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    TT LE usp 40 General Information / (1228.5) Endotoxin Indicators for Depyrogenation 1803 APPENDIX Additional References McCullough KZ. Structuring a depyrogenation study. In: MeCullough KZ, editor. The bacterial endotoxins test: a practical, ‘Quid, River Grove It: Davis Healthcare Intemational, LLC: 2011. Mandaro RM. Chapter 5. Charge-modiied depth liters cationic-charge-moaiied nylon fers. : Meltzer TH, editor Fi tration in the pharmaceutical industry. New York: Marcel Dekker; 1986 (1228.5) ENDOTOXIN INDICATORS FOR DEPYROGENATION 1. INTRODUCTION 2. ENDOTOXIN AND LPS 3, APPLICATION OF ENDOTOXIN INDICATORS 4, PREPARATION AND USE OF ENDOTOXIN INDICATORS. 4.1 Methodology to Create a Laboratory-Prepared Endotoxin: Principles to Consider 42 Inocdation of Els 43 Recovery of Endotoxin fom Els 4.4 Choice of Test Methodology forthe Analysis of Els '5. ANALYSIS OF RESULTS OF DEPYROGENATION STUDIES |. INTRODUCTION Depyrogenation is defined as destruction or removal of pyragens (see Depyrogenaton (1228). For the purposes ofthis chap. ter, the terms “bacterial endotoxin” or “endotoxin” refer to a component ofthe outer cell membrane of Gram-negative bacte- Fi, which is known to induce a eb response in humans and other mammals. The endotoxin complex contains mary cell wall components ineluding, but not limited to, phospholipids, ipoproteins, and lipopolysaccharides. Lpopelysaccharie (LPS) isthe biologically active portion of bath naturally accurring and laboratory prepared endotoxin complexes. The USP Endotoxin Reference Standard (wich, by corwention, is abbreviated as RSE) and contro standard endotoxin (CSE) preparations pur- chase from lysate manufacturers and ather third party vendors are not endotoxins but rather are preparations of purified LPS “Endotoxin indicators” (Es are tools that are used (where required) in conjunction with physical measurements to analyze the effectiveness ofa depyrogenation proces, Es used to determine the effectiveness of dry heat depyrogenation processes ‘ate commonly purchased as giass vals that ate inaculated with a know level of LPS activity. This chapter expands the defini tion of Eto include any carrer, including glass Vials, inoculated with endotoxin or LPS that is used to challenge a depyrogena- tion process, scan be used to analyze the effectiveness of endotoxin remaval by washing, rinsing, cleaning, or by using Sepa- ration technologie, such a itration or chromatography. Cartes can be a variety of materials, including rubber stoppers to ‘asses topper-washing processes, bulk product to asest and validate processing steps, and stainless steel coupons to assess the cleaning of production ves. Puried LPS, suchas CSE obtained from lysate manufacturers or other third-party vendors, has historically been a convenient choice for ue a the analyte used in the preparation of Els, Havever, Els prepared in-house using laboralory-derived endotoxin ‘more closely mimic product contamination, and asa result can provide a more realist assessment of the depyrogenaling ca- pabilty of various production processes than does highly puried LPS. Ths chapter provides information onthe preparation land use ofthese more specialized indicators to assure both consistency and comparablity of data among method develop ‘met an validation studies, 2. ENDOTOXIN AND LPS ‘A bacterial endotoxin is defined inthe Invoduction. LPs the biologically active portion of the naturally occurring and labo- ratory prepared endotoxin complex. Highly purfied LPS, extracted from the natural endotoxin complex, is used to prepare the ‘ximary compendial RSE or secondary CSE preparations, such as those purchased from Limulus amebocyte sate (LAL) reagent ‘manufactorets. LPS consist of three dstinct regions 1. The structure of the hydrophobic lipid A portion of the molecule isthe most highly conserved among Gram-negative spe- cies and is responsible for most, not all of the biological activity of endotoxins 2. core oligosaccharide links the lipid A to the hydrophilic O-specific side chain or O-antigen 3. The hydrophiic O-antigen is a highly variable region that canfersserlagical specficty tothe organism and soften used ta distinguish strains of Gram-negative bacteria 1804 (1228.5) Endotoxin Indicators for Depyrogenation / General information usp 40 |When drug products and devices are contaminated with endotoxin, the contaminant isnot purified LPS but rather whole Gram-negative cells andjer cll wall fragments containing LPS. LPS and endotoxin ae therefore disimiar in many respects. The amphipathic nature ofthe LPS molecule [ke having both a polar (hydrophilic) end and a nonpolar (hydrophobic) end} enables it to form complicated, three-dimensional, aggregated structures in solution. The aggregated forms of LPS have the capacity to adsorb, or “stick”, to surlaces, and depending on the LPS formulation and the surace, extraction and detection ‘may prove dificult using conventional extraction methods (see below). The degree of