Chapter -2. Materials and Methods

Chapter -2 Materials and Methods 21

2.1 Validation of Bacterial Endotoxins Test Methods: Bacterial Endotoxins test (BET) method validation is used to document that a BET procedure will detect Endotoxin in a specific drug product or Medical device extract without interference. This discussion addresses the components of BET validation. Overview: The critical elements in a BET validation are the formulations of the test material, LAL reagent and CSE. LAL reagents of FDA-approved suppliers differ in composition and manner in which they are processed. Differences include metal cation, protein, buffer and surfactant content. Test materials that are water extracts, low-concentration solutions and ph neutral exhibit a similar compatibility profile in the various reagents, i.e., little or no detectable differences in interference properties. However, more complicated drug formulae, which have buffers, metal chelators and components that aggregate Endotoxin may react differently in each reagent. Objective: The goal of a BET validation is to find a valid, compatible range (concentration or dilution) for routine LAL testing of the material in question. The following summary of steps required to validate a BET method in today's regulatory environment and technically advanced state of LAL testing. Steps in BET Method Validation: State the objective. Describe the product. Determine the Endotoxin limit. Define validation experiments. Conduct physical measurements. Define validation conditions. Prepare the validation report. 22

2.1 Validation of Bacterial Endotoxins Test Methods Validation of a new BET method should follow a validation protocol which encompasses the basic validation steps and satisfies regulatory 83 and pharmacopeial requirements. Description of Test Material: The components of the test material must be described in detail. A description of a pharmaceutical includes the generic names, potency and fills volumes of the product and other product specifications. A method for rehydrating a lyophilized product is needed to determine the resultant potency. A description of a medical device includes the name and fluid pathway or parts of the device that contact tissue. 2.2 Endotoxin Limit (EL): The tolerance limit and maximum human dose per hour, as described in directions for use, are required to calculate the Endotoxin limit by the K/M formula. It is prudent to check clinical publications to determine if there are dosage patterns that exceed the labeled dose range. Pharmacopeial limits are used, if available. The current IP, USP, EP, BP,JP will contain the most comprehensive list of Endotoxin limits. If differences occur between pharmacopeia, it is prudent to use the most conservative one applicable in the marketing area. The category of the test material may fall in one of the unique EL classes such as a radioactive drug 78, infusion solution or intrathecal material. The EL for combination drugs, (multivitamins, etc.) are best determined by using the volume of the dose in the K/M formula. The Endotoxin limit is the keystone for designing LAL-test protocols that ensure the absence of unsafe levels of Endotoxin in parenteral products. Because Endotoxin is ubiquitous in nature, it was necessary to assign an allowable EL (safe amount of Endotoxin) for a health care product that was below the threshold dose of Endotoxin for pyrogenicity. 23

2.2 Endotoxin Limit The K/M formula The K/M formula was presented in the FDA LAL-test Guideline (1987) as a way to calculate the Endotoxin limit, as shown below. Endotoxin Limit (EL) = K (tolerance limit) M (maximum dose/kg) (hr.) The EL represents the maximum safe amount of Endotoxin that is allowed in a dose of specific drug. The tolerance limit (k) of 5 Endotoxin units (EU) /kg represents the approximate threshold pyrogenic dose in humans and rabbits from a purified LPS extracted from E.coli. The M in the formula takes into consideration that pharmacological effects are dependent on the amount of Endotoxin in the administered dose. If the Endotoxin tolerance limit (EU/kg) is divided by the maximum dose (mg/kg) per hour, a dose related limit can be calculated for drug products. The K/M formula enables one to calculate the extent of dilution required to test a specimen at its products-specific limit. The EL is inversely related to the dose of a drug. Seventy kg is the average human weight used to calculate an EL; however, if the pediatric dose per kilogram is higher than the adult dose; the pediatric dose is applied to the formula. It may useful to calculate an EL based on 350 EU per dose (EU/kg x 70 kg), such as determining the EL for a multi-component drug. The formula for calculating the Endotoxin limit by body surface area is the following: Endotoxin limit = (5 EU/kg) (70 kg) (dose) (1.8 m 2 ) Endotoxin limit for CSF Drugs: For parenteral drugs administered into cerebrospinal fluid (CSF) know as intrathecal administration, K is reduced to 0.2 EU/kg. this lower limit is based on the finding that intralecethal administration is the most toxic route for Endotoxin. 24

