R. Lander,* W. Manger, M. Scouloudis, A. Ku, C. Davis, and A. Lee
Merck and Co., I nc., Sumneytown Pi ke, P.O. Box 4, West Poi nt, Pennsyl vani a 19486 Free radi cal -based oxi dati on has been detected i n the normal operati ng regi me of the Gaul i n homogeni zer, demonstrati ng that cavi tati on occurs i n thi s i mportant i ndustri al bi oprocessi ng equi pment. Free radi cal generati on i s suppressed by i mposi ti on of back pressure, provi ng that such cavi tati on occurs i n the i mpi ngement secti on. The cal cul ated val ue of the cavi tati on number i s consi stent wi th submerged jet cavi tati on, wherei n a hi gh-speed jet exi ti ng from the val ve gap accel erates fl ui d i n the i mpi nge- ment regi on, creati ng the vacuum condi ti ons for cavi tati on. Usi ng pol ysacchari des as a model shear-sensi ti ve compound, thei r breakage pattern i n the homogeni zer was characteri zed by mol ecul ar si ze and pol ydi spersi ty and compared to those of fl ui d shear fl ows i n capi l l ary tubes and cavi tati ng fl ow from a soni c horn. The resul ts i ndi cate that breakage occurs pri mari l y by fl ui d shear, al though a contri buti on by cavi tati on i s al so apparent when back pressure i s appl i ed. Because bi ol ogi cal mol ecul es can readi l y react wi th free radi cal s and the al terati ons caused thereby are subtl e i n nature, a thorough eval uati on of the i mpact of free radi cal s i n upstream homogeni zati on i s warranted. Introduction The Gaul i n homogeni zer i s commonl y empl oyed i n the bi opharmaceuti cal i ndustry for cel l breakage to rel ease i ntracel l ul ar products for downstream capture and pu- ri fi cati on. Accordi ngl y, several i nvesti gators have sought to understand the mechani sm by whi ch the homogeni zer accompl i shes thi s task. Descri pti ons of the hi gh-pressure devi ce cover the gamut of shear and i mpi ngement (1, 2), pressure shock (3), and cavi tati on (4). A combi ned mechani sm i s pl ausi bl e si nce the homogeni zer pressure i s rel eased through both a val ve seat and i mpi ngement secti on. The work of Hetheri ngton et al . (5) has afforded correl ati ons between cel l breakage and parameters of the hi gh-pressure val ve seat and the i mpi ngement secti on, and they descri bed the underl yi ng mechani sm i n terms of shear. Breakage of Saccharomyces cerevisiae was correl ated wi th pressure, usi ng an exponent of 2.9. Recogni zi ng the i mportance of the pressure gradi ent across the val ve seat, Kl ei ni g and Mi ddl eburg (6) have, through numeri cal fl ui d computati on, correl ated cel l breakage wi th the cal cul ated gradi ent. These cal cul ati ons showed that the l ocal vacuum requi red for cavi tati on does not exi st i n the val ve regi on of the homogeni zer. Brook- man (7) al so fai l ed to fi nd evi dence of cavi tati on, usi ng an acousti c chi p and a starch-based reacti on wi th an i ndi cator. Fi nal l y, correl ati ons have been devel oped that defi ne the si ze reducti on of broken cel l fragments i n terms of gri ndi ng (8) or by empi ri cal rel ati onshi ps wi th operati ng parameters (9). Thi s study was desi gned to i nvesti gate the mechani sm- (s) of Gaul i n homogeni zati on, usi ng a pol ysacchari de (Ps) as a model compound. Hi gh mol ecul ar wei ght pol ysac- chari des are (a) sensi ti ve to breakage by fl ui d shear and sonochemi stry, (b) subject to free radi cal chemi cal reac- ti ons, and (c) better characteri zed than cel l s. Through hi gh performance si ze excl usi on chromatography wi th mul ti angl e l i ght scatteri ng and refracti ve i ndex detecti on (HPSEC/MALLS/RI ), both si ze and pol ydi spersi ty were uti l i zed as di agnosti c tool s to understand breakage mechani sms. Capsul ar pol ysacchari de 19F (Fi gure 1), deri ved from Streptococcus pneumonia and a component of the mul ti val ent vacci ne Pneumovax23 (Merck and Co.), was used for thi s i nvesti gati on. The nati ve 19F pol ysac- chari de has a mol ecul ar si ze of about 1 10 6 Da and a pol ydi spersi ty of 1.2. The abi l i ty to control the reducti on of si ze of pol ysac- chari des wi th the homogeni zer was demonstrated by Marburg et al . (10). I n thi s appl i cati on, a target val ue of pol ysacchari de si ze was achi eved by speci fi cati on of the homogeni zer operati ng pressure. Moreover, homogeni za- ti on afforded an i mproved, narrower si ze di stri buti on as compared to the nati ve pol ysacchari de. I n thi s study, the pol ysacchari de was subjected to a range of homogeni zer condi ti ons i ncl udi ng pressure and mul ti pass or recycl e operati on, as wel l as the appl i cati on of back pressure to expl ore cavi tati on. To separatel y characteri