Package Insert: Licensed Endosafe-PTS Cartridges …

1 Sample Channel ZoomPPC (Spike) Channel ZoomSTORAGE CONDITIONS AND PRECAUTIONSPTS Cartridges are relatively heat stable and should be stored at 2 - 25 C. Allow the Cartridges to come to room temperature beforeopening the pouch and testing. Prolonged exposure to temperaturesabove 25 C should be avoided. To minimize contamination of thesample wells, the cartridge should be used immediately once the foilpouched seal has been opened. Cartridges are for single-test use REQUIRED BUT NOT SUPPLIEDLAL Reagent Grade Water must be used during the initial qualificationof each lot of PTS REQUIRED BUT NOT SUPPLIEDP ipettor (Endosafe PTS 400 or equivalent) and sterile , endotoxin-free glass dilution tubes or sterile, disposablepolystyrene tubes (Endosafe T300 or equivalent) for sample collection or dilution if Mixer (if necessary).EQUIPMENT REQUIRED BUT NOT SUPPLIEDE ndosafe Portable Test System (PTS) Reader:The reader is a dedicated instrument that accepts the cartridge and runs the PTS LALtest.

2 The reader consists of an incubating chamber, a sample pump,four LEDs and four detectors, an alphanumeric key pad with built inLCD, and a microprocessor. The reader operates using standard ACpower or an internal rechargeable battery. Battery power also acts asautomatic backup power in case of AC power Multi- cartridge System (MCS):This reader is a dedicat-ed instrument that is equipped with five independent cartridge MCS instrument can test up to five Cartridges . The cartridgescan be run simultaneously or in a random access fashion. Each MCScartridge reader consists of an incubating chamber, a sample pump,four LEDs, and four , the PTS reader measures the reaction time in each archived standard curve specific for each lot of Cartridges is constructed using the log of the reaction time vs. the log of the concentration. The sample and spike values are calculated by interpolation of the standard curve using the reaction times. SeeCertificate of Analysis for the lot specific standard COLLECTION AND PREPARATIONS pecimen for testing must be collected and prepared using depyrogenated materials and endotoxin-free reagents.

3 Glassware must be depyrogenated by validated conditions, such as 30 minutesexposure at 250 is prudent to test for endotoxin those materialsthat cannot be heat sterilized or those which are sold without an endotoxin-free label. Use aseptic technique at all Suitability RequirementsAcceptance criteria for a valid assay consists of a positive productcontrol (PPC) recovery value of 50-200%, and a coefficient of variation(%CV) of less than 25% on reaction times for both sample and PPCchannels. Note: Samples should be tested (following the USP BET9recommendation) at a dilution (less than the MVD) necessary toconsistently eliminate interfering factors such as pH, ionic strength,and high background OF ASSAYSI nitial Qualification: Each new lot of Cartridges must be qualified uponreceipt. The initial qualification testing requires one cartridge with LALR eagent Water as a sample. The evaluation must demonstrate nodetectable endotoxin and acceptable spike recovery (50-200%).

4 Inhibition/ Enhancement:Absence of interference is demonstrated byachieving acceptable spike recovery (50-200%) on a given : A validation currently defined by the Guideline on Validationof the Limulus Amebocyte Lysate Test as an End-Product Endotoxintest for Human and Animal Parenteral Drugs, Biological Products, andMedical Devices, Dept. of Health and Human Services, FDA,December7requires three lots of product to be tested at the AMEBOCYTE LYSATEENDOSAFE -PTS CARTRIDGESUS License No. 1197 SINGLE-TEST, DISPOSABLE Cartridges FORENDOTOXIN DETECTIONFOR USE WITH ENDOSAFE PORTABLE TEST SYSTEMAND MULTI- cartridge SYSTEM READERSINTENDED USED isposable test Cartridges contain Limulus AmebocyteLysate (LAL), endotoxin, and synthetic color-producingsubstrate. They are intended for use with Endosafe Portable Test System (PTS) to perform quantitativedetection of endotoxins by kinetic chromogenic AND SUMMARYF rederick Bang observed that bacteria causes intravascular coagulation in the American Horseshoe Crab, Limulus collaboration, Levin and Bang5found that the agent responsible forthe clotting phenomena resides in the crab s amebocytes, or circulatingblood cells, and that pyrogens (bacterial endotoxin) triggered theenzymes involved in the clotting LAL test is the most sensitive and specific means available todetect and measure bacterial endotoxin, a fever-producing byproductof gram-negative bacteria, commonly known as pyrogen.

5 The basis ofthe test is that the endotoxin produces changes in the appearance ofLAL that are easily ,5 The simplicity and overall economyof the LAL Test encourages the testing of in-process solutions and rawmaterials as well as end-product drugs, devices, and biologics10. TheUSP Bacterial Endotoxins Test and the USFDA Guideline for LALtesting provide standard methods for validating the LAL Test as areplacement for the rabbit pyrogen ,9 BIOLOGICAL PRINCIPLESIn this assay, bacterial endotoxins initiate the activation of a cascadingseries of serine proteases in LAL. The last activated enzyme in this series, the pro-clotting enzyme, cleaves a peptide from an endogenoussubstrate called coagulogen. The modified substrate produces an opacity and gelatin in LAL that is easily detected. A synthetic analog tocoagulogen can also be used to quantitatively measure the endotoxinmediated activation of the LAL pro-clotting enzyme. This synthetic substrate undergoes cleavage, resulting in the release of the chromophore, p-nitroaniline (pNA).

