大环内酯酶

1. 概论 大环内酯酶是采用重组基因构建质粒的水溶性蛋白。分子量30.5KD，等电点5.84。根据大环内酯类药物（阿奇霉素、罗红霉素）的化学结构而构建的重组蛋白，目标蛋白纯度＞95%，其基因片断的活性中心，可破坏大环内酯类 药物的结构式中关键大环结构使得开环后失去抗菌性。 2. 应用 可以对注射用阿奇霉素、罗红霉素及口服制剂的抗菌性中和（灭活、破坏），从而使得无菌检验的阳性菌生长良好；使得微生物限度检查的阳性菌回收率 大于70%，确保检验工作的准确性。 做抗生素的无菌检查时，应用手工注射器吸取无菌水注入药物的西林瓶中，摇匀，溶解，再吸出。加入至0.9%氯化钠的500ml溶液中，摇匀，不可用培养器上的针头溶解，输入，避免高浓度的溶液通过滤膜，造成难以冲洗彻底。 这里，再次强调：做抗生素的无菌检查时，一定要手工注射器注入无菌水 溶解样品，再把溶解的样品转移至0.9%氯化钠的500ml溶液中摇匀，使得样品不会造成局部浓度过高，通过滤膜时难以冲洗彻底的情况。 3. 操作 无菌检验时，取冻干粉的酶加入无菌水5ml，摇匀，制成大环内酯酶的 稀释液，取2ml酶的稀释液放入1500ml的冲洗液摇匀。待冲洗液冲洗培养器的滤膜完成后，泵尽。用手工注射器在三个培养器的呼吸口刺入，各加1ml的 稀释酶，尽量让酶平铺在滤膜整个面，静置10分钟，使高浓度的酶与培养器 滤膜充分接触，以中和（灭活、破坏）滤膜上残留的大环内酯类药物，然后再 泵入相应的培养基，摇匀。阳性对照管加入1ml的对应试验菌。 4.微生物限度检查 取冻干粉的酶，加入无菌水2ml溶解，摇匀，制成大环内酯酶的稀释液。取1ml加入至500ml的冲洗液，摇匀。先湿润滤膜，把溶解后的样品溶液通过滤膜，依次用含有大环内酯酶的蛋白胨冲洗液，少量多次冲洗，尽量在滤膜上不积液。泵尽，将1ml的大环内酯酶加入过滤杯，湿润整个滤膜20分钟，让酶直接中和（灭活、破坏）滤膜上残留的微量大环内酯类药物，泵尽，把滤膜正面朝上放在平皿上并计算回收率。 5.验证 在3和4的工作前，须经使用单位的质量部门进行验证工作，并保存验证 数据。根据结果编写验证的标准SOP。 6.灭活试验 把抗生素的抗菌性去除，可称为中和（灭活、破坏）。 取20mg/ml的阿奇霉素、罗红霉素溶液1ml，置灭菌空试管，加1ml的 大环内酯酶0.2mg/ml，摇匀，在37℃水浴放置20分钟，加入胰酪大豆胨培养基25ml，振摇，再加入金黄色葡萄球菌（10的负6或100CFu/ml）的1ml，摇匀。放37℃培养5天，每天振摇试管，观察细菌生长情况。 另取上述不加1ml的大环内酯酶，同样操作，为阴性对照。 阳性管细菌应在5天内生长良好，阴性管应无菌生长。 7.法规要求 按中国药典规定：检验方法需进行方法学的验证，对抗生素药物可采用中和（灭活、破坏）去除其抗菌性，以确保检验方法确实可行，检验结果的准确度。大环内酯酶是 根据药典附录的无菌检查法 原则和抗生素实际特点而制备。 针对大环内酯类的药物有效。各制药单位再结合大环内酯类药物的产品进行方法学验证，完全符合法规要求。 8.产品的包装规格 大环内酯酶采用西林瓶、胶塞、铝盖包装，属无菌冻干剂蛋白。20单位/瓶，1mg/瓶。 9.含量测定 大环内酯酶的含量测定采用高效液相色谱的外标法计算，具有专属性强、 方法先进、科学、数据重现性好、准确。 1. Introduction Macrolidenase is a water-soluble protein that uses recombinant genes to construct plasmids. The molecular weight is 30.5KD and the isoelectric point is 5.84. The recombinant protein constructed according to the chemical structure of macrolides (azithromycin, roxithromycin), the purity of the target protein is > 95%, and the active center of its gene fragment can destroy the key macrocyclic structure in the structural formula of macrolides, resulting in the loss of antibacterial properties after ring opening. 2. Application It can neutralize (inactivate, destroy) the antibacterial properties of azithromycin, roxithromycin for injection and oral preparations, so that the positive bacteria of sterility test can grow well; The recovery rate of positive bacteria in the microbial limit test is greater than 70% to ensure the accuracy of the test work. When doing sterility check of antibiotics, use a manual syringe to absorb sterile water and inject it into the drug vial, shake well, dissolve, and then suck it out. Add to 500ml solution of 0.9% sodium chloride, shake well, do not dissolve with the needle on the incubator, input, avoid high concentration of solution through the filter membrane, resulting in difficult to rinse thoroughly. Here, it is emphasized again: when doing sterile examination of antibiotics, it is necessary to inject sterile water into the manual syringe to dissolve the sample, and then transfer the dissolved sample to 500ml solution of 0.9% sodium chloride, so that the sample will not cause local concentration too high, difficult to wash thoroughly through the filter membrane. 