2.2 Endotoxin Limit As little as 1 ml of a nuclear imaging agent that was contaminated by Endotoxin produced 68 aseptic meningitis when injected into CSF (Cooper and Harbert 1975). Intraspinal infusions for pain management are subject to this concern 62. Pyrogenic Response to Endotoxins: Analysis of Endotoxin challenge studies and inadvertent pyrogenic outbreaks shed light on the adequacy of ELs. Reports vary as to human sensitivity to pyrogen (Greisman and Hornick 1969; Hochstein, et al. 1994). A study of EC-5 reference Endotoxin in human male volunteers determined that the threshold pyrogenic dose (>1.0 F rise in 50% of volunteers) was 4.1 EU/kg. (Hochstein et.al 1994). Pearson (1985) observed that solutions containing native Endotoxin required at least 80 EU/kg to consistently cause pyrogenicity in rabbits, where the EU values were determined by LAL assays. Endotoxin Alert Levels: Pyrogenic reactions to parenterals made under current good manufacturing practice conditions are rare; however, hemodialysis clinics have has occasional problems (Cooper, 2000). FDA surveillance study (2526 drug samples) 20 years ago found detectable Endotoxin in 3% of products, one of which was pyrogenic in rabbits. Contamination was associated with products of natural origin and emanated from the bulk material (Twohy 1986). Fanning (2000) reported on centers for Disease Control and FDA epidemiological investigations of a cluster of Gentamycin-related pyrogenic reactions 67 which 210 occurred in 155 patients. The typical reaction was fever, chills, rigors and shivering that began within 3 hours of the start of infusion. Most reactions were not serious, but five (3%) required support in intensive care settings. The reactions were caused by Gentamycin made by the same bulk supplier. The Gentamycin has less than the EL of 1.7 EU/mg, but a trend toward administering the total dose once daily rather than in three divided doses slightly less that 5 EU/kg. The USP subsequently lowered the limit from 1.7 to 0.71 EU/mg in light of these data. This execution reinforces the merit of an (Endotoxin Allowable Limit) EAL for bulk materials. A suitable EAL is 4-to-5 times more stringent than the EL, provided the material is compatible with LAL at higher test concentrations 8. 25

2.3 Validation Experiments: The experiments needed to document non-interference must be anticipated at the outset to assure the task is done completely and expeditiously. The principal experiment for a drug product is an interference screen with positive controls to find the dilution or concentration that is compatible with one or more reagents. A measurement of ph is needed to determine if ph contributes to interference. Solubility may be an issue for colloids, suspensions or drugs immiscible with water. Experimentation may be required to devise a meaningful, reliable way to elute complex devices so that tissue sensitive parts or fluid pathways are evaluated. Influence of ph: A ph profile is an essential part of validation for products that are buffered or have a ph range other than neutral. As an example, an antibacterial agent, having a ph range of 9 to 11, was diluted with LRW. The ph was measured before and after mixing equal parts of each solution with LAL reagent. The results demonstrate the importance of dilution and use of buffered LAL for neutralization. Although the ph of the mixture of LAL and sample (SPL) diluted to 1:10 was within the USP allowable range, only concentrations 0.5 mg/ml allowed full recovery of the Endotoxin spike because of other interference mechanisms. Data must verify that the validated method neutralizes the most extreme ph. Use of buffers and surface active agents in validated methods is discouraged for the following reasons: 1) LAL reagents are extremely sensitive to ph, divalent cations and surface active agents. 2) The effect of these agents varies with each batch of drug product and LAL reagent. It is difficult to know whether an additive has overcome inhibition or just enhanced the recovery of the Endotoxin control. Reliance on LRW dilution to resolve inhibition simplifies the validated method and avoids. 26

