Limulus Amoebocyte Lysate Assay for Detection and Quantitation of Endotoxin in a Small-Volume Parenteral

Transcription

1 APPLID AND NVIRONMNTAL MICROBIOLOGY, Sept. 198, p /8/9-533/6$2./ Vol. 4, No. 3 Limulus Amoebocyte Lyste Assy for Detection nd Quntittion of ndotoxin in Smll-Volume Prenterl Product KIYOSHI TSUJI,* KATHY A. STINDLR, AND SUSAN J. HARRISON Control Anlyticl Reserch nd Development, The Upjohn Compny, Klmzoo, Michign 491 A Limulus moebocyte lyste gel-clotting method for the determintion of endotoxin in smll-volume prenterl product hs been described. Smple dilution with.1 M potssium phosphte monobsic buffer (ph 8.) effectively eliminted ssy interference, wheres dilution with wter did not. The threshold pyrogenic dose for scherichi coli C-2 nd 127:B8 endotoxins ws determined to be 1. ng of endotoxin per kg of body weight. Not more thn 1. ng of endotoxin (the threshold pyrogenic dose) per the highest recommended humn dose or the USP pyrogen test dose per kg of body weight, whichever dose is more stringent, is logicl limit for the quntity of bcteril endotoxin in smll-volume prenterl products. xcellent correltion ws ttined when this criterion ws used to compre the Limulus moebocyte lyste ssy with the USP pyrogen test. 533 Limulus moebocyte lyste (LAL), n queous extrct of moebocytes from the horseshoe crb, Limulus polyphemus, rects with endotoxin to form gel or clot (7). Under stndrdized conditions, this rection detects picogrm quntities of endotoxin. The clotting rection is triggered when the LAL regent comes in contct with the lipopolyscchride (endotoxin) frction of the cell wll of grmnegtive bcteri. The endotoxin ctivtes n enzyme in the LAL regent which then rects with low-moleculr-weight clottble protein to form gel (17). Testing by LAL is expeditious, specific, nd convenient. The quntittive LAL test is more economicl nd requires smller volume of smple for testing thn does the qulittive United Sttes Phrmncopei (USP) pyrogen test. In ddition, lrge number of tests cn be performed by one individul in single dy. The LAL method hs been pproved by the Bureu of Medicl Devices, U.S. Food nd Drug Administrtion, s suitble test to replce the USP pyrogen test for finl relese of medicl devices (Fed. Reg. 42:57749, 1977). Unlike lrge-volume prenterls, the mjority of smll-volume prenterl products contin high concentrtions of phrmcologiclly ctive drugs. A drug concentrtion of 3 to 5 mg/ ml is not uncommon. The mjority of these potent drugs interfere with the physiology of rbbits; thus, smll-volume prenterl products tested by the USP pyrogen test must be diluted before dministrtion. Likewise, some drugs interfere with the LAL method, necessitting dilution or other modifiction to eliminte interference (8, 9, 14). This pper describes development of LAL methodology for smll-volume prenterl product contining spectinomycin. MATRIALS AND MTHODS Procedure. The experimentl protocol outlined by Hrrison et l. nd Wchtel et l. ws followed (3, 16). The LAL regent (Pyrotell; Assocites of Cpe Cod, Inc., Woods Hole, Mss.; lots nd ), sterile wter for injection (The Upjohn Co., Klmzoo, Mich.; lots 668FT, 5FX, nd 797GX), nd scherichi coli 127:B8 endotoxin (Difco Lbortories, Detroit, Mich.; lot ) were used. The endotoxin stock solution contined 5 jig of endotoxin per ml of sterile wter for injection. When the stock solution ws stored t 2 to 4 C, the endotoxin ws stble for t lest 1 yer. From this stock solution, fresh stndrds contining.1,.2,.4,.6,.8, nd.1 ng of