Endotoxin Detection Systems. Systematic Approaches to Quantitative Methods

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1 Systematic Approaches to Quantitative Methods

2 Quantitative Endotoxin Detection Systems deliver accurate results up to a 4-log concentration range. Quantitative systems are amazingly easy to use: simply add the sample and reagent, push a button and the system does the rest. Using our broad knowledge of endotoxin detection and regulatory requirements, Lonza has developed an integrated system to support quantitative endotoxin detection. Each system component has been validated and can be verified. This assures you of reliable, reproducible and accurate quantitative results. One Source, One Solution Each quantitative system incorporates 3 basic elements: Quantitative Endotoxin Detection Kits WinKQCL Endotoxin Detection Software Validated Incubating Microplate Reader These elements integrate seamlessly to meet your testing requirements. Whether you are releasing product for shipment, checking raw materials such as WFI or testing for endotoxin at the point of use such as in dialysis, our products provide you with the information, confidence and consistency to make critical decisions. 2

3 State-of-the-Art Kinetic LAL Kits: Turbidimetric and Chromogenic Lonza offers a range of endotoxin testing options to fit your requirements. The kinetic Limulus Ambocyte Lysate (LAL) assays, including our hallmark Kinetic- QCL product, can lead the way toward peak endotoxin testing efficiency and productivity. PYROGENT is a quantitative kinetic assay used for the detection of Gramnegative bacterial endotoxin. A sample is mixed with the reconstituted LAL reagent, placed in the reader and automatically monitored over time for the appearance of turbidity. The time required before the appearance of turbidity (Reaction Time) is inversely proportional to the amount of endotoxin present. In the presence of a large amount of endotoxin, the reaction occurs rapidly. The concentration of endotoxin in unknown samples can be calculated from a standard curve. PYROGENT Source LAL LAL Kinetic-QCL Data Quantitative Quantitative Type Kinetic Turbidimetric Kinetic Chromogenic Format 96-well plates, incubated 96-well plates, incubated Standards Usually 3 to 5 points Usually 3 to 5 points run in duplicate run in duplicate Negative Controls LAL Reagent Water LAL Reagent Water in duplicate in duplicate Assay Controls One set of spiked samples One set of spiked samples in duplicate per sample in duplicate per sample Range 0.01 EU/ml EU/ml EU/ml - 50 EU/ml Assay Time Approx. 1 hour Approx. 1 hour Temperature 37º C +/- 1º C 37º C +/- 1º C Instrumentation UV-Vis plate reader UV-Vis plate reader Detection Wavelength 340 nm 405 nm Software WinKQCL WinKQCL Kinetic-QCL is a quantitative kinetic assay monitored over time for the appearance of a yellow color. Based on the same mechanism as PYROGENT -5000, the time required before the color change (Reaction Time) is inversely proportional to the amount of endotoxin present. The concentration of the endotoxin in unknown samples is calculated from a standard curve. The PYROGENT Turbidimetric Kit is available in 100 Test (N383) and 200 Test (N384) kits. Each contains lysate, reconstitution buffer and endotoxin for preparing standard dilutions. The Kinetic-QCL Kit (50-650U) includes co-lyophilized lysate/substrate reagent, endotoxin for preparing standard dilutions and LAL Reagent Water sufficient to perform 192 tests. 3

