Application Note
Microplate-based endotoxin testing in GLP/GMP environments using a colorimetric assay
- Sensitive and specific method for detecting endotoxin contamination in the production process
- Automatic interpolation of endotoxin concentrations of unknowns from the standard curve based on onset times
- Complete solution for data acquisition and analysis
Introduction
Monitoring for contaminants is a critical step during the production process in the pharmaceutical and medical device industries. A frequent contaminant, endotoxin, can cause fever, inflammation, headache, nausea, and even death. Found in the cell wall of gram negative bacteria, endotoxin has routinely been detected by the sensitive and specific Limulus Amebocyte Lysate (LAL) assay. In the presence of endotoxins, LAL coagulates through an enzyme-mediated cascade, which has then been traditionally quantified based on gelation and turbidity.
The Pyrochrome® Assay from Associates of Cape Cod, Inc. (ACC) contains a synthetic substrate with paranitroaniline chromogen (pNA), which enables spectrophotometric quantitation. Clotting enzyme is (indirectly) activated by endotoxin. The enzyme cleaves the substrate, liberating the pNA, which absorbs light at 405 nm. The absorbance (A405) can be measured by the plate reader and is proportional to the endotoxin concentration in the sample.
Maintaining data integrity is a high priority for companies using instruments/ software to capture and store data in environments striving to meet regulatory requirements, including FDA 21 CFR Part 11. Transferring data among multiple software applications for collection, analysis, and presentation can introduce errors and complicate traceability. Additional overhead is also created in supporting, validating, and training personnel on multiple applications.
For validation of the Molecular Devices VersaMax™ Absorbance Microplate Reader for use with the Pyrochrome Assay, data acquisition and analysis were performed with SoftMax® Pro GxP Software.
Preconfigured method protocols specifically written for the Pyrochrome Assay and other ACC assays are available for SoftMax Pro Software and take advantage of built-in analytical tools for automating pass/fail criteria. Additionally, features of SoftMax Pro GxP Software support GLP/GMP guidelines by including user-assigned permission levels to read data, modify settings and formulas, and electronically sign files.
This application note describes a typical setup for a standard curve in the Pyrochrome Assay. The chromogenic test was conducted using a VersaMax Absorbance Microplate Reader. Data were analyzed and graphed using SoftMax Pro GxP Software.
Materials
- VersaMax Microplate Reader (Molecular Devices cat. #VERSAMAX)
- SoftMax Pro GxP Software version 5.4 (Molecular Devices cat. # SMP54-GXP- 10UL)
- Pyrochrome Assay chromogenic test reagent (ACC cat. #C1500)
- Control standard endotoxin (ACC cat. #E010)
- Pyroplate 96-well microplate (ACC cat. #CA961)
Methods
Before dispensing reagents to the microplate, the VersaMax Reader was powered on and SoftMax Pro GxP Software launched.
After selecting the “Pyrochrome Kinetic” method protocol from the drop-down menus in SoftMax Pro GxP Software, a new data file was created onscreen.
- Test and reagent information can be entered at any time in the designated Notes sections. Entering text and modifying formulas are subject to assigned user permissions.
- With appropriate user permissions, the Plate Template assigning the samples to the plate layout may also be modified at any time to reflect the proper orientation within the plate. Settings and calculations in SoftMax Pro GxP Software cannot be accessed unless the corresponding permission is assigned to a user.
The incubator temperature was set to 37°C.
The Pyrochrome Assay reagent was reconstituted with 3.2 mL reconstitution buffer, mixing with a gentle swirling motion to avoid excessive foaming and decrease in sensitivity.
A series of dilutions from the control standard endotoxin solution were prepared in LAL reagent water. The final concentrations used to generate a standard and curve were 50, 5, 0.5, 0.05, and 0.005 EU/mL. 50 µL from each dilution series was transferred in triplicate to the 96-well Pyroplate® microplate, which is certified free of interfering endotoxin and glucan contamination.
