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  • A Manufacturer's Guide To Depyrogenation

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    Surendar Kesavan
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    DEPYROGENATION GUIDE

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    A Manufacturer's Guide to Depyrogenation

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    DEPYROGENATION GUIDE

    Direitos autorais:

    © All Rights Reserved
    Baixe no formato PDF ou leia online no Scribd
    Sinalizar o conteúdo como inadequado
    243 visualizações6 páginas

    A Manufacturer's Guide To Depyrogenation

    Enviado por

    Surendar Kesavan
    DEPYROGENATION GUIDE

    Direitos autorais:

    © All Rights Reserved
    Baixe no formato PDF ou leia online no Scribd
    Sinalizar o conteúdo como inadequado
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    A Manufacturer’s Guide to Depyrogenation — the inactivation or re- moval of bacterial endotoxins — can be achieved in many ways. The depyrogena- tion method to be used is determined by both the physiochemical nature of the en. dotoxin and the nature of the material to be depyrogenated. The following feature ar cle describes the various depyragenation processes that are currently available; this | information can help the manufacturer se- lect the most appropriate process. uacurers of pharmaceutical products ae responsible Missive ett recs rnp ren that tet product wil not produce Tove reactors shenaiminatredtopatens- Alu potnia yori con tEminans ot pharmacetcls ean be eter mirobal or monm Era theone of mon concerto pharaeutal manufiaatrs 'themntconmon andy fart rs potent pen = haste Mendon Endotoxin an neprl component of te outer el sure of gram-negative bacteria (Figure 1). Toe endotoni shed rom rang beers date rccased mo eewironmen hen teria de and decompose Hecnusepranenepatie bacteria are found unversy iar, water ands tation cor ‘mony contaminates the vw ater ana processing equipment Shed nthe manufacturing of paraneals nits nar ste, tats endotoin acl that consists of tc: an pron: th compl acterized by se nepave charge. ht sab. and hgh molecular weit ‘When ily ports, endxonn aos no ora tia ‘Marlys Wears is manager of microbiologs/psragen technology at Baxter Healthcare Corporation, Box 490, Route 120 and Wilson Ra. Round Lake, IL 60073. Frederick Pearson Ill is vice presi dent of scientific operations at Cell Technolog’, Inc. 1658 Vltec Lane, Boulder, CO 80301, *To wnom correspondence should be addressed. Depyrogenation Marlys Weary* and Frederick Pearson 111 its chemical composition i referred to as a lipopolysaccharide (LPS) Depyrogenation can be achieved by inactivating or remo ing endotoxin. depending onthe physiochemical nature ofthe LPS, The Physiochemical Nature of LPS LPS consists of three distinet chemical regions: the innermost ip 1 A. the intermediate core polysaccharide, and the outermost O Specific polysaceharide side chain that is responsible forthe par Heularendotonin'simmunospecifiity (Figure 2). Lipid A. Lipid A is composed af a simple disaccharide of gl cosamine, winich is highly substituted with amine- and ester linked long-chain fatty acids (Figure 3). The most common Aamine-linked fatty acid is B-hydroxymyristic acid. a I-carbon, Straight chain. Ester-linked faty acids commonly found include capric. lauric. myristic. palmitic, and stearic acids — thats, stu ‘ated. straight-chain faity acids with even numbers of carbon a ‘om in their chains. The lipid A moiety is responsible for pyroge icity. as well as for complement activation. B-lymphocyte mitogenicity. and most ofthe 30-plus other biological activites, associated with endotoxins, Endotoxin tends to exist in high molecular weight aggregates parly because of lipid A’ hydrophobicity. The lipid A component Of LPS rotates away from the aqueous environment. and bilayer sheets or vesicles with diameters of <0.1 jm are formed. These structures apparently ate stabilized by the surrounding divalent, calcium and magnesium cations that are attracted tothe negatively charged LPS molecule Inthe absence of surface-active agents, bacterial endotoxin in an aqueous environment normaly have meoiecular weights enceeding 1.000.000 daltons. But i the stabilizing calcium and magnesium ions are removed from the endotoxin’s environment. the bilayers break down into what seem to be micelles with molecular masses in the 300,000- 1.000.000 dalton range. Inthe presence of surfaces tiveagens such as detergents or bile salt, these micelles can be fur ther broken down to molecular masses between 10,000 daltons and 20.000 daltons. Each stage is completely reversible. Irthe surface active agent is removed by dialysisand the divalent cations are rein troduced to the LPS. first micelles ad then membranous structures reassemble. Below 10,000 daltons, an LPS of irreversile size &x isis that has no biologial activity (3). Figure 1. Diagram ofa gram-negative cell membrane, including the et eee O-speciic side chains Upopolysaccharice Lips Phosoholipid Protein Lpopretein ‘Outer membrane Perilasmic space | inner memorane == Cytooiasm jotoxin ipopolysaccharide and outer membrane, Endotoxin can be inactivated by detoxifying or by destroying the LPS molecule, Endotoxincan be removed froma substance by ‘sing methods based on its affinity for binding 1o various surfaces, ‘or its physical characteristics. such as size, molecular weight, and electrostatic charge. Some of these depyrogenation methods. however, are suitable only for materials that can tolerate