4 The Future of Endotoxin Detection Systems PyroGene The WinKQCL Endotoxin Detection Software PyroGene is a new method for quantitative endotoxin detection based on the activation of Recombinant Factor C (rfc), the endotoxin sensitive protein that initiates the traditional Limulus Amebocyte Lysate (LAL) cascade. Benefits of PyroGene No animal utilization. PyroGene uses recombinant technology and does not rely on the horseshoe crab for its active ingredient. Greater endotoxin specificity. PyroGene is activated only by endotoxin, thus eliminating falsepositive reactions due to glucans. Less lot-to-lot variability. Genetic engineering allows far more control over the manufacturing process leading to greater lot-to-lot consistency. PyroGene works through a single-step end-point assay that measures the enzymatic cleavage of a fluorogenic substrate. The reaction is run in a 96- well microplate and measured at time zero and after a one-hour incubation in a fluorescence reader using WinKQCL. PyroGene represents a reliable, environmentally friendly alternative to LAL. The PyroGene Kit (50-658U) includes rfc enzyme, buffer, fluorogenic substrate, endotoxin for preparing standard dilutions and LAL Reagent Water sufficient to perform 192 tests. Our leading edge software is designed to run on Microsoft Windows and can help you manage, analyze and report quantitative endotoxin detection assay data. Provides user-access security, electronic signatures, audit trails and data archiving Allows compliance with 21 CFR Part 11 (V2.00 and higher) Trending Database helps analyze assay performance over time Share the database over a network in a client/server type of interface Export assay results to LIMS and PDF Use one software package to support all Lonza quantitative methodologies Backup your data automatically and archive records Customize and use your own set of accessories There are no complex calculations to make. The quantitative system saves time and effort with every assay you run. Reduced technician time and higher test throughput means greater lab efficiency and that translates into bottom-line savings for your entire endotoxin detection operation. Simple to Setup and Test Your Samples Each analyst can select the type of test and enter standards and products with corresponding lot numbers and expiration dates. The USP/FDA Endotoxin Limits Database puts USP and FDA endotoxin limits for over 400 pharmaceutical products right at your fingertips for easy incorporation into your WinKQCL product Accessories Template Manager Endotoxin Test 4

5 files. And if you need to customize the product and accessory database to your specific requirements, it is easily done with WinKQCL. The custom microplate design gives you total versatility in the placement of standards and samples on the plate. Once a template has been created, it can be stored for future use and editing. The Run feature allows the user to perform an endotoxin detection assay corresponding to the selected template, and then collects the run readings. On completion, results will be stored in the Catalog of Assays. Standard curves from Kinetic-QCL and PYROGENT assays will be stored for later use. And with WinKQCL You Get the PowerCurve Advantage Since the relationship between reaction time and endotoxin concentration is not linear, PowerCurve was developed to graph and calculate results. When your standard curve is fitted with this polynomial model, you get a truer picture of the relationship between reaction times and endotoxin levels, more accurate spike recovery, and more consistent results with dilutions of the same sample. You can also compare these results to the standard log/log linear correlation using the default linear regression feature. WinKQCL Meets FDA Requirements WinKQCL provides user access security, electronic signatures, audit trails, and archiving of data files. WinKQCL Endotoxin Detection Software meets FDA requirements for electronic records and electronic signatures under 21 CFR Part 11 regulations. A Reviewer can examine and electronically sign records and an Analyst can create templates, perform tests and electronically sign records. True Networking Is Available WinKQCL (V3.00 or higher) is designed to operate either as a standalone or a networked system. In a network environment, all terminals interfaced with WinKQCL will save and access data from a common database on the server. The network can reside on a company wide server, a laboratory server or simply reside on one lab computer with others linked. Calculator Provides User Confidence With this easy-to-use tool, the analyst is able to confidently and quickly calculate dilution factors, maximum valid dilutions, minimum valid concentrations and endotoxin limits for both pharmaceutical products and medical devices. Data Portability and Record Keeping Are Enhanced WinKQCL supports the output of Adobe Acrobat files along with the ability to backup records on command or automatically based on time and date. Records can also be exported to LIMS via a parsable XML file. Specific blocks of records can also be archived and cleared from the working database to help in database maintenance and management. This interfacing solution ensures that the accuracy and integrity of your data is maintained while allowing for additional calculations, combining data from different sources, and sophisticated reporting and archival functions. And as part of a disaster-recovery tool, the restore feature is used to restore and merge databases. Calculator 5