endotoxin per ml of sterile wter for injection or buffer were prepred for use ech dy. The United Sttes reference endotoxin, lot C-2, ws obtined from the Bureu of Biologics, Food nd Drug Administrtion, Bethesd, Md. Smple preprtion. Smples were prepred t the finl concentrtion of 5 mg of spectinomycin per ml of.1 M potssium phosphte monobsic buffer. The ph of the solution ws mesured nd djusted when necessry with pyrogen-free HCI or NOH to ph 6. to 8.. One-tenth milliliter of solution ws utilized in the gel clot LAL ssy. The.1 M potssium phosphte monobsic buffer ws prepred by weighing pproximtely 13.6 g of pyrogen-free KH2PO4 into pyrogen-free 1-liter volumetric flsk nd diluting to volume with sterile wter for injection. The ph ws djusted to pproximtely ph 8. with either pyrogen-free NOH or HCI. KH2PO4 ws depyrogented by dding 1% (vol/vol) Downloded from on December 24, 218 by guest

2 534 TSUJI, STINDLR, AND HARRISON u S) ( uj 'U I r 2.3 F 1.7 F 1.1 F.5 F _- ~~~~~~~~~~~~~~~ o,- GM - Oe coo -- Om_- _- Q I OS NDOTOXIN DOS (ng/kg) LN SCAL FIG. 1. Rbbit body temperture increses recorded fter dministrtion of grded endotoxin (. coli 127:B8) doses. Dshed lines re 95% confidence bnds bout the regression line. ctivted crbon (Drco G-6; ICI, United Sttes Inc., Wilmington, Del.) to the buffer solution. The solution ws utoclved for 6 min nd filtered through.22-,im Millipore depyrogented filter pprtus (Millipore Corp., Bedford, Mss.) into pyrogen-free vessel. The ph ws redjusted, when necessry, with either pyrogen-free NOH or HCI to pproximtely 8.. The buffer ws utilized to dilute the endotoxin stock solution to demonstrte tht the buffer does not ffect the geltion endpoints. The buffer ws lso tested s negtive control. Pyrogenic dose determintion. To correlte the results of the LAL test with the USP pyrogen test, the threshold pyrogenic dose (TPD) ws sttisticlly determined by use of the USP pyrogen test. A group of 86 rbbits ws injected with five grded doses of. coli 127:B8 endotoxin:.5, 1., 5., 1., nd 3. ng per ml of sterile sodium chloride solution per kg of rbbit body weight. To determine the TPD for the United Sttes reference endotoxin, lot C-2, 71 rbbits were similrly dministered grded doses of C-2 in sline. Body tempertures were recorded t hourly intervls for minimum of 3 h fter injection s outlined by the USP pyrogen test (15). The verge febrile response for ech dose ws liner function of the nturl logrithm of the endotoxin dose. The verge pyrogenic dose (APD) ws clculted s the dose t which the rbbits tested responded with verge temperture increses of.4625 C. This vlue,.4625 C, ws derived from the most stringent definition of pyrogenicity (3.7 C for eight rbbits), defined by the United Sttes Phrmcopei (15). The TPD, defined s the lower 95% confidence intervl of the APD, ws clculted from the liner regression nlysis. APPL. NVIRON. MICROBIOL. RSULTS AND DISCUSSION TPD. Both the USP pyrogen test nd the LAL ssy cn be used to detect endotoxins; however, only the LAL test cn rpidly nd precisely quntify endotoxin levels. The minimum quntity of endotoxin tht is pyrogenic in rbbits, the TPD, must be determined so tht the LAL ssy cn be correlted with the rbbit dt. Humns nd rbbits re reported to respond eqully on per-kilogrm-of-body weight bsis to threshold pyrogenic quntities of endotoxin (2, 6). Thus, the minimum quntity of endotoxin tht is pyrogenic to humns, the TPD, cn be determined by the USP pyrogen test (15). A grph of the nturl logrithm of the endotoxin (. coli 127:B8) dose versus the verge febrile response of the 86 rbbits is presented in Fig. 1. The APD nd TPD were clculted to be 1.43 ng nd 1.1 ng of endotoxin per kg of body weight, respectively. Similrly, the APD nd TPD were clculted for the United Sttes reference endotoxin, lot C-2, to be 1.37 ng nd 1.5 ng of endotoxin per kg of body weight, respectively. A grph of the nturl logrithm of the endotoxin (lot C-2) dose versus the verge febrile response of the 71 rbbits is presented in Fig. 2. Thus, the TPD of the two. coli endotoxins ws pproximtely 1. ng/kg of body weight. This vlue is in greement with the finding (1. ng/kg of body weight) of Helth Downloded from on December 24, 218 by guest

3 VOL. 4, 198 LAL FOR NDOTOXIN IN SMALL-VOLUM PARNTRALS r 1.18 I "S.. * -o eso.- o.e.m,, _ NDOTOXIN DOS (ng/kg) LN SCAL FIG. 2. Rbbit body temperture increses recorded fter dministrtion of grded endotoxin (United Sttes reference endotoxin, lot C-2) doses. Dshed lines re 95% confidence bnds bout the regression line. TABL 1. ffect of vrious concentrtions of spectinomycin in buffer on LAL geltion endpoints Concn of spec- ffect' of endotoxin concn (ng of endotoxin per ml of buffer): Spectinomycin lot tnemy/ml of buffer) Bufferb A 5 B 5 CD 5 5 LU U) i- ul I- : ul.54 F both humns nd rbbits is 1. ng/kg of body weight for n. coli endotoxin (2). Hochstein et l. lso estblished the threshold pyrogenic dose of. coli endotoxin in rbbits to be 1. ng/kg of body weight (5). Preprtory testing. To dequtely test smples by the LAL method, the smples must not inhibit or enhnce the geltion response or otherwise interfere with the LAL ssy. Unlike lrge-volume prenterls, the mjority of smll- & cp S , Formtion of gel tht remins firm when inverted through 18; -, no firm gel formtion. b The buffer utilized in this study ws.1 M potssium phosphte monobsic buffer, ph pproximtely 8.. Industry Mnufcturers Assocition collbortive study which used. coli 55:B5 endotoxin from Difco Lbortories (4). From the sttisticl nlysis, it ws concluded tht the two. coli endotoxins, 127:B8 nd 55:B5, re equivlent to the United Sttes reference endotoxin, lot C-2, nd tht the TPD cn be defined s 1. ng of. coli endotoxin (with reference to C-2) per kg of body weight. GreisInn nd Homick reported tht the threshold pyrogenic response for s see Downloded from on December 24, 218 by guest

4 536 TSUJI, STINDLR, AND HARRISON volume prenterl products contin high concentrtions of phrmcologiclly ctive drugs. A mjority of these potent drugs interfere with the physiology of rbbits; thus, the products must be diluted before dministrtion in the USP pyrogen test (12). Some drugs lso interfere with the LAL test, necessitting dilution or other modifiction to eliminte interference (8, 9, 14). Smples of spectinomycin were initilly diluted with sterile wter for injection t finl concentrtion of 5 mg of spectinomycin per ml, the level employed for USP pyrogen testing. The ph of these smples ws djusted with pyrogen-free NOH to rnge of between 6 nd 8. The smples were then spiked with.5 nd.1 ng of endotoxin per ml of smple preprtion. In ll cses, the formtion of gel ws inhibited, even though the LAL regent ws cpble of detecting less thn.5 ng of endotoxin per ml. The interference my be due to high N+ content in the testing smple, since these smples required reltively lrge mounts of NOH for neutrliztion. Sullivn nd Wtson (13) stted tht NCl concentrtions greter thn.154 N decrese the sensitivity of the lyste. Dilutions of spectinomycin