Simplified endotoxin test method for compounded sterile products

technical sheet
Compounded Sterile ProductsJames F. Cooper, PharmD, FAPhA The USP describes two tests for microbial contamination of a Limulus amebocyte lysate (LAL) reagent, FDA approved, is Compounded Sterile Product (CSP). The sterility test screens used for all USP endotoxin tests. Two types of endotoxin tests for live, infectious bacteria. The Bacterial Endotoxins Test (BET) are described in the USP <85> BET. Photometric tests require detects unsafe levels of microbial cell wall debris, from live or a spectrophotometer, endotoxin-specific software and printout dead Gram-negative bacteria, that causes fever and symptoms capability. The simplest photometric system is a handheld of septic shock. Until now, the BET required skilled analysts unit employing a single-use LAL cartridge that contains dried, and manipulation of cumbersome reagents. This discussion pre-calibrated reagents; there is no need for liquid reagents describes a uniquely simple system for assuring that CSPs are or standards. The FDA-approved unit is marketed under the free of endotoxin (pyrogen) within limits set by the Pharmacopeia name of Endosafe®_ PTS™. The device requires about 15 (USP) or consistent with current scientific opinion.
minutes to analyze small amounts of sample, a 25-μL aliquot from CSP diluted in a sterile tube, and to print out results. In contrast, gel-clot methods require a dry-heat block, calibrated Endotoxin is extremely potent, is heat stable, passes sterilizing pipettes and thermometer, vortex mixer, freeze-dried LAL membrane filters and is present everywhere bacteria are or reagents, LAL Reagent Water (LRW) for hydrating reagents and have been present. Of greatest concern are intraspinal infusion depyrogenated glassware. In this clot test, diluted sample and solutions because this route of administration is about 1000 liquid reagents require about an hour for sample and positive- times more potent for endotoxin than is IV injection.1 Non-sterile control preparation and an hour’s incubation in a heat block; powders, components and water are the most likely sources of results are recorded manually.1 Thus, the simplicity and speed of the automated system make it ideally suited to the pharmacy setting.
An endotoxin limit of 5 EU/kg/hr or 350 EU per adult (70 kg) wasscientifically established to avoid the fever and hypotension from The LAL-endotoxin reaction is an enzymatic cascade that IM or IV injection of endotoxin contamination.2 Time is a factor requires specific conditions for optimum reaction, including because there are mechanisms in the liver and blood that neu- neutral pH, optimum concentrations of mono-valent and divalent tralize endotoxin. However, there are no clearance mechanisms cations and low concentrations of most drug substances. in intraspinal spaces, so the IT (intrathecal) endotoxin limit is Fortunately, the BET has great sensitivity that allows substantial much more stringent. An IT limit of 0.2 EU/kg/hr was arbitrarily dilution of test materials without sacrificing safety with respect to set without supporting experimental data. A more scientifically sound IT endotoxin limit of 14 EU per day avoids the serious mortality and morbidity associated with the signs of meningitis that are induced by endotoxin in IT spaces.1 An Endotoxin Unit (EU) is a unit of biological activity of the USP Reference Endotoxin Standard.
BET for CSPs Intended for Intraspinal Infusions Intraspinal mixtures: As directed above, make a 1:80 The challenge posed in testing a CSP is preparation of a dilution for infusions that contain ≤20 mg/mL of total drug test sample that is 1) diluted sufficiently to avoid inhibitory components and make a 1:280 dilution for > 20 mg/mL, total. or interfering factors and 2) is tested in a method sufficiently The intraspinal endotoxin limit is always met when the test result sensitive to guarantee that test results are safely within an IT endotoxin limit of <14 EU per day. Validation data for testing intraspinal analgesics and antispasmodic agents by Endosafe® Table 1 lists the maximum daily dose published by a panel LAL reagents was published and peer reviewed.1 The study of experts for long-term intraspinal administration.3 The reported that dilution of intraspinal infusions to < 1 mg/mL and recommended intrathecal limit of 14 EU/day was divided by the testing in a sensitive system gave results within the desired panel’s maximum dose, in mg/day, to generate the drug-specific endotoxin limit. Validated test methods for individual as well as endotoxin limit. The calculations were done on a daily basis to combinations of intraspinal medications were described.
be compatible with daily dosage, to increase the BET sensitivity and to better reflect the kinetics of cerebrospinal fluid flow and An endotoxins test by the Endosafe®_ PTS™ requires precalibrated LAL cartridges, a PTS™ reader, sterile dilution tubes, fixed-volume pipette with sterile pipette tips, and a test Table 1: Endotoxin Limits for Intraspinal Infusions sample that is diluted to a BET-compatible level. A suitable tube for dilutions is a 14-mL Polystyrene sterile tube, product # 2057 by Becton Dickinson. The most convenient time to sample a CSP is just prior to sterilizing filtration of the final product. The following guideline for dilution-prior-to-testing applies to opioids and antispasmodic drugs commonly used for intraspinal dilution by transferring 25 μL of CSP to 2 mL SWI (Sterile Water *Maximum daily dosage published by an expert panel for long-term for Injection or Irrigation used in compounding) or LRW in a sterile 14-mL tube. The sensitivity (LOD, limit of detection) of an ∞EL for IT based on 14 EU/mL/Day divided by the maximum daily assay will be 4 EU/mL, when using a cartridge with a standard Intraspinal infusions > 20 mg/mL: Make a 1:280 dilution by transferring 25 μL of CSP to 7 mL SWI or LRW in a sterile 14-mL tube. The sensitivity (LOD, limit of detection) of an assay will be 14 EU/mL, when using a cartridge with a standard curve of 0.05-to-5 EU/mL.
