Introduction Charles River Laboratories revolutionary FDA licensed endotoxin detection system, the Endosafe Portable Test System (PTS), is a rapid point of use test system that provides quantitative test results in less than 15 minutes. The technology utilizes licensed LAL reagents in a disposable cartridge using the PTS for a completely contained, real time endotoxin assay. The Endosafe® PTS cartridges are licensed by the FDA allowing users to test raw materials, in process samples as well as final products. The flexibility of the PTS allows it to be used in conventional quality control settings or as a point of use test to support the drug development process.

Traditional LAL Methods The FDA now considers the PTS cartridge in the same category as traditional licensed LAL methods: Endosafe® - Gel-Clot Endosafe® Endochrome - Endpoint Chromogenic Endosafe® -KTA -Kinetic Turbidimetric Endosafe® Endochrome-K- Kinetic Chromogenic Endosafe® PTS – Rapid LAL Method

Traditional Methods The unique single cartridge design of the PTS eliminates the need for costly disposables and timely preparation of standard curves. The equipment needed for traditional LAL methods are listed on the right. While the traditional tests have many advantages, they do have limitations such as having to prepare standard curves with each assay, using costly disposables and long prep and incubations times Reagents and Equipment: LAL Vials Control Standard Endotoxin (CSE) LAL reagent water Depyrogenated Dilution Tubes Pipettors for delivery of accurate spike and sample volumes Depyrogenated Reaction Tubes (10 x 75 mm) or Microplate Water Bath, Heat Block, or Micro-plate Reader and computer with software

Endosafe® Portable Test System (PTS)

What is the PTS ? A revolutionary point of use LAL testing system Represents a unique new platform for LAL testing Utilizes: Portable hand held spectrophotometer LAL, CSE, and Synthetic Substrate all dried onto a single cartridge

PTS Hand Held Unit Device Includes: Features: Pumping system Spectrophotometer Imbedded software to calculate and store sample data Features: Precise thermal management Photo detectors at 395 nm Unique algorithm for absorbance changes imparts unit stability and consistency

PTS Cartridge Single use Polystyrene cartridge with 4 channels Configured to meet current regulatory requirements: Duplicate sample testing Duplicate positive product controls analysis All of the reagents required for a chromogenic analysis are dried onto a single cartridge. A sample layout of the cartridge is shown on the next slide. Formulations have been optimized for: LAL drying Substrate drying Endotoxin recovery Rapid results

Cartridge Set Up – Duplicate Sample, Duplicate Spike Pre-made Endotoxin Spikes LAL Reagent Chromogenic Substrate Optical Cells Pump Connections Sample reservoirs

Cartridge Diagram

How PTS Works During initial QC testing of each lot of cartridges we determine: Standard reaction times using RSE endotoxin Spike value (PPC) These values can be found on the Certificate of Analysis for each lot of cartridges.

Reaction Diagram 5.0 Eu/mL 0.5 EU/mL 0.05 EU/mL Color change Onset Times Time Reaction Time

How PTS Works The values for the standard reaction times and spike concentration are entered into the reader in the form of a Calibration Code which is also found on the Certificate of Analysis. The software plots the reaction times determined for each well against the known values to give sample value and spike recovery just like with traditional kinetic LAL assays. The next slide shows the location of this information on the C of A.

Endosafe® PTS Training Tips Prior to Testing

Cartridge Temperature All LAL reagents have been optimized to run at 37° C. We recommend that your samples and cartridges are at room temperature when starting your assay. Cartridges should come to room temperature in approximately 10-15 minutes if stored refrigerated at 2-8°C.

Cartridge & Sample Temperature Using cartridges or sample not at room temperature can cause issues with dissolution of the reagents and lead to invalid results. Condensation forming on a cold cartridge can interfere with the transmittance of light through the optical cell.

Handling the Cartridge Endosafe recommends Several tips for cartridge handling. Remove cartridge from pouch by touching the edges of the cartridge.