or con. form to exacting conditions. The pharmaceutical manufacturer ‘can choose from among the following methods, matching an ap propriate method to each product. Inactivation of Endotoxin ‘Acid and base hydrolyses. Depyrogenation by means of alkaline ‘or acid hydrolysis inactivate lipid A and thus effectively reduces, ‘or eliminates the biologic activity of LPS. Lipid A is linked tothe core polysaccharide by 2-keto-8-deoxyoctonie acid (KDO), an cight-carbon sugar acid that is unique wo bacterial LPS. Acid hy drolysis acts on the acid-lable ketosidic linkage to separate lipid specie core posacenarde | —potsacchaide upen Region! Regontt Regn Figure 2. Geneva structure of a tipopalysaceharide: i= Polysaccharide chain, common polyeac- Id A toxic egion, Reprinted trom Aseptic lf amaceutes! Nanaia cureay et ester Pr {A from the remainder of the LPS molecule. Because the released KDO and its atached core polysaccharides act as solte carriers for the molecules lipid portion, the free lipid Ais insoluble in squcous systems and is pyrogenic activity is reduced or elim nated. Galanos etal. demonstrated. however. that when fee lipid ‘A was combined with bovine serum albumin. the pyrogenic ‘equaled that of intact endoconn (3). Acid hydrolysis also my act ‘on the lipid A fraction. altering the molecule’ conformation and ‘masking necessary functional sts, or st may cleave off fatty acid ‘molecules at different rates, further affecting lipid solubility and thus pyrogenicity. Acid hydrolysis, using 0.08 N HCI for 30 min a 100 °C (4) oF 1.0% glacial acetic acid for 2-3 hat 100 °C (5) has been used fr depyogenation, Unlike acid hydrolysis, allatine hydrolysis does aot reduce o eliminate pyrogenic activity by removing KDO oF fatty acids. In alkaline hydrolysis. the major chemical and biological alterations Of the degraded molecule result fom the saponification of fatty acids (6) In 1956, Neter et al. reported that neither heat alone (100 *C. 24 h, pF7.2) nor hydrolysis in 0.25 W/ NOH at $6." for 6 min appreciably reduced the pyrogenicity ofthe LPS of E's herichia coi or Salmonella aborus equi (7). But exposure 0 (0.25 N NaOH at 56 °C for Th produced 8 moderate loss of pyro enicty for the E.coli LPS and 2 marked reduction for the 5 ‘abortus equi LPS. Niwa etal. (6) reported that depyrogenation is enhanced when LPS undergoes alkaline hydrolysis with 0.1 [NaOH in either 955 ethanol or 80% dimethylsulfoxide Oxidation. As eary as 1912, Hort and Penfold reported that, Salmonella tsphasa cells lost their fever-producing capacity after they were washed in H.O, (8). The exact mechanism of H.0.'s action on LPS isnot known, but peroxidation ofthe fatty acids in the lipid A region of LPS has been suggested. In 1945, Campbell and Cherkin reported tht a gelatin solution was rendered nor- Dyrogenic when boiled with0.1 MH.O. for2 hor autoclaved with 0.044 H.0. at 116 °C for 20 min (9). Tauband Har used H.0.t0 detoxify pyrogens in sterile Water for Injection (WF) USP. not. HONKY FA-O-cH ° Figure 2. Lipid A structure. Reprinted ttom the cover of Pyrogens (1868), by F.C. Pearson Il, courtesy of Marcel Deter ine, ‘mal saline. and dextrose-saline solutions (10), They found the ‘most effective treatment to be boiling in the presence of 0.1% H.O. for 2h. Under these conditions. the final solution also ay free of peroxide. This procedure was successfully adapted by Menezel for large-scale depyrogenation of infusion solutions at a Tel Aviv hospital (11), Cave and Novitsky demonsteated tht the inactivation of endo toxin by H.O. depends on time, pH, and concentration (12). Us ing as Idle ay 27% H.O. a1 68 °C for | h. these researchers ob served ~90% reduction of endotoxin, When the H,0. con centration was inreased to 27°, virtually 100% destruction was achieved with's Onidative dspyrogenaton offers several advantages over acid and base treatments. Hydrogen peroxide is safely handled. can be ccasily eliminated from solution, and apparently inactivates endo toxin under nonextreme condition (or example, low concent tion of H.0. allow temperature). H.O,'s ciet disadvantage isthat ‘tmay adulierate a solution or product. Other reported oxidative ‘methods may offer advantages in specifi applications” These ‘methods include treatment with molecular oxygen (13). hype 2, endo toxin aggregates are negatively charged and behave like anions Thus. they can be removed by cations adsorbents, suchas asbes~ ‘os, which has a positive charge at pH values of <8 3. Inaddivon tothe electrostatic atraction, the fine stongly branched asbestos fibers provide a large surface area tha allows the creation of @ epi fiter Because the pores in depth filters are in random and tortuous configuration, they retain panicles throughout the ma tix by mechanical means such as sieving, sedimentation, inerst: tial impingement, and interception, Thus, depth fiers can retain ‘ore particles before clogging than can screen filers ofthe same Fating, Pyrogen adsorption efficiency, however, depends on the concentration and molecult sie ofthe cssolved substances, The effectiveness of asbestos fers is lost with high molecular weight Protein solutions because of “adsorptive” displacement. In recent eats, FDA has restricted the use of asbestos filters for processing Small and large volume parenterals. and depth iers comaning ‘atrils other than asbestos have become widely used “Membranes produced from polyamies (nylon) or wth arsine ‘covalently bonded to their surfaces exhibit net positive charges in aqueous solutions of

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