Preparation and Use of Endotoxin Indicators for Depyrogenation Process Studies

TECHNOLOGY/APPLICATIONS Preparation and Use of Endotoxin Indicators for Depyrogenation Process Studies LAL Users GroupL> Preface charged membrane surfaces, hydrophobic attraction, or various other methods. Depyrogenation by inactivation on This article was developed by a subcommittee of the heat stable surfaces is usually accomplished by long expo LAL Users Group, an organization of scientists dedicated sure to high temperature, and otherwise by acid/base to the development and application of Limulus Amebo hydrolysis, oxidation, alkylation, moist heat, or ionizing cyte Lysate technology. Comprised of members from vari radiation. There are several excellent references available ous pharmaceutical and medical device manufacturers which include detailed discussions on the particulars of and other related industries, the primary objective of th~ each method (1-3). group is to guide the membership into the routine usage of Verification of dry heat depyrogenation may be sup LAL methodology. This includes the coordination of com ported by data showing the relationship of thermal treat pendia! requirements, vendor ;user relationships, routine ment to endotoxin inactivation. Studies by Tsuji, Robert handling procedures, and reagent evaluation. son and co-workers ( 4-7) demonstrated that the inactiva LAL Users Group Endotoxin Indicator Subcommittee: tion kinetics of lipopolysaccharide derived from various John Shirtz (Chairman), Burroughs Wellcome Co. gram-negative organisms were nearly identical to each Ed Seelig, Wyeth/ Ayerst Laboratories other, and that dry-heat D-values of intact whole cells at Karen McCullough, LAL Consultant 210°C could be calculated. They further demonstrated Betsy Waldheim, Organon, Inc. that the inactivation curves for purified endotoxin could Tim Schimmel, Merck & Co. be made linear and that inactivation for any dry heat Joan Craig, Smith Kline & French Laboratories process could be predicted given the product heating John Dubczak, Baxter Healthcare curve. These studies served to demonstrate the correlation between purified endotoxin and whole cells and helped Background establish the foundation for using purified endotoxin in depyrogenation studies as a predictor of whole cell (both The introduction of endotoxin from gram-negative bac intact cells and cellular debris) inactivation. teria into the bloodstream has long been recognized as a Akers and co-workers (8, 9) carried this calculation potentially serious condition. These bacterial endotoxins, or pyrogens. are fever-producing substances present in the concept one step further by developing F-value require ments for the destruction of endotoxin. This approach is outer cell membrane which retain their potency upon lysis also detailed in the Parenteral Drug Association Techni of the cell. The health industry employs production proce cal Report #3 (10). In a more recent study, Ludwig and dures that include a means for depyrogenation to insure that parenteral products, medical devices. and compo Avis (II) reported F-values which were significantly dif ferent from those reported by Akers, and concluded that nents are free of these pyrogens. In addition, quality con substantial endotoxin reduction can be achieved using trol procedures usually include analytical methods for temperatures as low as 170°C. These experiments, howev process validation and monitoring to assure that pyrogens er, utilized a specially designed heating cell with precision have been eliminated during processing. Depyrogenation is defined as the elimination of all py control and were not intended to mimic a routine produc rogenic substances, including bacterial endotoxin, and is tion depyrogenation cycle. None of these studies, howev er, includes any detailed technique for preparing and re generally achieved either by removal or inactivation. The covering endotoxin challenges analogous to the traditional specific method to be used is determined by both the Biological Indicator which has been a valuable tool for physiochemical nature of the endotoxin and the nature of many years in sterilization validation and monitoring. the material to be depyrogenated. Depyrogenation by re The employment of an endotoxin challenge in depyro moval may be accomplished by reverse osmosis, ultrafil genation validation is an important complement to ther tration, affinity chromatography, dilution, through ad mal profile monitoring. In their chapter on Sterilization sorption to a specfic medium such as charcoal or positively and Sterility Assurance of Compendia! Articles, USP ( 12) recommends an endotoxin challenge be included with Received October 17, 1988. Accepted for publication January 19 any dry heat process intended to render glassware or 1989. containers free of pyrogens. They also note that the depyr "Author to whom correspondence should be addressed: Mr. John T. Shirtz. Burroughs Wellcome Co .