- A negative control of 50 µL LAL reagent water was added to the plate in triplicate.
- 50 µL of reconstituted Pyrochrome Assay reagent was added to each well as rapidly as possible (optionally with a repeating pipette).
Note: Alternatively, 100 µL of each standard/control can be used while maintaining the 1:1 standard:lysate ratio.
The microplate was immediately placed in the microplate drawer, and the measurement process initiated by clicking on the “Read” button in the SoftMax Pro GxP Software. The software then automatically performed the following:
- Prior to the read, a proprietary automix motion mixed the contents of each well thoroughly without splashing.
- Absorbance data at 405 nm were acquired every 10–15 seconds for one hour for maximum sensitivity and displayed in real time. The interval and run times can be adjusted as per recommended incubation time for a specific reagent lot number.
- An automatic data reduction was applied to calculate the onset time in each well, i.e., time for each well to reach a specified optical density (OD) threshold. The onset OD specified was set to 0.03 but can be modified.
- A log-log standard curve was constructed using obtained onset times for concentrations of at least 3 standards (as per USP/FDA).
Note: Well assignments must be made through the plate template for the graph to auto-populate.
- The slope, y-intercept, and correlation coefficient were reported in the Graph legend and Summary Results section.
Results
To demonstrate the suitability of the VersaMax Microplate Reader to perform the Pyrochrome Assay, the resulting standard curve was produced by plotting the log of the mean onset time in seconds against the log of the concentration (Figure 1).
Figure 1. Pyrochrome assay standard curve. Pyrochrome Assay log-log standard curve generated in SoftMax Pro GxP Software version 5.3. Standard curve was composed of five dilutions of control standard endotoxin in triplicate.
The standard curve is noteable in three respects: the high r2 coefficient (0.992), small standard deviations for replicates (y-error bars), and the capability of the VersaMax Reader to reach the stated maximum sensitivity of the Pyrochrome Assay (0.005 EU/mL).
The method protocol of SoftMax Pro GxP software was also designed to automatically flag results used to determine the individual assay validity:
- The absolute value of the correlation coefficient |r| should be at least 0.980.
- The positive product control should be within 50–200% of the nominal concentration of the equivalent standard.
- The endotoxin concentration of negative controls (as determined by the extrapolation of the standard curve) should be considerably lower than that of the lowest standard concentration.
Conclusion
The generation of a linear standard curve with low variance between replicates and sensitivity down to the assay’s stated lower limit of detection demonstrates the performance of the VersaMax Microplate Reader and Pyrochrome Assay for detecting endotoxin in test samples. As a result of colorimetric changes, SoftMax Pro GxP Software automatically interpolates the endotoxin concentrations of unknowns from the standard curve based on the onset times. Interpolations can be restricted to the range of onset mean times for the standard curve.
While all work carried out in the data file of SoftMax Pro GxP Software is audit trailed, after the results have been reviewed, the file can be locked against further changes regardless of assigned permissions by electronically signing the file. Multiple signature fields are available to indicate various roles, such as reviewer and approver.
The Pyrochrome Assay together with the VersaMax Microplate Reader offers a sensitive and specific method for detecting endotoxin contamination in the production process. The all-in-one data acquisition and analysis of SoftMax Pro GxP Software for use with the VersaMax Reader minimizes the risks and costs associated with transferring data between multiple software packages for analysis and reporting.
Additional features in SoftMax Pro GxP Software for assisting in GLP/GMP compliance under regulations such as FDA 21 CFR Part 11 for electronic records, maintain the integrity of data by controlling the permissions of authorized users and establishing traceability for significant actions.