6 PNA is a yellow color that is measured photometrically at 385-410 nm. With the aid of a spectrophotometer, a kinetic colorimetric assay may be done, in whichthe early onset of color can be detected and precisely - - - - - - EnzymeEnzymeSubstrate - - - - - - Peptide + p-nitroanilineThe Endosafe - PTS cartridge and its interface with the reader havebeen designed to mimic currently Licensed Endpoint Chromogenic andKinetic Chromogenic Methods by measuring color intensity directlyrelated to the endotoxin concentration in a sample. Each cartridge contains precise amounts of FDA Licensed LAL formulations, chromogenic substrate, and Control Standard Endotoxin (CSE). cartridge REAGENTSEach Endosafe - PTS cartridge contains four channels to which LALreagent and a chromogenic substrate have been applied. Two of thefour channels also contain an endotoxin spike, and serve as the positive product controls. The use of two Sample Channels and twoSpike Channels is consistent with current FDA Licensed quantitativeLimulus Amebocyte Lysate (LAL) methods.

7 The PTS reader can beused to detect endotoxin in variety of StationReagent Station12 OpticalCellPumpConnectionEndotoxinSample ReservoirOpticalCellPumpConnectionEndoto xinSubstrateReagent StationReagent Station13 LALR eagent Station2 SampleReservoirEndotoxinLALS ubstrateOptical CellInformation OnlyInhibition is usually concentration dependent and can be overcome bydilution with LAL Reagent Water. The most common sources of inhibition are 1) conditions that interfere with the enzyme activity due toionic strength and/or pH, and 2) those that alter the dispersion of theendotoxin (positive) the positive product control fails and apH related problem is suspected, the pH of the test specimen shouldbe measured to assure a pH within the range of - Use an endotoxin-free TRIS buffer (Endosafe BT101, BT103 or equivalent) if pH adjustment is necessary. Do not arbitrarily adjust the pH of unbuffered Valid Dilution: The Food and Drug Administration hasestablished endotoxin limits of 5 EU/kg for intravenous drugs and for intrathecal limits for compendial items havebeen limits may be used to determine the extent of dilution that may be used to overcome an interference problem withoutexceeding the limit endotoxin Maximum Valid Dilution (MVD) is calculated by formulae presented in the previously mentioned documents and other , 9 For drug products that have a published limit, the MVD may be calculated by the following formula:MVD = Endotoxin Limit X Product PotencyLambdaEL = K/M, where K= 5EU/Kg and M= Maximum Dose per kg ofbody weight administered perhourProduct Potency = concentration of productLambda = sensitivity (lowest point on the archived curve) of test cartridgeFor example, the compendial limit for Sterile Water for Irrigation (SWI) is EU/mL.

8 If a test cartridge with an archived standard curve containing the lowest level of EU/mL of endotoxin is used to test this product, where the potency is 1 mL/mL, the MVD equals 1 , SWI may be diluted up to 1:5 to resolve potential inhibition (onepart to a total of five parts LRW).Routine Tests with the Endosafe - PTS Cartridges :See the User s Guide supplied with the Endosafe PTS or theEndosafe MCS reader for complete operations, procedures, , , A Bacterial Disease of Limulus Polyphemus , Bulletinof Johns Hopkins Hospital, Nr. 98, p. 325 (1956). , , Levin, J., and Wagner, , Endotoxins as a Cause of Aseptic Meningitis after Radionuclide Cisternography ,Journal of Nuclear Medicine, Nr. 16, p. 809 (1976). , , Levin, J., and Wagner, , QuantitativeComparison of In Vitro and In Vivo Methods for the Detection ofEndotoxin , Journal of Laboratory and Clinical Medicine, Nr. 78, (1971). , , The LAL Test versus the Rabbit Pyrogen Test for Endotoxin Detection: Update 87 , Pharmaceutical Technology, Nr.

9 11(6), p. 124 (1987). , J., and Bang, , Clottable Protein in Limulus: ItsLocalization and Kinetics of Its Coagulation by Endotoxin ,Thromb. Diath. Haemorrh., Nr. 19, p. 186 (1968). , , Resolving LAL Test Interferences , Journal ofParenteral Science and Technology, Nr. 44:1, p. 13, (1990). on Validation of the Limulus Amebocyte Lysate Test asan End-Product Endotoxin test for Human and Animal ParenteralDrugs, Biological Products, and Medical Devices, Dept. ofHealth and Human Services, FDA, December Guidance for Human and Veterinary Drug Products andBiologicals, Dept. of Health and Human Services, FDA, July15, Bacterial Endotoxins Test. In The , 26threvi-sion, p. 2023-6, The United States Pharmacopeial Convention,Inc., Rockville, MD., 2002 10. McCullough, Process Control: In-process and Raw MaterialTesting Using LAL. Pharmaceutical Technology, 12(5) (1988).11. Weary, Understanding and setting endotoxin limits. Journal of Parenteral Science and Technology, 44:1, p.

10 16 (1990).PATENT Patent No: US D472,324 Patent No: US 7,329,538 B2 Other patents By: CHARLES RIVER ENDOSAFEDiv. of Charles River Laboratories, Wappoo Road, Suite 43 BCharleston, SC 29407, USAPHONE NUMBER: 843-402-4900 FAX NUMBER: 843-766-7576 PIPTS2008 Information Only