were next prepred in.1 M potssium phosphte monobsic buffer. The smples were spiked with grded doses of the. coli endotoxin nd tested by LAL (Tble 1). Since the geltion endpoint of the endotoxin in the smple contining 8 mg of spectinomycin per ml mtched the endpoint of endotoxin in the buffer, the interference shown with the LAL test in sterile wter for injection ws eliminted. Complete inhibition of geltion occurred for the smples contining 9 nd 1 mg of spectinomycin per ml. Since the concentrtion of spectinomycin employed for USP pyrogen testing in rbbits is 5 mg of spectinomycin per ml, this concentrtion ws selected for LAL testing. Correltion of the LAL test with the USP pyrogen test. Thirty-five lots of spectinomycin were tested by LAL t 5 mg of spectinomycin per ml of.1 M potssium phosphte monobsic buffer. Two sets of positive controls were prepred for ech lot by spiking the smples with.5 nd.1 ng of the. coli endotoxin stndrd per ml. In ll cses, the lowest spiked level of endotoxin promoting gel ws the.5-ng/mlspiked positive control. USP pyrogen tests were performed on the 35 lots of spectinomycin to determine the correltion between the LAL test results nd the USP pyrogen test results. In ll cses, the smples were well below the threshold pyrogenic level of endotoxin; the nonpyrogenicity of the smples APPL. NVIRON. MICROBIOL. ws confirmed by the USP pyrogen test results (Tble 2). ffect of spectinomycin on endotoxin pyrogenicity. Since no nturlly pyrogenic lots were found, further testing ws needed to ssure tht "pyrogenic" USP test would correlte with n LAL vlue greter thn the cceptnce TABL 2. Correltion of the LAL test with the pyrogen test ndotoxin level detected by LAL USP pyrogen test re- Lot (ng per 5-mg/ml sults (mximum temp dose of spectino- increse, C)' mycin)h 1 LT.6.,.,.1 2 LT.6.1,.4,. 3 LT.6.2,.,.1 4 LT.6.,.2,.1 5 LT.6.,.1,.2 6 LT.6.5,.4,. 7 LT.6.,.1,. 8 LT.6.3,.,.1 9 LT.4.,.,.2 1 LT.4.1,.3,.1 11 LT.4.,.,.2 12 LT.4.,.2,.2 13 LT.4.3,.,.4 14 LT.4.,.,.1 15 LT.4.,.,. 16 LT.4.,.1,. 17 LT.4.,.,.1 18 LT.4.1,.2,.4 19 LT.4.,.1,.1 2 LT.4.,.,.1 21 LT.4.1,.1,.1 22 LT.4.5,.,. 23 LT.6.,.2,. 24 LT.4.1,.,.1 25 LT.6.,.,.5 26 LT.6.1,.,. 27 LT.4.,.,. 28 LT.4.,.,. 29 LT.6.,.,.2 3 LT.6.,.,. 31 LT.6.,.2,. 32 LT.4.,.,. 33 LT.4.,.,.1 34 LT.4.,.2,. 35 LT.4.,.,. The positive control for ll lots ws.5 ng of endotoxin per ml of smple preprtion. Smples were spiked with.1 nd.5 ng of endotoxin per ml of smple preprtion. The lowest endotoxin concentrtion tht gelled is listed. b The threshold pyrogenic level of endotoxin is 1. ng of endotoxin per kg. ' If no rbbit demonstrted n increse of.6 C or more bove its respective control temperture, nd if the sum of three individul mximum temperture increses did not exceed 1.4 C, then the smple ws considered nonpyrogenic (15). Downloded from on December 24, 218 by guest

5 VOL. 4, 198 criterion. Determining the effects of spectinomycin on the pyrogenicity of endotoxin ws lso necessry to evlute possible product synergism or inhibition. Solutions of spectinomycin were prepred t the USP nd LAL testing concentrtion of 5 mg of spectinomycin per ml of sterile sodium chloride solution, spiked with grded doses of. coli lipopolyscchride (.5, 1., nd 5. ng of endotoxin per ml of solution), nd dministered to the rbbits ccording to the USP pyrogen test (15). ight rbbits were tested t ech dose level, nd two such pyrogen ssys were performed, resulting in group of 48 rbbits tested. The dt obtined were similr nd could be sttisticlly