aggregation af the purified molecule is aso affected by the conditions to which the LPS is exposed, Factors such as temperature, pH, salt concentration, divalent cat fon concentration, chelating agents, and detergents can have a profound effect on the biological activity and stability of LPS in Solution. Purilied LPS preparations used for depyrogenation studies should not contain any “les” or excipients. The exci pients that are commonly used inthe formulation of CSE have been shown to reduce the heat resistance of LPS (1) and may Intertere in the recovery of LPS because ofa caramelized excipient that has been post-processed by dry heat Endotoxins contaminating parenteral products may exhibit greater stability of activity insolation and les surface adsorption than purified LPS, As wel, the detection of endotoxin may be les influenced than LPS by aggregation, disaggregation, of oth- «er conformations inckiced by some product matrices. Information on principles to cansier when preparing endotoxin in the Taboratory can be found below 3. APPLICATION OF ENDOTOXIN INDICATORS The choice ofan El shouldbe relevant to the process being validated. For physical depyrogenation, such as dry heat, the carrier materia forthe El may be a srlace such ss a glass val or appropriate coupon material with documented heating char- acteristic similar to the materials Being processed, and onto which a known quant of LS or endotoxin has been inoculated For stopper washing/depyrogenation studies, stopper carers are inoculated with known levels of LPS or endotoxin. For raw ‘material or process intermediates that ae inherently contaminated with assayable levels of endotoxin activity, there may not bbe need to add LPS or endotoxin to validate endotoxin reduction in the manufacturing process, as the level of contamina tion may be sutfcient to accurately measure activity upstream and downstream ofthe depyrogenating sep(). For processes using raw materials or for upstream intermediates that are net contaminated with endotoxins, the use of ei ther the USP RSE or CSE, which are both highly purified preparations, may not reflect the actual removal or reduction potential Of the product stream depyrogenation steps) under challenge. For these purposes, endotoxins harvested from Gram-negative cultures may be more suitable for depyrogenation processes typicaly found in biopharmaceutical product streams. The cell ‘wall fragments and outer membrane consituens asociated with these endotoxins represent realistic challenges to process ‘operations such as ultrafitration, afinty chromatography, and the use of charged media membranes or columns ‘Challenge studies for LPS or endotoxin removal in process streams should be conducted atthe laboratory or lot scale so as rot to introduce high levels of endotoxin or LPS into the actual production environment. 4, PREPARATION AND USE OF ENDOTOXIN INDICATORS 4.1 Methodology to Create a Laboratory-Prepared Endotoxin: Principles to Consider lass vial Els purchased from third-party vendors do not need further preparation before use, These indicators are labeled ‘with a nominal value of inoculated LPS, and the label claim should be confirmed upon receipt to assure that there is sufficient activity (endotoxin unit, oF EU) available forthe stud, ‘There i not one “best” or “standard” method for preparing endotoxin in the laboratory, but one example ofa published ‘method forthe preparation of laboratory-prepared endotoxin may be found in Bowers and Tran (2). Regardless ofthe ‘methodology for preparation, the folowing recommendations should be considered to properly and consstently pr: duce, identiy, and maintain laboratory-prepared endotoxin for ue as a tool for depyrogenation studies. An appropriate Gram-negative bacterial strain rom a recognized eulture collection is a good choice for preparing alboratry-derwved endotoxin, Altematvely, a Gram-negative organism isolated from a facility, water system, raw material, of product that i identified to the species level that has been shown to be genetically stable and that i properly maintained, may ako be considered, Establishing the identity and baseline genetic fingerprint of an environmental organism wl assure that subse {quent preparations are consistent. +The laboratory should create detaled procedures or laboratory work instructions for culture maintenance and endotoxin preparation o assie consistency between batches of endotoxin, For example, endotoxin may be wolated from a culture (of Gram-negative bacteria, according to the method of Bowers and Tran (2) ora well-documented variation of that meth- fod, Whatever the methodology for growth and endotoxin isolation that is developed by the laboratory, the methodology "Should be documented and used consistenty + Consistent with good microbiological practice, the culture and maintenance ofthe cells used to produce a laboratory- ‘prepared endotoxin should be consistent with Microbiological Best Laboratory Practices (1117). Instructions on 1) the prop fer maintenance of the