3. Operation During sterility test, take the enzyme of freeze-dried powder and add 5ml of sterile water, shake well to make the diluent of macrolisterase, take 2ml of the diluent of enzyme and add 1500ml of rinsing solution and shake well. After the rinse solution has washed the filter membrane of the incubator, the pump is exhausted. A manual syringe was used to Pierce the respiratory mouth of the three incubators, and 1ml of diluent enzyme was added to each of them, and the enzyme was spread on the entire surface of the filter membrane as far as possible. The enzyme was left in full contact with the filter membrane of the incubator for 10 minutes, so as to neutralize (inactivate or destroy) the residual macrolides on the filter membrane, and then pumped into the corresponding medium and shook well. Positive pairs were treated with 1ml of corresponding test bacteria. 4. Microbiological limit test Take the enzyme of freeze-dried powder, add 2ml sterile water to dissolve, shake well to make the diluent of macrolide enzyme. Add 1ml to 500ml of rinse and shake well. Wet the filter membrane first, pass the dissolved sample solution through the filter membrane, and rinse with peptone containing macrolidenase successively, a small amount of multiple times, and try not to accumulate liquid on the filter membrane. After the pump is finished, 1ml of macrolide enzyme is added to the filter cup, and the whole filter membrane is wet for 20 minutes, so that the enzyme directly neutralizes (inactivates and destroys) the trace macrolides remaining on the filter membrane. After the pump is finished, the filter membrane is placed face up on the plate and the recovery rate is calculated. 5. Verify Before the work of 3 and 4, the quality department of the user unit shall carry out the verification work and save the verification data. Write a standard SOP for validation based on the results. 6. Inactivation test Removing the antibacterial properties of antibiotics can be called neutralization (inactivation, destruction). Take 1ml of azithromycin and roxithromycin solution of 20mg/ml, place in sterilized empty test tube, add 1ml of macrolisterase 0.2mg/ml, shake well, place in 37℃ water bath for 20 minutes, add 25ml of soy peptone medium with buttermilk, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, shake, and shake. Add another 1ml of Staphylococcus aureus (10 minus 6 or 100CFu/ml) and shake well. Culture at 37℃ for 5 days, shake the test tube every day, and observe the growth of bacteria. The above macrolidenase without adding 1ml was also taken as a negative control.The positive tube bacteria should grow well within 5 days, and the negative tube should grow sterile. 7. Regulatory requirements According to the provisions of the Chinese Pharmacopoeia, the test method needs to be verified by methodology, and the antibiotic drugs can be neutralized (inactivated, destroyed) to remove their antibacterial properties, so as to ensure the feasibility of the test method and the accuracy of the test results. Macrolidenase is prepared according to the principles of sterility test in the pharmacopoeia appendix and the actual characteristics of antibiotics. Drugs that target macrolides are effective. The pharmaceutical units combined with the products of macrolides to conduct methodological verification, which fully met the regulatory requirements. 8. Packaging specifications of the product Macrolidenase is packaged with cillin bottle, rubber plug and aluminum cap, which is a sterile lyophilized protein. 20 units/bottle, 1mg/ bottle. 9. Content determination The determination of macrolidenase is calculated by external standard method of high performance liquid chromatography, which has strong specificity, advanced method, scientific, good data reproducibility and accuracy.

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