简介
污染物监测是制药和医疗器械行业生产过 程中的关键环节。其中一种较常见的污染 物—内毒素，它可以引起发烧、炎症、头 痛、恶心，甚至死亡。内毒素是在革兰氏 阴性细菌的细胞壁中被发现的，目前可以 利用敏感的、特异性的鲎试剂 ( LAL ) 法对 其进行检测。
在内毒素存在的情况下，LAL 通过酶介导 的级联反应产生的凝聚现象，传统方式是 我们可利用其凝聚产生的浑浊度变化对其 进行定量检测。
Pyrochrome 检测试剂是美国科德角公司 (ACC) 推出的一款内毒素定量检测产品， 其组成含有对硝基苯胺 (pNA) 的合成底物， 由于颜色变化后可用于吸光度检测，凝集 化酶是由内毒素 ( 间接 ) 激活的酶裂解底 物，释放 pNA，其在 405 nm 处有很强的 光吸收值，我们在微孔板读板机上进行检 测，OD值高低与样品中的内毒素浓度成正 比。对于企业来说使用仪器和软件必须 在相应的合规环境来获取和存储数据， 维护数据完整性是一项非常重要和迫切的 任务，符合 FDA 21 CFR Part11 法规要 求环境，如果使用多个软件用于数据的 收集、分析并进行数据传输的话可能会 导致潜在错误，并让可追溯性变的复 杂。此外还额外产生应用支持、多个应 用程序上验证和培训人员的费用等。
使用经过验证 Molecular Devices 的 Versa Max™ 光吸收读板机和 Pyrochrome 检测 试剂进行相应检测，结合 SoftMax®Pro GxP 合规软件进行数据采集和分析。
SoftMax Pro 软件内置针对该内毒素检测 试剂盒的检测模板，无需设置，直接读数 后自动化获得数据并判断结果是否通过。 此外，SoftMax Pro GxP 合规软件符合 GLP/GMP 指南要求，针对不同用户之间 对软件进行的设置、公式修改、数据生 成、电子签名文件生成都有明确分级管理 的功能。
本篇应用介绍了如何针对该试剂盒在 VersaMax 光吸收酶标仪上进行参数设置、 SoftMax Pro GxP 软件上获取标准曲线并 绘制曲线图表过程。
材料
- VersaMax 微孔板读板机 (Molecular Devices cat. #VERSAMAX)
- SoftMax Pro GxP 软件 (Molecular Devices cat. # SMP54-GXP-10UL)
- Pyrochrome 检测试剂盒 (ACC cat. #C1500)
- 内毒素质控标准品 (ACC cat. #E010)
- Pyroplate 96 孔板 (ACC cat. #CA961)
方法
首先打开 VersaMax 读板机并在电脑端登 陆 SoftMax Pro GxP 软件，然后在微孔板 中加入试剂。
随后在 SofMax Pro GxP 软件的模板文件 菜单中选择“Pyrochrome Kinetic”检测 方法，可以在电脑上看见新的数据类型文 件被创建。
检测方法和试剂信息可随时输入软件内置的记事本中，当然进行相应操作用户必须具有输入文本和修改公式的权限。
当具有相应的用户权限时，则可以编辑试 剂样品真实的放置在微孔板区域及梯度稀 释关系，我们将其分配到微孔板任何区域 并 可 以 随 时 进 行 修 改 。 除 非 用 户 具 有 SoftMax Pro GxP 软件的设置和计算功能 权限，否则将无法进行相应操作。将仪器 内部温度设置至 37 摄氏度。我们使用 3.2 ml 溶解缓冲液对 Pyrochrome 试剂进行 充分溶解，均匀轻度混匀以避免产生大量 泡面降低检测灵敏度。
我们使用 LAL 试剂专用水对标准品内毒素 溶液进行一系列梯度稀释，用于制作标准 品曲线溶液的最终的浓度是 50、5、0.5、 0.05 和 0.005 EU/ml，每个浓度下的标准 品溶液制备三个重复以 50 μl 体积加入值 微孔板中，当然要确保整个反应体系中不 存在内毒素和葡聚糖污染物的干扰。
- 50 μl LAL 试剂专用水溶液以三个复孔的 方式加入微孔板中作为阴性对照