organism, 2) growth conditions, including any requirements to prepare media, nutient require ‘ments, and time/temperature of incubation; 3) methods for cryopreservation or lyophilization for master cll banks and working cell banks; 4 storage ofthe endotoxin, once prepared including concentration, vessel type, and volume; and 5) TT LE usp 40 General Information / (1228.5) Endotoxin Indicators for Depyrogenation 1805 master batch production records to assure consistency in subsequent studies should be writen, managed via change con- tro, and fellowes * Once isolated, the relative activity of the endotoxin preparation should be established by comparing is activity toa known LPS standard such as RSE, or a CSE that has been standardized against the RSE. Determination of activity in volves diluting the endotoxin preparation and assaying the dilutions against an LPS standard curve such that the re- suleof the dilution falls within the range ofthe referenced standard curv. A with the CSE standard used in the bac {eral endotoxins test (BET) assay ftom Bacteriol Endezoxns Test (85), the activity ofthe endotoxin may vary, depend ing onthe lot of lysate and lot of PS used forthe anayss. Its recommended, consistent withthe assignment of potency for the CSE, that activity of an endotoxin preparation be evaluated for each ysate manulacturer,Isate lt, land test method (gal, kinetic urbidimetric, or kinetic chromogenic) in use inthe laboratory. ‘The activity ofthe stock endotoxin preparation in EU/mLis reported as (Test result in EU/ml) x (lution factor = EU of the starting endotoxin preparation ‘Once activity has been determined, and i appicable tothe stxdy design, a standard series of lutions ofthe newly prepared endotoxin should demonstrate onset times that result in slope and intercept values that are consistent With the standard curve parameters ofthe RSE/CSE standard using the same lot of lysate. This demonstrates that the activity of endotoxin preparation dilutes and reacts withthe lysate in a manner that is similar to LPS, Characterization of the endotoxin preparation should als include data on the stability ofthe preparation, because Stability is critical tothe comparison of data from one study tothe next. Ifthe endotoxin preparation i stored, sto. age parameters including the concentration of the preparation in EU/miL, the composition of the vessel, the tempera {ure of storage, and the length of storage, should be defined. An expiration date should be asigned based on deter mined stability 4.2 Inoculation of Els To prepare an Ein house, inoculate endotoxin or LPS onto an article (carte) that wil serve as the substrate for the El. Cat- e's for Els can be anything that is subject to depyrogenation such as: vias (or dry heat depyrogenation), stoppers (fr stopper ashing), stainless stee! coupons (for vese cleaning), or product (lr depyrogenation of process stream). “The simplest way to inoculate these indicators i to adda small volume af a highly concentrated solution of endotoxin or LPS tothe carrer, The volume and concentration of added endotoxin o LPS should be caeulated to add at least 1000 EU to the carrie, although higher or lower concentrations may be justified based on historical data on the endotoxin content of the ‘material. [NoTe—The remaining discussion assumes a 1000-EU inoculum, but the principles hold for ay level of inital inocu- lum For noniiqud carrier, the endotoxin i “fixed” of dried onto the carrer substrate. This fixing step mast easy accom= plished by drying in a unilreciona airflow cabinet or hood, although other drying methods including vacuum drying, yo Dhilzation, and other fixation methods could be used. In depyrogenation challenge stucies, once afxing method is chosen, it should not change in subsequent studies to assure comparability of results Before using the Elsa recovery procedure, consist- ing fa reconstitution or extraction method, should be developed and verified for consistency (3). For liquid carers such as bulk product, the level of inoculation in EUYIml. should be justified based on “worst case” challenge for the depyrogenation step under study, meaning thatthe highest concentration of endotoxin that could be in the upstream product, based on process knowiedge and historical endotoxin values, should be used. Such justification should take all contsi- bating factors nto account, including but not limited to: Gram-negative bioburden in raw materials and bulk; endotoxin con- {ent in raw materals including water, contributions by product contact surfaces; and the effect of hold times, particularly for ronsterie bulk 4.3 Recovery of Endotoxin from Els ‘Touse fists necessary to recover and quantify the activity ofthe endotoxin or LPS from both unprocessed indicators (controls) and from processed indicators (., those that have been through the depyrogenation proces). LPS tends to adorb to surfaces and may aggregate or disaggregate in some product matrices; therefore, recovery of activity from Els made with LPS often not 100% af