Table 2 indicates how IT infusion mixtures would be prepared Simple solutions or very dilute drugs may be tested with little for endotoxin testing by the PTS™ system. When no endotoxin or no dilution. The dilutions recommended below should avoid is detected, the results are reported as <4 or <14 EU/mL, test inhibition, as evidenced by recovery of the positive control, depending upon the extent of initial dilution of an aliquot from an and give results that are within the endotoxin limit (see below). The validated test dilutions detailed in Table 3 and discussed throughout this document apply to Endosafe® LAL reagents Table 2: Summary of CSP Dilution and Results for and may not apply to reagents from other sources. The dilution required for a therapeutic CSP is related to the concentration of the active ingredient, as detailed below: Intravenous or IM solutions ≤ 20 mg/mL: Make a 1:100 dilution by transferring 25 μL of CSP to 2.5 mL SWI or LRW in a sterile 14-mL tube. The specific drug or mixture endotoxin limit may be determined by applying the calculations suggested below or existing methods.
Intravenous or IM solutions > 20 mg/mL: *Data for coded IT medications and concentration in mg/mL; where, MS = morphine sulfate; CL = clonidine HCL; BP = bupivicaine HCL; Make a 1:400 dilution by transferring 25 μL of CSP to 10 mL SWI HM = hydromorphone HCL; FN = fentanyl citrate.
∞Sensitivity for LAL cartridge = 0.05 EU/mL (PTS™ System) BET for CSPs Intended for IV or IM injection Make a 1:200 dilution by transferring 25 μL of CSP to 5 mL of A great variety of CSP medications are prepared, from sodium SWI or LRW in a sterile tube. A glucans blocker may be needed chloride injection to steroidal or antibiotic suspensions, so that the development of a simplistic dilution scheme is challenging. Summary of CSP Dilution and Results for Representative CSPs *Data for IM or IV medications in mg/mL.
∞Sensitivity for LAL cartridge = 0.05 EU/mL (PTS™ System) Understanding and Calculating Endotoxin Limits interference. The PTS™ result was no-detectable-endotoxin and The adverse effects of endotoxin are dose-dependent, so valid recovery of the positive control, giving a report of <20 EU/ the objective of meeting endotoxin limits is to assure that any mL. Therefore, a maximum dose 10 mL exposes the patient to potential contamination is within safe limits, that is, below the <200 EU per 10 mL of medication, which is less than the USP’s threshold for pyrogenic reactions. These adverse effects are also dependent on route and rate of administration. If an IV drug is infused over a period of time that exceeds one hour, the dose may be divided by the number of hours to determine the dose- The dilutions specified herein were recommended to avoid per-hour. Validation of BET methods, including extent of pre-test inhibitory test conditions, where recovery of the positive control dilution, is done after endotoxin limits are established, based on is < 50%. There is one known non-specific activator of LAL factors such as dose, infusion rate and route of administration. reagent that produces enhancement or endotoxin-like reactivity, Endotoxin may be expressed in EU/mg of a specific medication usually accompanied by enhanced recovery of the positive or EU/mL of a mixture of medications in solution. The term control of < 200%. This agent is a β-D-glucan that is found in pyrogen-free may be applied to a CSP that contains endotoxin yeast cell wall and in cellulosic materials.4 Therefore, suspending agents that contain CMC or other cellulosic materials may give a falsely high endotoxin value. If not solved by dilution, this Manufactured injectable products are made in specific enhancement interference may be avoided by using an concentrations, which simplify the calculation of endotoxin limits. endotoxin-specific reagent supplied by Charles River.
However, CSPs vary in drug concentration and composition of mixtures, as required by prescription, which complicates the calculation of limits. Therefore, this discussion presents a method of determining the safety of a medication by grouping 1. Cooper JF and Thoma LA. Screening extemporaneously them by concentration, knowing that interfering conditions are compounded intraspinal injections with the bacterial endotoxin concentration dependent. The objective is to assure that an test, Am J Health-Syst Pharm, 59:22426-33. Dec. 15, 2002.
adult patient (specified as 70 kg.) receives less than 350 EU per 2. Williams KL. 2001. Endotoxins, 2nd Edition, Marcel Dekker, dose per hour of contamination in an IV or IM CSP or less than 14 EU/mL/day in an intraspinal infusion. Pediatric doses would have to meet a limit of 5 EU/kg of medication. The dilutions 3. Bennett G, Burchiel K, Buchser E et al. Clinical guidelines for recommended here apply validations done with Endosafe® LAL intraspinal infusion: report of an expert panel. J Pain Symptom reagents, which are buffered and otherwise highly interference resistant; reagents from other LAL suppliers may not give valid results under these test conditions.
4. Cooper JF, Weary ME, and FT Jordan. 1996. The impact of non-endotoxin LAL-reactive materials on Limulus amebocyte To exemplify how this approach works, let us consider a CSP lysate analyses. PDA Jour Parent Sci Tech. 51:2-6.
mixture that has a maximum dose of 10 mL, injected as a bolus of medication. The mixture contained >20 mg/mL of drug substances, so a dilution of 1:400 was made to avoid test USA T: + 1 877 CRIVER 1 • E: [email protected] • www.criver.com EUROPE T: + 00 800 15 78 97 43 • E: [email protected] 2008, Charles River Laboratories International, Inc.

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