• P.O. Box 1887. Greenville. NC 27834- ogenation process should demonstrate a minimum 3 log 1887. cycle reduction in the difference between the recoverable Vol. 43, No. 3 I May-June 1989 109 endotoxin level and the residual endotoxin level.' The but may also be performed with an endotoxin-free pipet.· specifics of preparation and recovery. however. are left up syringe, or with any other similar equipment which can to the user. accurately and consistently deliver the desired volume. It Considering the numerous variables that the prepara has been suggested that the use of a surfactant may also be tion, handling. and evaluation of an endotoxin challenge included in the inoculum diluent if it has been demonstrat can involve. the following is presented as a guide to facili ed to enhance recoverability of the endotoxin yet not tate the use of such challenges. The primary application of interfere with the depyrogenation process. the challenge, or "Endotoxin Indicator," would be for dry It is generally recognized that the number of recover heat depyrogenation, but the concepts could be applied able endotoxin units may vary from the theoretical inocu and/or modified for various other endotoxin reduction lated level, depending on a variety of factors such as techniques. endotoxin concentration in the original inoculum (the more potent the inoculum, the higher the recovery), glass Endotoxin Indicator type and shape, drying method. recovery process, etc. Drying or fixing of endotoxin onto representative chal An Endotoxin Indicator is a carrier to which a known lenge components may be conducted in one of several amount of endotoxin is added, and is used to measure ways. The easiest is to subject the inoculated units to endotoxin reduction or inactivation in a depyrogenation ambient laminar flow, although the use of an elevated process. It serves i.n a capacity similar to a Biological temperature (not exceeding 50°C), such as in an incuba Indicator in a sterilization process and is used to further tor, may be preferred to hasten the process. Vacuum dry demonstrate heat penetration and distribution data estab ing or lyophilization have been reported to be very effi lished by physical measurement. Endotoxin Indicators are cient fixation methods for both the quick drying of endo prepared from a purified lipopolysaccharide which has toxin onto challenge units, and for excellent recovery of been adequately characterized against the current Refer endotoxin once fixed ( 13, 14 ). ence Standard Endotoxin (RSE). This material may be obtained through a number of sources including licensed Preliminary Testing lysate manufacturers or bacteriological media distribu tors as a lyophilized stock. It is incumbent upon the user to confirm the validity of the Endotoxin Indicator before its use as a tool for the Carrier evaluation of routine depyrogenation processes: 1) The endotoxin concentration both in the spiking Different surfaces and materials may contribute in solution (the "initial spike") as well as in the dried varying degrees towards the effectiveness of the depyro reconstituted control vial ("recoverable endotoxin") genation process. Therefore, the unit which serves as a must be determined. It is also recommended that carrier for the endotoxin should be the same as or chemi each laboratory determine the percent recoverable cally similar to that which is being depyrogenated. A endotoxin from each type of container used as an universal carrier, such as a small colored vial or planchet, indicator, as recovery may vary widely depending on may be developed for numerous applications provided the the chemical composition and configuration of the thermal characteristics of the universal carrier have been unit employed (13, 14). shown to be at least as resistant to the depyrogenation 2) A key issue in the fixing process remains the ques process as the item being studied. Depyrogenation of the tion of whether any additives, such as surfactants carrier prior to its use ( 12) is necessary to minimize the used in the spiking solution to increase endotoxin possibility of false positives due to intrinsic endotoxin recovery, are likely to enhance the depyrogenation contamination. All inoculated units must be clearly iden capability of the process being studied (thus in a tified for easy retrieval after processing. false negative sense enhancing the overall outcome of the validation exercise on the challenge units Inoculation only). This concern would usually impact only those The lyophilized endotoxin should be reconstituted and processes wherein the properties of the additive may diluted with a sufficient

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