害虫试验:对父母产品中发热反应风险的科学评估

父母用药、生物制品、疫苗、细胞疗法、输血溶液和与血液接触的医疗器械,必须达到最高的安全标准,因为它们直接被输入血液或组织。 这些产品中的多发性污染会给患者造成发作反应,导致严重的临床条件. 肾上腺素检测 对一种产品或材料在体内诱发易发性反应的潜力进行生物评估。 我们的实验室根据 2.6.8级博士 和 USP 第151条 根据GLP原则,与一批有经验的毒理学家合作。

核素试验的科学基础和重要性

皮罗根斯是引起体内发热反应的物质. 这些物质可能包括细菌内分泌毒素(从克拉姆-阴性细菌的细胞壁上释放出脂蛋白),由克拉姆-阳性细菌衍生出细胞壁成分,病毒污染物,真菌衍生物质,颗粒物质,甚至某些化学剂. 发热反应可导致高发热,寒冷,下垂,化粪休克等,在罕见情况下可导致死亡. 因此,仔细评估了脂原污染—特别是静脉注射的产品—这一点至关重要。

肾上腺素测试提供了一种综合安全评估,不仅涵盖已知的内分泌毒素,而且还涵盖非内分泌的肾上腺素。 在这方面,它与LAL(Limulus Amebocyte Lysate)测试有着根本的不同,该测试只能检测出克氏阴性细菌内分泌. 一些生物制品、细胞疗法和复合制剂可能含有脂原素,而脂原素试验无法检测;因此,脂原素试验对这些产品是不可或缺的。

蛋白质检测的重要性也源于监管要求. 药典专著明确规定具体产品必须进行何种检验. 例如,虽然对生物产品、疫苗和细胞疗法一般必须进行脂原测试,但LAL测试可能足以提供简单的化学配方和输液解决方案。 然而,根据产品的性质和制造工艺,可能有必要进行两次试验。 全氟辛烷磺酰胺检测结果直接用于产品放出,监管审批流程,患者安全.

Comparative Analysis of Ph. Eur. 2.6.8 and USP <151> Standards

2.6.8级博士 (欧洲药典)和 USP <151> (美国药典)是两种国际公认的主要参考物,用于蛋白质检测。 这两项标准都采用了相同的基本方法:在兔子模型中测量静脉注射后直肠温度的增加. 然而,实验设计和解释标准存在一些显著差异.

在这两项标准中,测试都是对健康的成年兔子(最好是新西兰白兔)进行的. 最初使用三只兔子;如果结果无定论,则再用另外5只兔子(共8只兔子)进行试验. 试验物质通过耳静脉静脉注射;注射体积一般为每公斤10毫升,不过在产品专用专著中可能规定出不同的体积. 温度是直取测量的;在注射前后的特定间隔记录读取(一般每30分钟3小时).

两种标准的初步温度标准相类似:个体兔的初始温度必须在38.0至39.8°C之间,而动物之间的差别不得超过一等. 然而,接受和拒绝标准存在差异。 根据Ph. Eur. 2.6.8,如果三只兔子的总气温升高为1.15°C或更低,则该产品被视为无脂;如果总增量超过2.65°C,则该产品含有脂激素并被测试视为失败. 这两种值之间的结果被认为值得怀疑,再用另外5只兔子重复测试. 根据USP 第151节,接受标准为1.4°C,拒绝标准为2.8°C. 在"八兔"试验中,接受标准为:"博士"为 2.80°C,"USP"为 3.7°C.

The table below provides a comparative summary of the key characteristics of the Ph. Eur. 2.6.8 and USP <151> standards.

P基测试和LAL测试之间的密钥差异

皮热测试和LAL测试是两种评价方法,它们常常被混淆,但根本不同. LAL测试是 试管 仅能检测克氏阴性细菌内分泌毒素(利波利沙克查里德)的方法。 这一测试所依据的原则是:从马蹄蟹(英语:Limulus polyphemus)的血细胞中得到的利沙酸盐在出现内分泌毒素时表现出了凝血反应. LAL测试是快速的,经济的,并提供定量结果;然而,它无法检测出非内分泌的活性激素.

另一方面,色素测试是 阴道 方法,涵盖所有种类的花生。 利用这项试验可以检测出细菌内分泌毒素、格拉克阳性细菌产品、病毒污染物、真菌衍生物质、颗粒和化学多肽。 因此,对蛋白质的检测是强制性的,特别是对生物产品、疫苗、细胞疗法和复杂制剂。 然而,脂原测试需要使用动物,价格更昂贵,耗时更长,不能提供定量结果(它只产生定性结果,表明脂原的存在或不存在).

选择使用哪种测试取决于产品的性质以及药典专著中规定的要求. 在某些情况下,可能有必要同时进行这两项试验。 例如,当需要评估生物产品的内分泌限值和总的多活性特征时,补充使用LAL和多活性素试验。

单体活性试验(MAT):一种现代替代品

欧洲药典接受 单体活性测试(MAT) 作为某些条件下的多糖性试验的替代品。 MAT是一个 试管 试验,以测量人类单细胞在活性物质反应下生产细胞样(特别是IL-1β、IL-6和TNF-α)。 这种方法覆盖了所有种类的花生,并消除了动物检测的需要. MAT被视为一种有希望的替代品,特别是生物产品和细胞疗法。

However, product-specific validation and regulatory approval may be required for MAT to replace the pyrogen test. The suitability of MAT for each product must be evaluated based on the product’s composition, manufacturing process, and potential sources of pyrogens. Our laboratory offers MAT as an alternative to pyrogen testing and provides scientific consultation to determine the most appropriate testing strategy for your product.