Handling the Cartridge Once removed, try to touch only the “handle” end of the cartridge (curved edges) to place the cartridge into the reader.

Handling Tips Touching the sample reservoirs Touching optical cells This can introduce contamination which will be pulled in with the sample. Touching optical cells A fingerprint or smudge on the optical cell can cause problems with the transmittance of light through the optical cell.

Location of Sample Reservoirs and Optical Wells Optical Cells Handle

Sample Preparation Endosafe stresses that your lab use only materials known to be endotoxin free and non-interfering to the assay to prepare or dilute your sample. Keep in mind that many accessories that are labeled “Pyrogen Free”, may only be tested to levels of .25 EU/mL Accessories can include water, tips, pipettes, and dilution tubes. CRL can help identify the appropriate materials.

Sample Preparation Samples should be prepared according to your SOPs or product validation if applicable. Dilutions used for alternative methods should apply to the PTS Sample preparation may include dilution, heat treatment, pH adjustment…

Sample Interference LAL is an enzymatic assay and is subject to interference from the sample Turbidity, color, pH, protein concentration, chelating compounds and detergents all can disrupt the reaction Dilution is your friend! Use as much dilution as acceptable for a robust assay Understand and utilize Endotoxin Limits and Maximum Valid Dilutions (EL)

What is an Endotoxin Limit? The level of “allowable” endotoxin that can be in a product Endotoxin limit is based on: Route of administration Threshold pyrogenic dose Dose of the product One cannot validate a product without a corresponding endotoxin limit

Calculating Endotoxin Limits for Drug Products Some endotoxin limits have been calculated and can be found in USP monographs For non-compendial items and new drugs, the endotoxin limit must be calculated by the user Formula for the calculation of endotoxin limits: K M

Calculating endotoxin limits: K K = the threshold pyrogenic dose in endotoxin units/kg Experimentally determined in rabbits, confirmed in humans K = 5 EU/kg for drugs with a route of administration other than intrathecal K = 0.2 EU/kg for drugs administered intrathecally

Calculating Endotoxin Limits: M M = maximum human dose of product/kg of body weight in a single hour period If the pediatric dose/kg is higher than the adult dose, then the pediatric dose/kg = M For new drug products or drug products that have no compendial limit, the user must read the product insert to determine the most stringent (i.e. maximum) dose

Example: Sodium Oxacillin K 5.0 EU/kg 0.2EU/mg M 25 mg/kg Administered intramuscularly Maximum human dose = 25 mg/kg K 5.0 EU/kg 0.2EU/mg M 25 mg/kg = =

Maximum Valid Dilution (MVD) Most products interfere with the LAL test The easiest way to overcome interference is through dilution MVD provides an upper bound for dilution that still provides for endotoxin detection at the endotoxin limit

MVD Calculation Where: MVD = (Endotoxin Limit)(Concentration of sample solution) l Where: Endotoxin Limit = K/M Concentration of sample solution = concentration in units/mL l = confirmed label claim sensitivity of the GC lysate or lowest point on the referenced standard curve If the limit is in EU/mL, the concentration of the sample solution is not needed *****MVD is unitless…it is a dilution factor*****

Observations on the MVD MVD = (Endotoxin Limit)(Concentration of sample solution) l Endotoxn Limit (K/M) is a constant, and is specific for a particular drug product with a particular route of administration and a particular dose. Concentration of the sample solution is a variable. Generally, it is the concentration in the final presentation, but may vary - especially with raw materials, in process samples, etc. For drugs administered on a mL/kg basis, the concentration of the sample solution = 1 Lambda is a variable, and depends on the sensitivity of the test.