6 Built-In Trending Module Allows You to Study Assay Variables A product trending database enables you to trend endotoxin results in individual products over time. Using the trending feature, product results can be recalled and compared, within individual analysts, reagent lot numbers, or other variables. Queries can be saved for future use and results exported to Microsoft Excel for further analysis. When a problem occurs in the laboratory, WinKQCL provides help to lead you through a logical investigation of irregularities that might have occurred. The documentation can be a great asset when generating an Out-of-Specification Report. System and Validation Tests Prior to running an assay, the reader should be setup and verified for Optical Density, System Test, Uniformity and plate alignment. WinKQCL includes the software support to ensure that the system is working properly. Electronic Signature Trending Four Tests to Choose From For the quantitative methods, WinKQCL allows four different tests: Routine, Inhibition/ Enhancement, RSE/CSE and Initial Qualification Routine Test: This test calculates the concentration of endotoxin in samples by comparison to the performance of a series of endotoxin standards. As part of a Routine Test, the user has the option to include a Positive Product Control (PPC) as a monitor for product inhibition or enhancement. A PPC is a sample of product to which a known amount of endotoxin spike has been added. The software automatically calculates the amount of endotoxin recovered in the PPC, allowing for a comparison to the known amount of endotoxin spike. Inhibition/Enhancement Test: This test is designed to determine what level of product dilution overcomes inhibition or enhancement. A Positive Product Control (PPC) must accompany each product dilution. The software calculates the amount of endotoxin recovered in the PPC for comparison to the known amount of endotoxin spike. From this comparison, it can be determined which product dilutions are non-interfering. RSE/CSE Test The RSE/CSE test is designed to determine the potency of a Control Standard Endotoxin (CSE) in terms of the concentration units of the Reference Standard Endotoxin (RSE). One or more sets of Control Standard Endotoxin dilutions are compared to the Reference Standard Endotoxin dilutions and the mean potency values are calculated in EU/ ml, EU/ng or other user-specified units. Initial Qualification Test This test is used to qualify technicians and perform a qualification of reagent procedure for each new lot of reagents. It is designed in accordance with FDA Guidelines. 6

7 Incubating Plate Readers Two plate readers have been validated as part of our quantitative endotoxin detection systems. These 96-well plate readers feature sample compartments with uniform temperature from well to well. Both readers seamlessly interface with the WinKQCL software. Kinetic-QCL and PYROGENT assays For the kinetic LAL assays, the ELx808 reader is highly reliable and easy to use. It features fully-automated reading of 96-well plates. This product can also be used as a standard spectrophotometer. It has built-in memory capability to store up to 55 assays, test results and standard curves. On-Site Service and Preventive Maintenance contracts are also available to ensure that your instrument is always working properly. PyroGene assay The FLx800 reader is part of the quantitative endotoxin detection system that features the recombinant Factor C fluorescent endpoint assay. The assay is measured with excitation/emission wavelengths of 380/440 nm. In addition to fluorescence, this reader can be used as a luminometer. The instrument can read from the top or bottom from 6-well up to 384-well microplates. 7

8 WinKQCL Software V3.1 Capabilities Types of Endotoxin Detection Tests Assays Supported User Maintenance Templates AER On Screen Monitoring Trending Data Storage Network Capable Export of Data Routine, Inhibition/Enhancement, RSE/CSE, Initial Qualification Kinetic - PYROGENT and Kinetic-QCL End-point - PyroGene Multi-level user identification Maintains Supervisor, Analyst and Reviewer accounts Password Protection Custom templates can be setup for products, accessories, assays, trending queries Can be saved for later use More than one assay can be run on a microplate through a convenient template addition tool Specifications, such as correlation coefficient and slope, can be set by the user for various assay parameters Assays can be monitored while in progress Search historical data and plot data using a wide range of variables Save trending queries and export trend results to Microsoft Excel All data stored in a MicroSoft SQL Server database Store a list of user-defined product names, units of measure, release limits and non-inhibitory dilutions Access to USP endotoxin limits Store a list of accessories including lot number, manufacturer name, calibration due and expiration dates Store a list of user-defined 96-well templates indicating where the blanks, standards, products and positive product controls are placed Archive standard curves Backup your data automatically on a one-time or recurring basis and archive records Merge V2.00 and higher backups and restore as one database Can share the database over a network using a true client/server application Export to LIMS using a compatible XML file Export Assay Reports as PDF or Microsoft Excel files 21 CFR Part 11 Ability to electronically sign records Compliance Three levels of security: Supervisor controls user ID, system settings and other security issues, a Reviewer can examine and electronically sign a record and an Analyst creates templates, performs tests and electronically signs records Audit logs to monitor the use of electronic records and security issues Validation Endotoxin Calculator Audit Reader and Analyst performance Perform multiple reader performance verification tests from the software Assists in making dilutions and calculating MVD s, MVC s and endotoxin limits 8