2.3 Validation Experiments 3) Lot-to-lot variation in recovery. A ph-related interference may be avoided by selecting a properly buffered LAL. Of course, there are exceptions to the caution against buffers. For example, anticoagulant solutions containing citrate cannot be tested without both magnesium replacement and neutralization. As previously discussed, many raw materials present neutralization and dissolution problems that may require additives. Compatibility Profile: A compatibility profile is an extension of a preliminary screen for inhibition. The sole purpose of this profile is to find the compatible concentration (CC) of the drug entity that doesn't inhibit or enhance Endotoxin recovery. The traditional way to find compatibility is to test a series of 2-fold dilutions of a sample and identify the range where the Positive Product Control (PPC) is fully recovered. The 2 PPC in gel clot methods only assures 50% recovery; a more informative method is a screen with at least 2 LAL manufacturers to gain comprehensive knowledge about interference and discover how efficiently dilution resolves interference. 1. The LAL-compatible concentration for most potent pharmacological agents is usually 5 mg/ml or less. Assays of 2-fold dilutions in an 8-to- 0.03 mg/ml series usually reveal the compatible range. 2. Sensitivity is not an issue during preliminary tests because sensitivity may be adjusted after compatibility studies are analyzed. For simplicity, one may select a 0.125 EU/mL/ 0.0625 EU/mL or 0.0312 EU/mL for a gel-clot screen. A compatibility profile provides valuable information about the interference characteristics of a test material. First, the profile reveals how efficiently dilution with LRW resolves interference conditions. Most drugs require a 100- fold dilution to go from no recovery to ideal compatibility with LAL. Some require a 4 to 10-fold dilution or greater to go from 50 to 100% recovery. 78 Also, the presence of unsuspected glucan contamination may be revealed by evaluating enhancement of PPCs in kinetic studies. Finally, the profile may show that one LAL method is much more suitable than the others. 27

2.4 Procedure for LAL test: Equal volume of test sample and LAL reagent is added in a test tube of 10 X 75mm and incubate this mixture at 37± 1 C for 60±2 min. Then invert the tube by 180 and look for Gel formation. If a gel inside the test tube able maintain its integrity after inverting tube to 180 then it is a positive reaction which indicates presence of Endotoxin in the sample. Other than this any condition is considered as negative which indicates absence of Endotoxin in the sample. Reagents required to carry out LAL test 1. Lyophilized LAL reagent, Sensitivity (λ EU/mL) 2. Control Standard Endotoxin(CSE), for control curve and PPC 3. LAL Reagent Water (LRW). Control Standard Endotoxin (CSE) Preamble, Bacterial Endotoxins Test, Pharmacopoeial Forum 26: 218 19, 2000 The use of in-house standards shown to be equivalent to USP Reference Standards is permitted under the requirements for alternative methods in the General Notices. CSE is the in-house standard. SOP is in compliance if it requires. 1. Acceptance of CoA from the CSE vendor 2. Completion of a label claim verification Accessories required to carry out LAL test 1. Vortex mixture 2. Heating Block 3. Depyrogenated Glass test tubes for Assay (10X75mm) 4. Depyrogenated Glass test tubes for dilutions 5. Micropipette 6. Sterile micropipette tips 7. Timer (Stop watch) 28

