FDA原文 针对生物制品快速无菌检测方法的研究

快速检测的法规研究----以确保高风险产品安全

美国马里兰州，罗克维尔市，FDA,生物制品评价与研究中心 

快速无菌检测的需求

食品药品监督管理局要求所有注射用生物制品，例如疫苗，使用药典要求的无菌检查法经无菌检测，以确保产品上市安全。该方法是观察细菌、酵母菌和真菌在液体培养基中生长时引发的浑浊度变化，以检测到极少数量的微生物。然而这种方法提供检测结果需要14天时间。两周的时间是对生物制品放行的一个重要限制因素，特别是流行病疫苗和短效期产品。

FDA合规性和生物制品质量办公室（TheOffice of Compliance andBiologics Quality，QCBQ）的质量部门（DPQ）着手研究评价快速微生物检测方法，以缩短生物制品无菌检测时间这一重要问题。

以下内容作为OCBQ专家研究的一部分，比较了几种可替代的快速方法，以及固体和液体培养基微生物检测能力，包括在生物制品中存在有机汞成分作为防腐剂抑制细菌及真菌的情况，以及不存在抑菌剂时的情况。 

比较快速无菌检查法与药典无菌检查法

微生物梯度稀释至10ml冲洗液A中，包括存在或不存在100ppm防腐剂的情况，评价药典无菌检查法（薄膜过滤及直接接种法）和三种快速微生物检测方法，对被稀释微生物的敏感性和检测速度。       

药典无菌检查法

    

使用液体培养基的薄膜过滤法使用液体培养基的直接接种法

 

用于药典无菌检查法的培养基：硫乙醇酸盐流体培养基（FluidThioglycollateMedium,FTM）和胰酪大豆胨液体培养基（TrypticSoy Broth，TSB）

质量部门（DPQ）研究小组根据药典方法选择了快速无菌检查方法，其提供7天的检测时间。研究人员评价了快速方法，及其对多种微生物的生长性能。

Rapid Milliflex^®

●  固体培养基的生长：TSA（大豆胰酪琼脂培养基Tryptic soy agar）,SDA（沙氏葡萄糖琼脂培养基Sabouraud dextrose agar ）,SBA（Schaedler血琼脂培养基Schaedler blood agar）

●  先使用薄膜过滤；薄膜上加入含有ATP-荧光物质的溶剂

●  ATP荧光通过光电成像技术检测菌落；成像后的图片显示在电脑端。培养基上的菌落在肉眼可见以前即可被成像系统识别。

 

Rapid Milliflex^® ATP生物发光成像与固体培养基实际生长的菌落对比

 试验总结

a. 药典方法与Rapid Milliflex^®方法对最低限量的微生物水平呈现同等的敏感性

b.  Rapid Milliflex®方法检测最低限量的痤疮丙酸杆菌，厌氧的普通拟杆菌检出时间为5天，药典方法检出时间为9-10天

c. Rapid Milliflex^®方法检出药典促生长试验要求的菌种1CFU/10ml，检出时间为5天内，通常为（1-3天），与使用10-100CFU/ml的检出时间类似。

d. 固体培养基（TSA,SDA,SBA）比起液体培养基，检出药典促生长试验要求的菌种速度更快。

e. 抑菌剂存在时，使用Rapid Milliflex^® 方法加入ATP-荧光物质后，SBA支持药典促生长试验要求的菌种，而TSA,SDA效果不理想。

更多关于Milliflex^® Rapid微生物无菌检查方法适用性及验证详情，请点击阅读原文咨询

  

Milliflex^®  Rapid微生物快速检测计数系统

• 大多数微生物的检测时间在4～48小时，无菌检查时间在3～5天

• 基于标准化的Milliflex^® 过滤技术

• 创新ATP*生物发光成像系统（*三磷酸腺苷）

• 微生物计数图像分析系统

 

Milliflex^® Rapid微生物快速检测计数系统

Milliflex^® Rapid 快速图像分析

 

