评价方法就与传统疫苗有所不同。mRNA疫苗通过将编码目标抗原的mRNA递送到宿主细胞中, 利用宿主细胞的翻译机制产生抗原蛋白, 进而诱导免疫反应。由于其独特的作用机制, mRNA疫苗的效价评价方法需要结合mRNA的特性、递送系统的效率以及抗原蛋白的表达和免疫原性等多方面因素。因而其效价测定可涉及上述多种类型的检测方法。如采用毛细管电泳、定量PCR检测mRNA完整性及纯度, 采用动态光散射、透射电子显微镜、体外转染实验等评价递送系统效率, 采用ELISA、抗体中和试验评价免疫原性, 采用Western blot、流式细胞术、免疫荧光染色等检测抗原蛋白的表达^[11-13]。随着技术的进步和标准化评价体系的建立, mRNA疫苗的效价评价将更加科学、高效, 为其广泛应用提供有力支持。

3　体外方法的建立及其基本原则

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目前, 相当一部分疫苗使用动物体内方法进行效价测定的原因如下: 体内实验可直接反映疫苗的生物活性, 但除此之外体外方法不能对疫苗进行彻底表征或未能全面了解疫苗的CQAs也是重要原因之一。然而, 随着疫苗在开发周期中的日益成熟, 疫苗效价的体外测定方法通常会应运而出, 从而取代体内方法。自21世纪初期QbD理念提出后, 其影响范围日益扩大, 与传统理念相比, QbD旨在将质量构建到生产/分析过程中, 其为制药领域带来的优势有目共睹。因此, 疫苗体外效价测定方法亦可以QbD理念为基石而进行建立, 其流程大致如下^{[1, 14]}: ①根据所需开发的体外测定方法相关产品的CQAs及其预期用途建立该方法分析目标概况(analytical target profile, ATP); ②对候选方法进行选择, 与传统方法相比, QbD方法最大的不同点在于方法质量及商业性能(method quality and business performance) 均已提前设定而非经验所得; ③对所选方法进行风险评估, 此步骤是降低风险的首要阶段, 可对影响分析方法测定的影响因素进行识别和控制; ④确定方法的设计空间, 多因素设计(design of experiments, DOE) 是此步骤最有效的常用的工具之一; ⑤确定方法的验证和控制策略, 主要包含2个关键元素: 一是分析方法关键指标的控制, 二是方法常规使用中有效监测工具的确定, 前者可通过风险评估确定, 后者通过稳健性DOE (robustness DOEs) 的结果确定; ⑥对分析方法进行维护, 此程序可对分析方法的控制状态进行检测和评估, 从而为方法质量性能, 甚至是商业性能提供实时检测(a real-time view)。

此外, 需要强调的是: 由于疫苗效价测定的复杂性及固有的高变异性, 在方法设计阶段应全面考虑可能的变异来源, 包括: 试剂、标准物质、检测时间及温度、细胞培养代次、季节、检测人员、关键设备等。此外, 设立方法优化期对于建立此类方法也尤为重要。为使得疫苗效价测定结果更加可靠, 可在方法建立阶段将方法性能(method performance) 的可接受标准固化在检测方法中。这一标准的建立可利用稳定的参考品得以实现, 例如: 在方法建立时, 使用适宜、稳定的标准物质进行多次测定, 并对测定结果进行分析, 由此建立该方法各种测量参数的具有统计学意义的范围, 包括曲线拟合参数、重复性、日间变异等。这一标准的设立可用于判断实验是否成立, 并在出具测定结果前做出客观合理的决策^[1]。

有关疫苗体外效价测定的基本原则如下。

3.1　体外方法应能科学合理地检测出与生产过程控制影响效力相关的差异

研究数据应证明体外替代方法可控制疫苗的CQA, 并维持待签发批次疫苗质量与临床试验或常规使用安全有效批次间的相关性。方法替代后, 可能需通过重新设置适当的CQA的质量标准以保持工艺的一致性^[2]。

3.2　建立的新的疫苗体外测定方法必须进行方法学验证^{[15, 16]}

验证主要取决于两方面: ①被测试疫苗的类型; ②分析方法的特定程序。验证指标一般包括但不局限于: 特异性(specificity)、精密度(重复性、中间精密度、重现性)、准确度、线性、范围、耐用性(方法建立时即可进行) 等。方法验证的接受标准应为验证计划的一部分, 应根据测定方法特点和验证目的预先进行设定^[17-19]。

体外测定法不仅应稳健、精确, 还应具备与制造需求相匹配的测试通量。此外, 由于疫苗通常在各国进口时需由监管机构进行测试, 因此测定方法的可转移性也非常重要^[4]。

3.3　疫苗体外测定方法对疫苗效价的改变应具有足够的灵敏度

疫苗体外测定方法应能够检测出可引起人体免疫原性降低的低效疫苗, 即对疫苗效价的降解具有足够的敏感性。基于疫苗生产的现状, 由于良好的生产一致性的存在使得低效疫苗较难获得。因此可采用不同浓度, 或不同类型压力条件下的样品(或不合格产品) 进行检测, 以评估体外方法对样品稳定性的检测能力^{[5, 10, 20, 21]}。其中高温、理化破坏是常用的实验手段, 但需要注意的是: 尽管冷冻过的疫苗在人体内的免疫原性会降低, 但由于佐剂的存在, 其体外效价并不一定会发生明显变化。此外, 对于某些特定疫苗产品, 由于效价过高的疫苗可引起免疫原性增强, 从而在人体中产生不良反应, 因此同样需要被检出^[15]。

3.4　参考品(reference preparation)