2.4 Procedure for LAL test Procedure to carry out control curve Reconstitute CSE referring to CoA. Dilute the CSE to 2λ, λ, 1/2λ, 1/4λ where λ is sensitivity of Lysate. Add LAL to above dilutions and also in Blank (LRW). Incubate tubes at 37± 1 C for 60±2 min. Tubes LRW CSE LAL Blank 100µL 1------ 100µL 2λ ------ 100µL 2λ 100µL λ ------ 100µL λ 100µL ½λ ------ 100µL ½λ 100µL ¼λ ------ 100µL ¼λ 100µL Table: 2.1 Results of Control Curve 1. Valid I II III Blank -- -- -- 2λ(0.25EU/mL) ++ ++ ++ λ(0.125eu/ml) ++ ++ -- ½λ(0.0625EU/mL) -- ++ -- ¼λ(0.03125EU/mL) -- -- -- Table: 2.2 2. Invalid I II III Blank ++ -- -- 2λ(0.25EU/mL) ++ ++ -- λ(0.125eu/ml) ++ ++ -- ½λ(0.0625EU/mL) ++ ++ -- ¼λ(0.03125EU/mL) ++ ++ -- Table: 2.3 Calculation of Geometric Mean Formula: GM end point Concentration = antilog (Σe/ƒ) Where, Σe = Sum of log of Endpoint Concentrations ƒ = Number of Replicates 29

2.4 Procedure for LAL test Calculation: GM = log (0.125) + log (0.125) + log (0.0625) + log (0.0625) 4 = Anti[(-0.9030) + (-9030) + (-1.2041)+ (-1.2041)] 4 = Antilog [(-1.0536)] = 0.0883 EU/mL If GM endpoint is between 2λ and l/2 λthen results are acceptable and test acceptance criteria are met. Depyrogenation: The best way to make Endotoxin-free preparations is to avoid introduction of contamination 72-74. Rinsing with water for injection is an effective way to dehydrogenate and reduce particulars in vials destined for a terminal sterilization process. Dry-heat sterilization 76 is effective in rendering glassware and heat-stable salts or materials free of Endotoxin. The USP suggests that a dehydrogenation cycle should reduce an Endotoxin indicator by at least 1000-fold (3-log) reduction as measured by LAL methods. 7 Endotoxin indicators contain known amounts of Endotoxin in 1-to-10 thousand EU/vial. A cycle validation study requires placement of indicator vials throughout a dryheat oven that is filled with a typical load of materials, exposure to a specified heat cycle and LAL assay to determine Endotoxin reduction. Exposure to 250 C cycles for 30-to 60 minutes (dwell time) is common. For more details, refer to the section that discusses the BET for monitoring aseptic processing. 30

2.5 Endotoxin Reference & Control Standards: Endotoxin Reference Standards: The early years of LAL were troubled by poor reproducibility between labs because of non-uniform potency for Endotoxin preparations. The industry and FDA recognized that a single, well characterized Endotoxin compound was needed to standardize the test it would be used to establish the sensitivity of LAL reagents and control routine testing. The search for a standard was complicated by the fact that potency varied with method of purification, bacterial origin and formulation. Rudbach was engaged by the USP and FDA to resolve this issue. The objective of his efforts was selection and preparation of a reference Endotoxin that was free of biologically active proteins, had a representative biological activity could be chemically characterized and would remain stable upon lyophillization 8. A 30 gram batch of reference Lipopolysaccharide was prepared from E.coli 0113:H10:K (-) that met these criteria. This LPS was used to prepare EC preparations, which serve as RSE. The reference Endotoxin was designated E.C. A 30,000-vial lot was made by Don Mills at Mallinckrodt and designed EC-5 with a potency of 10,000 EU/vial. Bulk EC was dried under vacuum at 50 C and dissolved in a solution of 2 % lactose and 2% polyethylene glycol 6000 prior to filling and lyophillization. The standard was divided between the FDA and USP and designated as EC-5 and RSE, respectively. A human pyrogenicity study using EC-5 confirmed that the 5 EU/kg Endotoxin limit was realistic 9. Problems of uniformity and standardization returned early in this decade when the European Pharmacopoeia adopted as its standard a liquid, highly refined Endotoxin known as BRP (Biological Reference Preparation). The WHO, USP and FDA collaborated on preparation and characterization of a new international standard under the direction of steven poole in the UK. 49 six thousand vials were used in 3 sub lots; one became RSE lot G of the USP and EC-6 for the FDA and the balance was designated as IS, International standard preparation 94/580. An international collaborative study ascertained uniform potency throughout the lot and an equivalent potency to EC-5. Therefore, the new lot was assigned and activity of 10,000 IU per vial. All LAL methods and standard form of EP and JP were used in the 31