the nominal or measured spike value. This section addresses the methodology for recovery and poss: ble strategies for addressing recoveries that may be observed in challenge testing In the case of commercaly available Els prepared with LPS, Une manulacture's directions for extraction and recovery should be followed. With such products, there shouldbe tle dtfcuity in achieving recovery within a factor of 2 ofthe labeled LPS concentration. f recoveries within the specitied range cannot be achieved, the manuiacturer should be contacted for techaical assistance, For Els made in-house using LPS, the composition of carers, such as plastics, can alec recovery or result in inconsistent recovery because of adsorption. For these carers, there fs no prelabeled concentration to very. inthis case, the expected recovery should be based on the measure ofthe activity of endotoxin or LPS added tothe atcle and the volume of extraction fluid used to recover i. The actual (measured) activity inthe extract should then be compared to the measured activily of the {endotoxin or LPS added to determine the percentage of recovery. TT LE 1806 (1228.5) Endotoxin Indicators for Depyrogenation / General information usp 40 For example, considera stock endotoxin or LPS preparation containing a measured activity of 100,000 EU/mL that is used to prepare in-house El. I a volume of 50 iL of ths preparation i ned on the surface of each of a number of 1O-mL vial, the known amount of activity added is 5000 EV. I te recovery/extraction is performed in 5 ml of water for BET, andthe recovery 15 100%, the expected activity in the extract soltion is 1000 EU/mL. 5,000 Eu/via $Sml extraction solution/vial 1000 EU/mL however, the measured activity alter extraction is 200 EU/mL as opposed to the expected 1000 EU mL, the efficiency of the extraction method is 20%. Recovery of endotoxin of LPS from noniquid Els prepared in-house can follow recommendations fr the extraction of medi ‘al devices in preparation for LAL testing. Medical Devces—Bctera! Endotoxin and Pyrogen Tests (161) states, “The standard extraction method is to soak or immerse the device or flush the fluid pathway with extracting fluid that has been heated to 4371.0, keeping the extracting fui in contact with the relevant surface(s) for NLT Ih.” The volume uted for reconstitution ‘or extraction should be appropriate forthe material, size, and shape ofthe EL, recognizing that a volume to0 low may not efficiently recover the endotoxin or LPS and that excessive volumes will unnecessarily dilate the endotoxin or LPS thal has been extracted. the recovery of added endotoxin or LPS is variable, an alternate extraction method may be developed and validated. This ‘may include agitation or mixing, sonication or alternative extraction solutions. A combination of extraction in 0.01% sodium laurel sulfate, sonication, and vertex mixing fs ane such approach that has been reported to be more effective than extraction in water for medical devices (4-6). Other extraction methods are summarized by Bryans eta. (7) and in ANSWAANMI standard S172:2011 (). [Another stuation concerns liquid endotoxin of LPS preparations that ae used either to validate a depyrogenation process in a proces stream or to investigate the destruction or removal of endotoxin or LPS in a manufacturing process. In these case, the initial concentration of the stock quid endotoxin o LPS solution should be measured belore its added tothe sytem o& process If some of this preparation is added (“spiked”) to a bulk proces solution that is then subject toa particular process or treatment, the activity of endotoxin or LPS in this bulk solution should be measured and recorded as the starting activity. leis, important to determine whether changes in the endotoxin or LPS activity ofthe processed solution are due to effects ofthe process and not to instability ofthe LPS or endotoxin in the solution. The stability ofthe activity ofthe LPS or endatoxn in {hese preparations should be verified over a period appropriate to the proposed use ofthe preparation. — ‘As with the spiking method (choice of endotoxin/LPS and “Fixing” process), whichever reconsitution/extracton procedure is chosen should be verified for consistency and should be used forall subsequent studies to assure comparability f esl. 44 Choice of Test Methodology for the Analysis of Els ‘Any of the test methods described in (85) can be used for the analysis of processed and unprocessed Es. As with the rest of the methodology, its highly recommended that an assy (kinetic turbidimetic, kinetic chromogenic, or gel cot assay) be chosen during method development and used consistently throughout the inl study and in subsequent studies to assure that data are comparable. The use of alternate astay is permissible, provided that they are validated to asure that they are equivalent to or nonsnferor to the standard compendia assays 5. ANALYSIS OF RESULTS OF DEPYROGENATION STUDIES To evaluate the effectiveness of a depyrogenation proces, the