Milliflex^®  Rapid微生物快速检测计数系统是一款自动化检测系统，可对生产过程中可过滤样品进行快速微生物无菌检测。借助ATP成像技术，可短时间内对活的、可繁殖的微生物进行检测。

 

FDA原文如下：

SeemaParveen,Simleen Kaur, James L. Kenney, William M. McCormick, Rajesh K. Gupta

Center forBiologics Evaluation and Research, FDA, Rockville, MD

The Needfor Rapid Sterility Test

The Food and DrugAdministration requires that all parenteral biological products undergosterility testing using the compendial sterility method to ensure that productssuch as vaccines are safe when they reach the market. This method is based onthe observation of turbidity in liquid culture media due to the growth ofbacteria, yeast, and fungi and can detect extremely small numbers ofmicroorganisms. However, this method takes 14 days to provide results. Thistwo‐week period can be a significant limiting factor in the timely release ofbiologicals, particular for pandemic vaccines and products with short shelflives. Researchers in the Division of Product Quality (DPQ) in the Office ofCompliance and Biologics Quality (OCBQ) are working to resolve this problem byevaluating rapid microbial methods that significantly reduce the time neededfor sterility testing of biologicals. As part of that effort a team ofresearchers in OCBQ compared the ability of several alternative rapid tests todetect microorganisms in either solid or liquid media in the presence andabsence of thimerosal, the organomercury compound used as an antibacterial andantifungal preservative in certain biological products.

 

ComparingRapid Sterility Methods to the Compendial Sterility Method

Microorganismswere spiked into 10 ml of Fluid A with & without 100 ppm thimerosal, andthen evaluated for detection by Compendial Sterility Methods (Filtration andDirectinoculation method) and Rapid Sterility Methods (Milliflex DetectionSystem, BacT/Alert, BACTEC). Three rapid microbial methods and the compendialsterility method were comparatively evaluated for the sensitivity and speed ofdetection of spiked microorganisms.

 

Media Used in Compendial Sterility Method:

Fluid Thioglycollate Medium and Tryptic Soy Broth

 

The DPQ team developed criteria for an alternate rapid sterility method tobe equivalent in sensitivity and accuracy to the Compendial Method, butprovides results within seven days rather than two weeks. The researchersevaluated the ability of three alternative methods to meet the criteria andcompared the results to the standard compendial sterility method. To comparethe various methods they spiked growth media with various organisms and usedthe methods to detect, isolate and identity the spiked organisms.

 

AlternativeMethods

Rapid Milliflex^® 

      Growth on solidmedia: Tryptic soy agar (TSA), Sabouraud dextrose agar (SDA), and Schaedlerblood agar (SBA) 

•       Inoculum filtered by membrane; membrane sprayed withATP‐releasing and bioluminescence reagents

•       Photons generated ATP bioluminescence are captured anddetected by photon counting imaging tube; picture displayed on computermonitor. Some colonies identified even before visual growth on medium.

Images generated by photons captured by Milliflex

ATP bioluminescencecompared to actual growth

on solidmedia.

 

Major Findings

•       Compendial method, Rapid Milliflex, BacT/Alert, andBACTEC showed equivalent sensitivity at detection of lowest spikedmicroorganism level.

•       Milliflex Detection System detected the lowest spiked of P.acnes and B. vulgatuswithin 5 days versus 9‐10 days for compendialsterility method, BacT/Alert, and BACTEC.

•       Milliflex consistently detected spiked organisms at 1CFU/10 ml within 5 days (usually 1‐3 days). Similar results were obtained at 10and 100 CFU/10 ml.

•       Solid media (TSA, SDA, and SBA) showed faster growth ofspiked organisms (within 5 days) compared to liquid medium.

•       BACTEC and BacT/ALERT did not show growth in experimentsusing matrix containing thimerosal for most of the microorganisms.

•       Unlike TSA and SDA media, SBA supported growth of spikedorganism in the presence of thimerosal and after treatment of organisms withRapid Milliflex ATP‐Bioluminescence reagents.

 

Milliflex Detection System appearsto be a promising alternative to the compendial sterility test for filterablebiological products.