MVD Example: Sodium Oxacillin = (0.2 EU/mg)(10 mg/mL) = 8 Endotoxin Limit = 0.2 EU/mg Starting concentration = 10 mg/mL Gel clot test with a sensitivity of 0.25 EU/mL MVD = (Endotoxin Limit)(Concentration of sample solution) l = (0.2 EU/mg)(10 mg/mL) = 8 0.25 EU/mL Changing the test sensitivity to 0.06 EU/mL raises the MVD to 32

Endosafe® PTS Training Reader Setup and Entering Information

Certificate of Analysis The analyst will want to have the Certificate of Analysis (COA) accessible prior to running the test. The C of A includes the cartridge lot number and calibration code. A sample of the C of A is given on the following slide.

Reader Operation Tips The PTS unit can run on battery or AC power. Battery life is approximately 4 hours on a full charge. Make sure it is the right adapter! Charles River Laboratories recommends turning the reader off at night. Labs can keep the unit plugged in on the bench top if not in portable use. Use Dry electronics air to clean chamber periodically Schedule annual calibration with Charles River Laboratories.

System Start Up Press the MENU key on the PTS to turn the reader on. The reader begins a “System Self Test” and heats up to 37° C in approximately 5 minutes.

Photo Photo of reader during “System Self Test”

System Start Up Once the reader reaches 37° C the reader display will change from “SYSTEM SELF TEST” to “INSERT CARTRIDGE”

Photo Photo of reader displaying “INSERT CARTRIDGE”

Inserting Cartridge Insert cartridge firmly into the slot at the front of the reader with the sample reservoirs facing up.

Keypad Operation The alphanumeric keypad operates much like the keys of a cellular phone. Pressing the keys quickly will move through the letters or symbols above the key. If the key has not been pressed for a second or two the cursor moves over to the next space. The arrow keys can be used to back up and overwrite text already entered.

Entering Test Information Enter OID: Operator Identification Enter Lot #: Cartridge Lot # Lot number is found on the cartridge, cartridge pouch and C of A Enter Calibration Code: Found on C of A. This is a lot specific code. Once a calibration code has been entered for a particular lot number, the PTS will not ask you to input it again. Take great care to enter code correctly.

Entering Test Information Lot #: Lot number displayed on screen The screen confirms the cartridge lot # entered. Enter Sample Lot #: The analyst is given a field to enter their sample information Reader will accept any text entered.

Entering Test Information Enter Sample ID: Allows the analyst to enter further sample information. Enter Dilution Factor: Reader will only allow whole numbers to be entered. A 1:40 dilution is simply entered as 40 Undiluted samples are entered as 1 Reader displays “ADD SAMPLE; PRESS ENTER”

Dilution Note Entering the dilution factor into the reader will automatically correct for the dilution by multiplying the raw endotoxin value determined by the dilution factor. This gives the value for the original undiluted sample.

Dilution Example Dilution Factor Reporting value factoring in dilution ********* ENDOSAFE Test Record ********* V7.10 07/25/06 DateTime: .......... 10/10/06 @ 10:55:26 Device: ........................... 0662 OperatorID: ........................ BCP Cartridge: ................... Endotoxin Temperature: .. Start: 37.0C End: 37.0C Method: ......................... KX-122 Cartridge Lot#: ................ 6206162 Range: ......................... 5-0.05 Range Time: ............... Sec: 132-777 Onset Time (s): ..... >777 296 >777 316 Slope: -0.385 ........ Intercept: +2.390 Dilution: .......................... 100 Sample Lot: ..................... IP0003 Sample ID: ............................1 Sample Value: .............. <5.00 EU/mL Sample CV: ........................ 0.0% Spike Value: .............. 0.513 EU/mL Spike CV: ......................... 4.6% Spike Recovery: .................. 125% : Dilution Factor Reporting value factoring in dilution

Endosafe® PTS Training Addition of Solution to Sample Reservoirs

Importance of Accurate Volume Because of the speed of the assay and the small volume of sample being used, accurate volumes are critical. The PTS uses ¼ the amount of sample, is much quicker than traditional assays and is therefore less forgiving. Charles River recommends using a calibrated fixed-volume pipettor or qualifying the pipette to be used daily on an analytical balance.