9 WinKQCL Software V3.1 Hardware and Software Requirements Minimum hardware requirements for Client and Server Systems Requirement Client System Server System Operating System Windows XP Professional SP2 or higher; Windows Server 2003 SP1 or higher Windows Vista Business or Enterprise CPU Intel Pentium III 1GHz or higher Intel Pentium IV 2.26 GHz/533 MHz/512 kb or higher suggested Memory 256 MB RAM minimum, 512 MB recommended 1 GB RAM recommended (1.5 GB RAM or higher recommended for database size of 1 GB or greater) ELx808 Absorbance Reader Specifications Wavelength Range Filters Supplied Absorbance Range Temperature Control Read Method Light Source Dimensions Weight 340 to 900 nm 340, 405, 450, 490 and 630 nm to to 900 nm to to 400 nm 4 C above ambient to 50 C Kinetic under WinKQCL Control Tungsten Halogen Bulb 16-inches deep x 15.5-inches wide x 8.75-inches high (40.6 cm x 39.4 cm x 22.2 cm) 35 lb (15.9 kg) FLx800 Fluorescence Reader Specifications Wavelength Range Temperature Control Read Method Light Source Dimensions Weight 300 to 700 nm 4 C above ambient to 50 C Endpoint under WinKQCL Control Tungsten Halogen Bulb 16-inches deep x 15-inches wide x 9-inches high (40.6 cm x 38.1 cm x 22.9 cm) 30 lb (13.6 kg) 9

10 Ordering Information* Reagents PYROGENT Kinetic Turbidimetric Assay Cat. No. Size Sensitivity (EU/ml) N tests N tests Contains: 2 vials lysate (50 or 100 tests/vial) 2 vials reconstitution buffer 1 x 50 ng/vial endotoxin Kinetic-QCL Kinetic Chromogenic Assay Cat. No. Size Sensitivity (EU/ml) U 192 tests Contains: 8 x 24 tests/vial of lysate/substrate reagent 2 x 10 ng/vial endotoxin 3 x 30 ml vials LAL Reagent Water PyroGene Endpoint Fluorescent Assay Cat. No. Size Sensitivity (EU/ml) U 192 tests Contains: 2 vials rfc enzyme 2 vials rfc buffer 2 vials fluorogenic substrate 2 x 10 ng/vial endotoxin 2 x 30 ml vials LAL Reagent Water * Bulk quantities are available on a special order basis. 10

11 Ordering Information Software and Instrumentation: WinKQCL Software V3.1 Client/Server Version Cat. No Endotoxin Detection Systems Qualification Manual Cat. No WinKQCL V3.1 Software License Agreement (required for multiple workstations) Cat. No ELx808 Absorbance Plate Reader Cat. No FLx800 Fluorescence Plate Reader Cat. No

12 Contact Information North America Customer Service: Technical Service: Online Ordering: Europe Customer Service: 32 (0) Technical Service: 32 (0) (German Speaking) 32 (0) (French Speaking) or Online Ordering: International Contact your local Lonza Distributor Customer Service: , ext Fax: International Offices Australia Austria Belgium 32 (0) Brasil Denmark France Germany India Italy The Netherlands Spain Switzerland United Kingdom Lonza Walkersville, Inc. Walkersville, MD Microsoft, Windows, SQL Server, Excel, Windows Vista, Windows XP, and Windows Server 2003 are trademarks of Microsoft Corporation. Adobe and Acrobat are trademarks of Adobe Systems, Inc. Intel and Pentium are trademarks of Intel Corporation. ELx808 and FLx800 are trademarks of BioTek Instruments, Inc. Unless otherwise noted, all trademarks herein are marks of the Lonza Group or its affiliates. Copyright 2007, Lonza Walkersville, Inc. All rights reserved. BR-KLAL /07