参考品是许多疫苗效价测定中的关键组成部分, 其作用是控制可能随时间变化的实验因素, 并在临床证明有效性的疫苗批次和后续商业批次间建立联系。参考品应根据产品特性进行选择, 其中最为关键的一点是: 参考品应尽可能接近所使用的特定效价测定中疫苗样品的反应和特征, 即“同类对比”是其基本原则。因此, 虽然参考品不必与待测疫苗具有相同的制剂或基质制备, 但二者的剂量反应特性应相同。此外, 强烈建议建立一级和二级参考品。一级参考品作为“黄金参考品”, 用于标定后续二级参考品, 一级参考品的存在可更好地避免参考品批次更迭时出现量值传递的错误^[1]。首批参考品建立时应考虑但不限于以下内容^{[15, 16]}: ①来源应具有代表性; ②尽量选用纯度高的标物原料; ③标准物质的数量应充足, 可根据使用频率、稳定性、单支含量、原料获取的难易程度, 以及合理损耗等综合考虑; ④全面表征检测; ⑤如有可能, 同时使用体、内外测定法进行赋值; ⑥均匀性评估; ⑦采用加速降解和实时稳定性实验进行稳定性评估。

技术进步、QbD理念和3R理论的推广将持续使得疫苗生产商和监管机构改进和完善疫苗的检测手段, 从而更好理解和定义疫苗的CQAs。疫苗检测的工具和手段可能会不断发展, 但效价测定在疫苗质量控制中发挥的核心作用必将持续存在。随着科技的发展, 人们对疫苗作用机制了解日臻完善, 疫苗体外效价测定方法有望取代更多的动物体内方法, 甚至可能取代基于细胞的检测。然而, 鉴于疫苗及其相关检测的寿命周期较长, 因此替代之路必会荆棘载途, 疫苗工作者还需砥砺前行。

参考文献

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2025年第60卷第9期

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doi: 10.16438/j.0513-4870.2025-0169

接收时间：2025-02-20
首发时间：2026-09-20
出版时间：2025-09-12

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收稿日期：2025-02-20
修回日期：2025-06-11

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¹中国食品药品检定研究院, 北京 102629

²上海市食品药品检验研究院, 上海 201203

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谭德讲, Tel: 86-10-53851589, E-mail: tandj@nifdc.org.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Characteristics	In vivo method	In vitro method
Sensitivity	High, capable of reflecting the overall immune response in living organisms.	High, capable of detecting subtle changes in specific antigens or antibodies, but limited in simulating complex immune responses.
Specificity	Lower, influenced by individual animal differences and environmental factors.	Higher, capable of precise detection of specific antigens or antibodies with minimal external interference.
Reproducibility	Lower, results are affected by individual animal differences and experimental conditions, leading to higher variability.	Higher, experimental conditions are controllable, results are more stable, and variability is lower.
Experimental duration	Longer, involving animal breeding, vaccination, observation, and other time-consuming steps.	Shorter, simplified experimental procedures, and faster results.
Cost	Higher, involving costs related to animal breeding, experimental operations, and ethical reviews.	Lower, relatively lower costs for materials and equipment, and no expenses related to animal experiments.
Ethical considerations	Involves animal experiments, raising ethical concerns and not aligning with the 3R principles (replacement, reduction, refinement).	No animal experiments required, aligns with the 3R principles, and has fewer ethical concerns.
Applicable scenarios	Suitable for evaluating the overall immune response of vaccines, especially for simulating complex immune reactions.	Suitable for monitoring production consistency, detecting antigen content, and assessing specific immune responses.
Limitations	Results are significantly affected by individual animal differences, making it difficult to precisely control experimental conditions.	Limited ability to simulate complex immune responses, and potential interference in detecting certain vaccines (e.g., adjuvanted vaccines).
Regulatory acceptance	Traditional methods, widely accepted but gradually being replaced by in vitro methods.	Increasingly accepted by regulatory agencies but requires rigorous validation to ensure correlation with in vivo methods.

Characteristics

In vivo method

In vitro method

Sensitivity

High, capable of reflecting the overall immune response in living organisms.

High, capable of detecting subtle changes in specific antigens or antibodies, but limited in simulating complex immune responses.

Specificity

Lower, influenced by individual animal differences and environmental factors.

Higher, capable of precise detection of specific antigens or antibodies with minimal external interference.

Reproducibility

Lower, results are affected by individual animal differences and experimental conditions, leading to higher variability.

Higher, experimental conditions are controllable, results are more stable, and variability is lower.

Experimental duration

Longer, involving animal breeding, vaccination, observation, and other time-consuming steps.

Shorter, simplified experimental procedures, and faster results.

Cost

Higher, involving costs related to animal breeding, experimental operations, and ethical reviews.

Lower, relatively lower costs for materials and equipment, and no expenses related to animal experiments.

Ethical considerations

Involves animal experiments, raising ethical concerns and not aligning with the 3R principles (replacement, reduction, refinement).

No animal experiments required, aligns with the 3R principles, and has fewer ethical concerns.

Applicable scenarios

Suitable for evaluating the overall immune response of vaccines, especially for simulating complex immune reactions.

Suitable for monitoring production consistency, detecting antigen content, and assessing specific immune responses.

Limitations

Results are significantly affected by individual animal differences, making it difficult to precisely control experimental conditions.

Limited ability to simulate complex immune responses, and potential interference in detecting certain vaccines (e.g., adjuvanted vaccines).

Regulatory acceptance

Traditional methods, widely accepted but gradually being replaced by in vitro methods.

Increasingly accepted by regulatory agencies but requires rigorous validation to ensure correlation with in vivo methods.