residual activity tht is recovered from processed indicators i compared to the endotoxin or LPS activity of unprocessed contals. Typcally, te log, af the endotoxin or LPS activity meas Lured forthe processed El (or solution) is subtracted from the logy ofthe measured endotoxin or LPS activity of unprocessed Control indicators. The result of the subtraction is the log reduction that is attributable tothe depyrogenation proces. I there are multiple controls and/or samples of processed material (and there usually are), the most conservative approach isto sub: tract the highest log\, concentration recovered from the processed Es (or solution samples) from the lowest log, unprocessed contol endotoxin athvity For example ‘The activites in three unprocessed Els are 1286, 1000, and 1532 EU/mL, The activites in three processed Els ar 0.634, 0.512, and 0.496 EU/mL The log reduction is calculated as: og (1000) ~ tog 0.634 = 3 ~ (0.198) = 3.198 log reduction Historically, 2 23-ag reduction has been required by requlatory/compliance guidance, However, depending on the process and historical data, a 10g reduction may be either excesive or inadequate. For example, for glass vis witha low or non ‘measurable endotoxin content upan receipt, the requirement to continually and repeatedly revalidate with an acceptance cr terion of @3og reduction ofthe endotoxin spike of >1000 EU is excessive. Alteratively, a fermentation process with an endo- toxin content of =10° EU/mL in the cared culture supernatant will require more than 3-ag reduction to achieve sae levels Of endotoxin in te drug substance or drug product. Additional, given the sensitivity of BET assays, may not be necessary TT LE usp 40 General information / (1229) Steilization of Compendial Articles 1807 tw spike with 1000 EU to demonstrate a3-og reduction. For example, if te assay sensitivity is 0.005 EU/mL, one may choose to spike with $0 EU and demonstrate an uitimate recovery in the test articles of less than 0.05 EU/ml, which calculates to ‘greater than a 3-19 reduction. In ary event, the design of experiments including an appropriate specification for the log re- ‘duction of processed indicators and required test sensitivity to demonstrate the specie log reduction should be established and justified in a preapproved protocol forthe study. The total reduction, of course, may be achieved over several teps i a purification process. Thus, the necessary reduction soften achieved addtively over the course of multiple purification steps [REFERENCES Ludwig JD, Avis KE, Dry heat inactivation of endotoxin on the surface of glass. Parenter Sci Technol. 1990;44(1):4-12. Bowers K, Tran L. Creation of an in-house naturally occurring endotoxin preparation for use in endotoxin spiking studies and LAL sample hold time analyst. A Pharmaceut Rev. 2011;14(6):92-97. htp://wnw.americanpharmaceutiale- view.com/Featured-Artiies/37219-Creation-f-an-n-house-Naturaly.Occurting Endotoxin Preparation 4or-Use-in-Endo- toxin Spiking-Stadis-and-LAL Sample-Hold-Time-Analyss/ 3. LAL Users Group. Preparation and use of endotoxin indicator for depyrogenation process studies | Parenter Sci Technol 1989;43(3):109-112. 4. Berzolsky RN, Scheible LS, Willams KL, Validation of endotoxin removal from parenteral val closures. SioPharm. june 1994:58-66, 5. Ross VC, Twohy CW. Endotoxins and medical devices. Prog Clin Biol Res. 1985;189:267-28), {6 Twohy CW, Duran AP, Peeler JT Extraction of bacterial endotoxin from medical devices. | Parenter Sc Techno. 1986;40:287-291 7. Bryans TD, BraitwaiteC, Broad J, Cooper JF, Darnell KR, Hitchins VM, et al. Bacterial endotoxin testing: a report on the methods, background, data and regulatory history of extraction recovery efficiency. Biomed Instrum Technol. 2004;38(1): 73-78. 4 ANSIJAAMI standard S172:2011. Bacterial endotoxine—test methods, routine monitoring, and altematves to batch test ing. Afingion, VA: Association for te Advancement of Medical insumentaion; 2011. (1229) STERILIZATION OF COMPENDIAL ARTICLES BACKGROUND AND SCOPE This general information chapter provides an overview af the concepts and principles invalved in sterilization (by various ‘mades) of compensa articles that must be sterile. It includes information about supportive serlzation processes ublized in their preparation.’ Moce detailed recommendations are presented in speci information chapters for each sterilization mode: * Steam Steitzation by Direct Contact (1229.1 + Moist Heat Steriization of Aqueous Lguids (1229.2) * Monitoring af Boburden (1229.3) * Steiizng Fitton of Liquids (1229.4) * Biological Indicators or Steriizaion (1229.5) * Liquid Phase Stenkzation 1229.6) * Gaseous Sterizotion 1229.7) + Dry Heat Sterlization (1229.8) + Physicochemical integrators and Indicators for Sterilization (1229.9) + Ratition Steritza%en (1229.10) + Vapor Phase Steriization (1229.11 In the strictest definition of stery, a specimen is deemed sterie only when there isa complete absence of viable microor

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