Which Products Require Pyrogen Test?

Pyrogen testing is required for all products administered directly into the bloodstream or tissues that carry a risk of pyrogen contamination. Pyrogen testing is mandatory for parenteral drugs (injectable solutions, infusion fluids, intravenous drugs) because these products are administered directly into the bloodstream, and pyrogen contamination poses a life-threatening risk. Pyrogen testing is also mandatory for biological products (monoclonal antibodies, recombinant proteins, enzymes, hormones); the risk of pyrogen contamination is high in biological manufacturing processes, and the LAL test cannot detect all pyrogens.

Pyrogen testing is mandatory for vaccines (inactivated vaccines, toxoid vaccines, recombinant vaccines); since vaccines directly stimulate the immune system, the risk of inducing a pyrogenic response is high. Pyrogen testing is also mandatory for cell and gene therapies (CAR-T cells, mesenchymal stem cells, viral vectors); complex biological matrices can be a source of non-endotoxin pyrogens. Pyrogen testing may be required for medical devices (implants, catheters, dialysis machines, blood bags); in accordance with ISO 10993-11, device extracts may need to be evaluated for pyrogenicity.

Pyrogen testing is mandatory for dialysis and infusion solutions (peritoneal dialysis fluids, hemodialysis solutions, Ringer’s solutions); pyrogen limits are strictly controlled in products administered parenterally in large volumes. Pyrogen testing may also be required for certain topical products (ophthalmic solutions, certain wound care products); pyrogen control may be necessary for products applied to sensitive tissues such as the eyes and open wounds.

Conducting Pyrogen Tests in Our Laboratory

In our GLP-certified laboratory, we conduct pyrogen tests in accordance with Ph. Eur. 2.6.8 and USP <151> in full compliance with international standards. Our testing process begins with a comprehensive preliminary evaluation. The physicochemical properties of the test substance, available toxicity data, and the requirements in the pharmacopoeia monograph are reviewed. Based on this information, the most appropriate test protocol and test volume are determined.

Healthy adult New Zealand White rabbits are used for the test. A pre-test acclimatization period of at least seven days is implemented; this period allows the animals to adjust to laboratory conditions and minimizes stress factors. Rabbits weighing at least 1.5 kg are selected; their initial body temperatures should be between 38.0 and 39.8°C, and the difference between animals should not exceed one degree. Animals that have not been used in pyrogen testing within the past two weeks are preferred.

The temperature and humidity in the test chamber are maintained at constant levels (temperature: 20–24°C; humidity: 40–60%); noise and stress factors are minimized. The test substance is prepared as specified in the product monograph; all solvents, syringes, and equipment used are depyrogenized in advance. Injection is administered slowly intravenously via the ear vein; aseptic conditions are strictly maintained. Temperature measurements are taken using thermometers with calibration certificates or telemetry systems; measurement accuracy is ±0.1°C.

The observation period lasts at least three hours after injection; temperature is recorded every thirty minutes. The maximum increase relative to the baseline temperature is calculated and interpreted according to pharmacopoeia criteria. Veterinary supervision is provided during and after the test; appropriate intervention is performed in the event of a severe reaction. After the test, the animals are allowed to rest; a minimum of forty-eight hours is waited before reuse, and it is verified that the previous response was within acceptable limits.

One of our laboratory’s key advantages is that pyrogen test results can be directly integrated into your regulatory submissions. Complete study reports compliant with pharmacopeia templates meet the requirements of EU, U.S., and other international regulatory authorities. Additionally, by offering complementary and alternative methods such as the LAL test and the Monocyte Activation Test (MAT), we provide the most comprehensive pyrogen safety assessment for your product.

常见问题

One of the most common questions we receive regarding pyrogen testing is which standard to choose between Ph. Eur. 2.6.8 and USP <151>. The choice depends on your product’s target market. While the Ph. Eur. standard is used as a reference in the European Union and many international markets, the USP standard applies in the United States and certain other regions. For global markets, a testing strategy compliant with both standards is recommended.

We are also frequently asked whether the LAL test can serve as a substitute for pyrogen testing. For some products, yes; for others, no. Pharmacopoeial monographs specify which test is mandatory for specific products. As a general rule, the LAL test may be sufficient only for simple chemical formulations that carry a risk of endotoxins; the pyrogen test is required for biological products and complex matrices.

There is also curiosity about whether the Monocyte Activation Test (MAT) can serve as an alternative to the pyrogen test. Yes; the European Pharmacopoeia accepts the MAT as an alternative to the pyrogen test under certain conditions. The MAT measures cytokine production by human monocyte cells in response to pyrogenic substances and covers all classes of pyrogens. However, product-specific validation and regulatory approval may be required. Regarding the test duration, the test itself takes one to two days; however, including animal acclimatization, pre-screening, and reporting, the total duration is typically two to three weeks.

Pyrogen testing is one of the cornerstones of the safety of parenteral products and medical devices. With our tests fully compliant with Ph. Eur. 2.6.8 and USP <151> standards, we evaluate your products’ potential to induce a febrile response with the highest scientific rigor. Our GLP-certified laboratory, with its experienced staff and state-of-the-art infrastructure, is your reliable partner for pyrogen testing needs. To demonstrate your product’s pyrogen safety, contact us.