Technique Be cautious when drawing sample into pipette tip: be aware of bubbles or extra space at the end of the pipette tip as these can cause inaccurate volumes to be dispensed.

Proper Technique Hold pipette so that the tip is at an angle and not completely at the bottom of the sample reservoir.

Photo Photo of properly holding pipette when dispensing sample.

Improper Technique Holding pipette straight up and down at the bottom of the sample well. This can force some sample into the channel early and disrupt the reagent mixing stations. Any technique which is prone to splash out or bubbles.

photo Photo of improper technique (straight up and down at bottom of sample reservoir.

Splash Out Will render the volume inaccurate which can lead to invalid test results particularly in the spiked channels.

Photo Photo of sample splashed out onto cartridge or reader. Splashout

Bubbles Can cause false onset times because of the disturbance in the light transmittance. Can interfere with mixing of sample with reagents by separating the sample so that it is not homogeneous.

Photo Bubbles Photo of bubble in sample reservoir. Use HSA or DA??

Addition of Sample Once the sample has been entered correctly, press ENTER Sample will be drawn into the channels and mixed with the reagents. Display will count down mixing stage. System will begin counting up when the sample has entered the optical eye and measurement of the color change begins

Endosafe® PTS Training Retrieving Results

Retrieving Results Data from the assay can be retrieved several ways. The unit will store 100 results to allow data to be accessed at a later date.

Retrieving Results At the completion of the assay the reader will alternate displaying the following information on the LCD screen until the cartridge is removed from the unit: Sample EU/mL Sample % CV Spike EU/mL Spike % CV % Spike Recovery Remove Cartridge

Retrieving Results A complete test report can be printed at the completion of the assay if the lab is using the Epson printer. The report will automatically be printed upon completion of the assay. Data can be exported to a text file on the lab’s PC for further record keeping. A serial cable and software is provided with the unit for this purpose.

Endosafe® PTS Training Interpreting Results

Validity A valid test has: Spike Recovery of 50-200% Sample CV < 25% Spike CV <25% The following slides explain these parameters.

Spike Recovery Many substances can interfere with the LAL assay, rendering the reagent inactive. To determine if any interfering characteristics exist, each LAL assay must have a positive product control (PPC) to ensure that endotoxin would be detected if it were present in the sample.

Spike Recovery (continued) With traditional methods, the analyst is required to prepare the Positive Product Controls (PPC) and spike their samples. Analysts do not need to prepare PPC’s for the PTS. On the PTS cartridge, 2 of the 4 channels contain a known amount of endotoxin (spike) in addition to the LAL reagent. The reader measures spike recovery for each assay.

Spike Recovery (continued) The regulatory bodies have established that a gel-clot sample must be positive when spiked to a 2 λ concentration and spike recovery must be 50-200% for kinetic methods . If Spike Recovery is out of the valid range the sample should be diluted further or an alternate preparation should be considered such as dilution in buffer.

CV (Coefficient of Variation) CV = standard deviation / mean CV is a statistical measurement of how far apart replicates are. A CV of 25% or greater indicates a significant split in the 2 replicates and since the analyst cannot be sure which of the values is correct, the test is considered invalid and should be repeated.

Test Report ********* ENDOSAFE Test Record ********* V7.10 07/25/06 DateTime: .......... 10/17/06 @ 00:25:51 Device: ........................... 0811 OperatorID: ........................ GC Cartridge: ................... Endotoxin Temperature: .. Start: 37.0C End: 37.0C Method: ......................... KX-122 Cartridge Lot#: ................ 6206162 Range: ......................... 5-0.05 Range Time: ............... Sec: 132-777 Onset Time (s): ..... >777 360 >777 330 Slope: -0.385 ........ Intercept: +2.390 Dilution: ............................ 1 Sample Lot: .................... G053447 Sample ID: ......... LRW Sample Value: ............. <0.050 EU/mL Sample CV: ........................ 0.0% Spike Value: .............. 0.362 EU/mL Spike CV: ......................... 6.1% Spike Recovery: .................. 88% : Reaction time of 4 channels. 1 & 3 are sample channels, 2 & 4 are spiked channels. CV % of channels 1 & 3. Represents variation in reaction time of the two CV % of channels 2 & 4. Represents variation in reaction time of the two. Represents % of spike that was recovered in channels 2 & 4