2.5 Endotoxin Reference & Control Standards collaborative study. The WHO accepted the new IS for Endotoxin based on the equivalency finding, therefore one IU (EP) equals one EU (USP). This international acceptance of the IS Endotoxin is an important step in global harmonization for Endotoxin testing. FUNCTION OF THE REFERENCE STANDARD ENDOTOXIN (RSE) The RSE has three critical functions. Through its licensing process, the FDA requires all LAL producers to determine the sensitivity of LAL by an assay of a two-fold dilution series of RSE made from 1 EU/mL. The FDA has a sub-lot of the international standard, known in the FDA as EC-6, which LAL suppliers use for sensitivity assays (thus, 1 EU=11U). Secondly, the RSE is the primary standard used by LAL. Finally, RSE is used when a reference test is needed to resolve a disagreement over test results 50. Purpose of CSE preparations: Since RSE is expensive and potentially exhaustible, Control Standard Endotoxin(CSE) is produced by LAL-reagent suppliers for routine work. The sole purpose of a CSE is to serve as a surrogate for RSE during routine Endotoxin testing. It is not designed to assay the sensitivity of LAL. The roles of CSE are to confirm test validity by recovery of positive product controls and to verify control of test parameters (e.g., reagents, glass, accessories, analyst proficiency) through recovery of label claim. The consequences of inaccurate or improperly used RSE/CSE rations are costly. In gel-clot testing, the results of an inaccurate RSE/CSE ratio are: 1. Invalidation of results due to failure to verify CSE potency, or 2. Potential underestimation of Endotoxin contamination because the PPCs were strong enough to mask inhibition. The consequences of inaccurate ratios in kinetic LAL testing are even more serious because the CSE is used to generate the standard curve, which determines Endotoxin values in unknown. If the ratio is used incorrectly, there is a danger of under-or over-estimating Endotoxin contamination in a simple. Variability of CSE potency The potency assigned to EC-5 and EC-6 was 10 Eu/ng. (6) Analysts who were unaware of the variability problems associated with Endotoxin preparations expected CSE to have the same ratio. In response to this expectation, there developed in the 1980s a tradition in the industry whereby any RSE/CSE ratio 32

2.5 Endotoxin Reference & Control Standards in the range of 5 and 15 EU/ng was rounded off to ten. Although rounding off to a uniform RSE/CSE ratio may appear convenient, that practice is highly discouraged because it introduces error and added variation into routine testing. New LAL analysts are often concerned that the RSE/CSE ratio of one CSE varies with different LAL reagents. Variations related to the biological origins of LAL and CSE, and the limitations of titer-test analysis for each preclude the possibility of a precise, uniform ratio. The principal reason for CSE potency variation is that the two-fold dilution interval used to define reactivity results is an approximation rather than an exact number. Also, mg amounts of Endotoxin are weighed and greatly diluted, causing more variation. The gelclot method of the BET requires that CSE potency is found by testing a parallel, two fold dilution series of RSE and CSE. The geometric mean endpoint of the RSE series in EU/mL is divided by the GM endpoint of the CSE series in ng/ml to give a calibration ratio in EU/ng. Assignment of LAL reagent sensitivity from a 2-fold dilution assay introduces additional variation. For example, a reagent labeled 0.25 EU/mL means that the LAL consistently clotted at label claim; however, the sensitivity actually may approach 0.125 EU/mL. Since LAL sensitivity and CSE potency determinations are both done by 2-fold dilution, both are approximate values that may either add to or substract from the extent of variation. Every LAL/CSE pair is unique. Table: 2.4 LAL Lot A LAL Lot B RSE endpoint 0.125 EU/mL 0.125 EU/mL CSE Lot X endpoint 0.0125 ng/ml 0.0088 ng/ml Ratio 10 EU/ng 14 EU/ng In this example, label claim with RSE was verified in both cases, but there was subtle variation in the CSE endpoints. It s in the best interest of LAL users for vendors to provide ratios that are not rounded off because they accurately reflect the true relationship between a given LAL and CSE combination. When properly used, the ratio allows the CSE to properly substitute for RSE as a source of positive controls. 33