pooled. A grph of the nturl logrithm of the endotoxin dose versus the verge febrile response is presented in Fig. 3. From the liner regression nlysis, the APD ws clculted to be 1.19 ng of endotoxin per kg of body weight. A comprison of these sttistics with those of the endotoxin in sterile sodium chloride solution (APD = 1.43 ng of endotoxin per kg of body weight) (Fig. 1) indictes tht spectinomycin dministered in dose of 5 mg/kg does not influence the pyrogenic ctivity of the endotoxin. ndotoxin limits. The USP pyrogen test ws designed to mimic the dministrtion of the humn dose (milligrms of drug per kilogrm of body weight) per injection. However, drugs w CO ni- U'. U' I r F so LAL FOR NDOTOXIN IN SMALL-VOLUM PARNTRALS 537 O _- re phrmcologiclly ctive compounds, nd some must be diluted for testing by the USP pyrogen test to void rbbit sensitivity. Quite often, requirements for such dilutions re so extensive tht the rbbit dose is severl-fold less concentrted thn the humn dose. When pplied to smll-volume prenterl products, n endotoxin limit should control the totl, bsolute quntity of endotoxin ptient receives per injected dose rther thn the endotoxin per milliliter of drug solution, since the febrile response is function of the quntity of endotoxin injected per kilogrm of body weight (1, 2, 1). Administering constnt mount of endotoxin, 2 ng of. coli 127:B8 endotoxin per kg of body weight, in vrying volumes of sline confirmed tht the febrile responses of the rbbits were similr regrdless of the injection volume (Tble 3). The febrile responses were function of the totl, bsolute quntity of endotoxin injected per kilogrm of body weight. Thus the limit of endotoxin should be bsed on the totl quntity of endotoxin ptient receives per injection dose, e.g., the TPD, rther thn the endotoxin concentrtion, i.e., nnogrms of endotoxin per milliliter, s proposed by the Food nd Drug Administrtion (Fed. Reg. 45:3668, 198). The ultimte gol of ny endotoxin or pyrogen test is protection of the ptient by preventing the dministrtion of drugs nd other products _~~~ _- ~~ Go.1 _~~~~~~~~~~~~~~~~~- sc_m- - Go... so G-Z..oo Downloded from on December 24, 218 by guest NDOTOXIN DOS (ng/kg) LN SCAL FIG. 3. ffects of spectinomycin spiked with grded doses of. coli 127:B8 endotoxin on rbbit body temperture increses. Dshed lines re 95% confidence bnds bout the regression line.

6 538 TSUJI, STINDLR, AND HARRISON TABL 3. Results of dministering constnt endotoxin dose in vrying volumes by the USP pyrogen test" Dose (ng Avg of endo- Dose temp toxin per vol Temp increse ( C) in rbbits inkg of (ml) crese body wt) (OC) 2 1.,.7,.7,.2, 1., 1.1,.6, 6.3,.4, ,.7,.9,.4,.9,.4,.5,.56.4,., ,.1,.5,.2,.6, 1.,.5,.59.6, 1.1,.4 F vlue:.3 (no sttisticlly significnt difference). possessing high levels of bcteril endotoxins, which cn produce febrile response. Thus, the limit of endotoxin in smll-volume prenterl products, determined by the LAL test, should be expressed in terms of the TPD per the highest recommended humn dose or per the USP rbbit dose (milligrms of drug per kilogrm of body weight), whichever is the more stringent. This proposl is often much more stringent thn tht being pplied to medicl devices (Fed. Reg. 42:57749, 1977), which requires the LAL test for medicl devices to be t lest equivlent to the USP pyrogen test. The TPD hs been defined s 1. ng of endotoxin (United Sttes. coli reference endotoxin lot C-2) per kg of body weight. Greismn nd Hornick reported tht humns nd rbbits