Endosafe® PTS Training Troubleshooting

Invalid Spike Recovery Invalid spike recovery can be caused by your sample, accessories or pipettor. If dilution in LRW does not bring the spike recovery in range you will want to check the accuracy of your pipettor and check the quality of your accessories. If the pipettor is accurate (25µL +/- 1µL) you may be using inhibitory LRW.

Invalid Spike Recovery Dilution is the easiest way to overcome any type of LAL interference. If you do not have room for dilution within the MVD (Maximum Valid Dilution), you will need to look at alternate sample preparations such as the use of buffers.

Sample Inhibition (<50% recovery) Inhibition can be caused by: pH out of range Divalent cation reduction Enzyme modification Contact Charles River Laboratories Technical Service for more details on LAL inhibition.

Sample Enhancement (>200% recovery) The only known source of false positives for the LAL assay is Glucan. Most glucan interference can be eliminated using ES-buffer to dilute the sample. Contact Charles River Laboratories Technical Service for more details on LAL enhancement and Glucan.

Invalid CV It is recommended that the user remove the cartridge (very gently) to observe if either of the two instances may have caused the invalidity. Bubble in sample or optical well. Sample did not completely fill optical well (if the leading edge of the sample remains in the optical well this can cause a false onset time).

CSE Not Giving Expected Value Control Standard Endotoxin (CSE) and other non-primary standards cannot be used for endotoxin verification on the PTS system. In order to use CSE, a manufacturer must standardize CSE against the Reference Standard Endotoxin (RSE) on a lot specific basis to obtain an EU/ng ratio for that lot combination. We perform all testing on the PTS cartridges using the RSE which is the USP / FDA standard. RSE must be used to obtain appropriate values because there has been no RSE/CSE standardization performed for PTS cartridges as it is with LAL/CSE combinations.

Endosafe® PTS Training Error Messages

Photo Photo of “led out of spec”

“One or more test LEDs out of spec” “Abort Test CANCEL OR ENTER” The reader checks the intensity of each LED prior to letting the user run a test. If the reader detects that one or more LEDs does not have sufficient intensity to run an accurate test, this message will be displayed. The user should contact Technical Service to set up repair.

photo Photo of “sample not seen”

“Sample not Seen” The reader is able to look for a sample in each of the optical cells shortly after initiating a test (reader begins counting up). If a sample is not detected in one or more of the optical cells, the reader will alternate between “Sample not Seen” and “Test in Progress”

“Sample not Seen” (continued) The user can pull the cartridge out of the reader (very gently) and examine it to see if sample was pulled into each of the 4 optical cells. If this problem persists, please contact Technical Service for troubleshooting.

Endosafe® PTS Training Product Validation

Product Validation Validation for any LAL test is performed in 2 steps: Inhibition / Enhancement Screen Validation

Inhibition / Enhancement Screen The purpose of the I/E screen is to find a non interfering dilution or preparation at which to validate the sample for routine testing. Multiple concentrations should be made and tested between undiluted and the MVD (Maximum Valid Dilution).

Validation Prepare a dilution of each of 3 different lots of product at the optimal dilution found to be non interfering during the I/E screen. If all 3 tests are acceptable, the product has been validated at the tested dilution and may be tested routinely anywhere between this dilution and the MVD.

Validation A 1-lot validation of product may be performed under special circumstances. For example if only one lot or product has been made and other lots will follow at a later date.

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