- 50 μl 重悬 Pyrochrome 检测试剂快速 加入微孔板的每个孔中
注：或者每个标准品/质控品 1:1 比率关 系，100 μl 总体积：裂解液的比率
我们将相应的微孔板迅速放入微孔板读板 机中，然后点击 SoftMax Pro GxP，界面 正上方绿色的“Read”按键进行检测。软 件程序会执行相应设置。
- 在进行检测以前，程序步骤会要求仪器进行适当强度的自动混匀，确保液体混合均匀且不飞溅出孔外。
- 405 nm 处的吸光度值，我们每隔 10-15 秒收集一次数据，持续 1 小时，并实时 显示最大灵敏度，间隔时间和总检测时 间可以根据不同批次的试剂所推荐时间 进行相应的调整。
- 可以设置每个孔达到某一指定 OD 值的 时间，软件可以自动计算并显示出到达 相应OD值的时间，这个模板内置的是 达到 0.03 OD 值的时间点，当然可以根 据实际需求进行更改。
- 使用log-log方式进行标准曲线进行拟合数据，至少需要三个数据点。
注：在进行自动计算和曲线拟合前必须对相应孔进行相应定义。
- 斜率、y 轴截距和相关系数见图例和总 结结果的部分。
结果
获得的结果说明 VersaMax 微孔板读板机 可用于 Pyrochrome 试剂进行相应检测， 将平均达到 OD 值时间 ( 秒 ) 的均值与已知 浓度以 log-log 拟合方式作图，得到标准 曲线。( 图 1 )
***图 1 Pyrochrome 试剂制作标准品曲线。*此曲线为 SoftMax Pro Gxp 软件自动拟合生成，曲 线是由 5 个浓度标准样品在每个浓度 3 复孔下计算获得。
The standard curve is noteable in three respects: the high r2 coefficient (0.992), small standard deviations for replicates (y-error bars), and the capability of the VersaMax Reader to reach the stated maximum sensitivity of the Pyrochrome Assay (0.005 EU/mL).
内置于 SoftMax Pro GxP 软件的检测模 板，可用于自动确定样品的分析数值和判 断结果的有效性。
- 相关系数 R^2 值至少高于 0.980
- 阳性质控样品应在等效标准标品浓度的 50-200% 之间
- 内毒素浓度阴性质控样品 ( 由标准曲线 外推法确定 )，应大大低于最低标准浓度
结论
我们获得一条线性标准曲线，发现曲线的 复孔之间的 CV 值很小，结果反映出其检 测灵敏度很高，发现可以使用 VersaMax 和 Pyrochrome 试剂进行内毒素的检测和 分析，并证明其检测优异性能。借助于颜 色随着时间的变化，达到某一个制定 OD 值的时间，那么 SoftMax Pro GxP 软件可 通过标准品曲线自动计算出内毒素浓度， 可以限制在标准曲线的起始平均时间范围 内。SoftMax Pro GxP 软件对数据文件中 的所有工作流程及结果进行了相应的审计 追踪。在检查完结果之后，可以通过对文 件进行电子签名来锁定文件，防止发生进 一步的更改，而不管所分配的权限如何。 可以使用多个签名来指示各种角色，例如 审阅人和审批人等。
本文中通过 VersaMax 微孔板读板机和 Pyrochrome 试剂获得高灵敏和特异性检 测结果。使用了 SoftMax Pro GxP 软件的 VersaMax 微孔板读板机可进行数据采集和 分析，大大减低了在多个软件之间进行数 据传输、分析、生成报告等所产生潜在风 险和成本。
SoftMax Pro GxP 软件符合 GLP/GMP 法 规 ( 如 FDA 21 CFR Part 11 的电子记录、 审计追踪 ) 的要求，通过控制授权用户的 权限和建立重要操作的可追溯性来维护数 据的完整性。