2.5 Endotoxin Reference & Control Standards Certificate of Analysis (CoA) for CSE: Until this year, the CoA of specific LAL-CSE combination had a certificate that the RSE/CSE ratio was determined in accordance with the USP s <85> BET (Bacterial Endotoxin Test 54. Since ratio determination is not addressed by the current, harmonized BET, CSE suppliers make the ratio determination according to the previous USP 80 chapter <85>. Labeling of CSE in EU by other means is not meaningful. The CoA and apply correct information. The Endosafe CoA specifies how to rehydrate the CSE to a concentration that is easily diluted to 1 EU/mL. There are reports that some FDA inspectors have required the RSE/CSE ratio be made on site rather than accepting the CoA of the LAL supplier, but this issue should become a moot point if the FDA accepts the harmonized BET as the minimum requirement for routine LAL testing. Unique steps are taken at C.R. Endosafe to verify the accuracy of RSE/CSE ratios. Historical kinetic data are used to assure the potency of an EC-6 standard before using it for sensitivity or ratios studies. Also after a ratio is determined, a corroborative study is done, using the proposed ratio, to confirm label claim. For kinetic LAL reagents, the confirming assay must also recover RSE spikes. This extra effort assures accuracy. Preparation of the Endotoxin Dilution series and positive controls Interference controls: Endotoxin standards are used to demonstrate that there is no interference in a LAL assay that would mask Endotoxin contamination in a test sample 64. In gel-clot methods, the interference control is an amount of Endotoxin added to a test sample that equals 2 λ, double the labeled sensitivity of the LAL reagent. The most reproducible and interference-resistant way to prepare this control is to add 10µL of a 20-λ CSE solution to tubes designated a positive product controls (PPCs), just prior to LAL addition; this is sometimes referred to as the hot-spike technique. Mixing equal parts of 4λ and double concentrations of test sample represent an earlier method for making the PPC, but it requires preparation of a separate solution. 34

2.5 Endotoxin Reference & Control Standards Endotoxin dilution series (EDS): Natural Endotoxins remain dispersed in aqueous media for long periods of time; no vortex mixing is required. However, low levels of purified Endotoxins are not very stable to dispersion in water. Because of this instability, the most difficult aspect of LAL testing is successful preparation and maintenance from the EDS 84, a two-fold dilution of LPS from 2λ to 0.25λ. from the EDS is prepared the positive water control and a positive product control (PPS, interference control). LPS tends to aggregate into larger macromolecules and also bind somewhat to containers. Both effects limit the shelf stability of very dilute Endotoxin solutions (<0.1 EU/mL). It is imperative to follow the vendor s instructions for preparing Endotoxin solutions. New products were recently introduced to minimize the problems associated with daily preparation and storage of an Endotoxin standard dilution series. A liquid standard was presented, but it failed to meet the requirements of the BET for CSE because it is not an end-point dilutions series that confirms the potency of the PPC. Another product was presented that followed the BET and also has certified long-term storage. The CSE kit contains 50-mL Endotoxin diluents for ED s preparation that contains low levels of an Endotoxin dispersing agent and bacteriostatic agent. The kit lasts until consumed or for at least 4 months. 35