respond eqully on per-kilogrm-of-body weight bsis to threshold pyrogenic quntities of endotoxin, nd tht the TPD of. coli endotoxin for both humns nd rbbits is 1. ng/kg of body weight (2). The highest recommended intrmusculr dult dose of spectinomycin is 4 g (11), or pproximtely 6 mg of spectinomycin per kg per injection, ssuming n verge humn body weight of 7 kg. The limit of endotoxin, which is expressed in terms of the humn dose rther thn the lower USP pyrogen test dose, is thus 1. ng of endotoxin per 6 mg of spectinomycin. The results of this study, s well s of those in the literture (2-4), suggest tht criterion of not more thn 1. ng of endotoxin (referenced to lot C-2), which is the TPD, per the highest recommended humn dose or USP pyrogen test dose per kg of body weight, whichever dose is the more stringent, is logicl choice to limit the quntity of bcteril endotoxin in smll-volume prenterl products. APPL. NVIRON. MICROBIOL. ACKNOWLDGMNTS The technicl ssistnce of K. T. Gibson, C. A. Metzer, nd M. Aldrich is gretly pprecited. Sttisticl nlyses were provided by A. R. Lewis nd. D. Willims. LITRATUR CITD 1. Brun, H. A., nd V. V. Klein Some problems of pyrogen testing. Bull. Prenter. Drug Assoc. 14: Greismn, S.., nd R. B. Hornick Comprtive pyrogenic rectivity of rbbit nd mn to bcteril endotoxin. Proc. Soc. xp. Biol. Med. 131: Hrrison, S. J., K. Tsuji, nd R. M. nzinger Appliction of LAL for detection of endotoxin in ntibiotic preprtions, p In. Cohen (ed.), Biomedicl pplictions of the horseshoe crb (Limulide). Aln R. Liss, Inc., New York. 4. Helth Ilidustry Mnufcturers Assocition HIMA collbortive study for the pyrogenicity evlution of reference endotoxin by the USP rbbit test. Helth Mnufcturers Assocition, Wshington, D.C. 5. Hochstein, H. D., R. J. lfin, J. F. Cooper,. B. Seligmnn, Jr., nd S. M. Wolff Further developments of Limulus mebocyte lyste test. Bull. Prenter. Drug Assoc. 27: Keene, W. R., H. R. Silbermn, nd M. Lndy Observtions on the pyrogenic response nd its pplictions to the biossy of endotoxin. J. Clin. Invest. 4: Levin, J., nd F. B. Bng The role of endotoxin in the extrcellulr cogultion of Limulus blood. Bull. Johns Hopkins Hosp. 115: Mscoli, C. C., nd M.. Wery Applictions nd dvntges of the Limulus mebocyte lyste (LAL) pyrogen test for prenterl injectble products, p In. Cohen (ed.), Biomedicl pplictions of the horseshoe crb (Limulide). Aln R. Liss, Irnc., New York. 9. Newsome, P. M Penicillins nd the Limulus mebocyte lyste test for endotoxin. J. Phrm. Phrmcol. 29: Personeus, G. R Pyrogen testing of prenterl phrmceuticls, p In M. S. Cooper (ed.), Qulity control in the phrmceuticl industry, II. Acdemic Press, Inc., New York. 11. Physicins' Desk Reference rd ed., p Medicl conomics Co., Ordell, N.J. 12. Public Helth Service Code of Federl Regultions 21, Food nd Drug , prts Sullivn, J. D., Jr., nd S. W. Wtson Fctors ffecting the sensitivity of Limulus lyste. Appl. Microbiol. 28: Sullivn, J. D., Jr., nd S. W. Wtson Inhibitory effect of heprin on the LimuLlus test for endotoxin. J. Clin. Microbiol. 2: United Sttes Phrmcopei th ed., p Mck Printing Co., ston, P. 16. Wchtel, R.., nd K. Tsuji Comprisons of limulus mebocyte lystes nd correltion with the United Sttes Phrmcopeil pyrogen test. AppI. nviron. Microbiol. 33: Wtson, J. D., F. W. Vlois, nd S. W. Wtson ndotoxins: the Limulus mebocyte lyste system, p In A. W. Bernheimer (ed.), Mechnisms in bcteril toxinology. John Wiley nd Sons, Inc.. New York. Downloded from on December 24, 218 by guest