ze pol ysacchari de breakage by the mechani sms of fl ui d shear and cavi tati on, breakage was measured i n the model shear systems of capi l l ary tubes and by soni cati on, respecti vel y. Free radi cal formati on, whi ch accompani es the hi gh l ocal temperatures caused by cavi tati on (11), was di agnosed i n the homogeni zer by measuri ng the oxi dati on of potassi um i odi de sol uti ons. The breakage patterns were characteri zed by measure- ment of mol ecul ar si ze and pol ydi spersi ty, the l atter provi di ng a sensi ti ve di scri mi nati on of the breakage mechani sm. Experimental Procedures Materials. Unl ess otherwi se noted, reagents were anal yti cal grade or better and used as suppl i ed. Potas- si um i odi de (KI ) was obtai ned from Al dri ch Chemi cal Co. (Mi l waukee, WI ). Sodi um azi de and sodi um phosphate were suppl i ed by Fi sher Sci enti fi c Co. (Pi ttsburgh, PA). Mol ecul ar wei ght range pul l ul an standards for the 80 Biotechnol. Prog. 2000, 16, 8085 10.1021/bp990135c CCC: $19.00 2000 American Chemical Society and American Institute of Chemical Engineers Published on Web 12/21/1999 HPSEC/MALLS/RI anal ysi s were suppl i ed by Pol ymer Laboratori es (Amherst, MA). Ul trahi gh-puri ty water (resi sti vi ty > 18 M-cm) was generated by a Barnstead RO/DI Nanopure water puri fi cati on system (Dubuque, I A). Puri fi ed capsul ar pol ysacchari de (19F) was obtai ned from Merck Manufacturi ng Di vi si on (West Poi nt, PA). Molecular SizeandPolydispersityAssay: HPSEC- MALLS/RI. Hi gh performance si ze excl usi on chroma- tography wi th mul ti angl e l aser l i ght scatteri ng and refracti ve i ndex detecti on was used to determi ne number- and wei ght-averaged mol ecul ar wei ghts, pol ydi spersi ty, and effecti ve pol ymer radi us for pul l ul an standards and nati ve and si ze-reduced pol ysacchari des (12). Sampl es were anal yzed at 1-5 mg/mL concentrati ons i n water usi ng 50 L i njecti ons (Rai ni n AI -1A autosampl er, Woburn, MA). Mobi l e phase, 0.1 M sodi um phosphate wi th 0.05%sodi um azi de, pH 7.2, was conti nual l y pumped through a Waters Ul trahydrogel guard col umn and two 0.7 cm 30 cm Ul trahydrogel l i near col umns (Bedford, MA) at a fl ow rate of 0.5 mL/mi n usi ng a Di onex I P20 i socrati c pump (Sunnyval e, CA). Col umn effl uents were conti nuousl y moni tored wi th a mi ni -DAWN l aser l i ght scatteri ng photometer (Wyatt Technol ogy Corp., Santa Barbara, CA) and a Rai ni n Dynamax RI -1 di fferenti al refractometer (Woburn, MA). Li ght scatteri ng data were acqui red and processed wi th Wyatts ASTRette software. Thi s method affords a rel ati ve devi ati on of l ess than (5% (12). Detection of FreeRadicals: KI Oxidation. Accord- i ng to the method of El Pi ni er and Kol esni kove (13), free radi cal s can be detected quanti tati vel y by thei r oxi dati on of aqueous i odi de to mol ecul ar i odi ne. I odi ne, i n turn, i s conveni entl y measured by i ts UV absorbance at 350 nm usi ng a Beckman DU-520 (Ful l erton, CA). Gaulin Homogenizer. The homogeni zer was an APV Gaul i n model 15MR (Wi l mi ngton, MA). The homogeni za- ti on fl ow rate was 950 mL/mi n for al l experi ments. The val ve seat was a cerami c cel l di srupti on (CD) type, shrouded wi thi n an 11.0 mm i mpact ri ng. The appl i ed homogeni zer pressure ranged from 400 to 8000 psi . The vari ous experi mental protocol s for the homogeni zer are descri bed bel ow and i l l ustrated i n Fi gure 2. MW of Ps vs Pressure and Number of Passes. The homogeni zer was confi gured for di screte passes. Speci fi c homogeni zati on pressures were establ i shed wi th di sti l l ed water pri or to the addi ti on of the feed sol uti on. Pol ysac- chari de sol uti on was i ntroduced behi nd the water fl ush, and the product was col l ected i n bottl es. The batch was fol l owed by a water fl ush, and di l uti on was mi ni mi zed by maki ng the product cut on the basi s of the homog- eni zer dead vol ume. Conti nuous Recycl e: FreeRadi cal Measurement. Homogeni zati on recycl e experi ments were performed to study free radi cal formati on. A cool i ng coi l was i nserted i nto the Gaul i n feed tank to mai ntai n constant batch temperatures of 24-28 C duri ng the recycl e experi - ments. The hi gh reci rcul ati on rate rel ati ve to the vol ume of sol uti on i n the feed tank (1 L/mi n, 500-700 mL sol uti on) provi ded adequate mi xi ng. Homogeni zati on pressure was hel d constant, and sampl es were taken from the feed tank at predefi ned i nterval s. Useof Back Pressure: Cavi tati on i n I mpi ngement Secti on. Recogni zi ng that cavi tati on i s the resul t of l ocal vacuum caused by fl ui d accel erati on, control experi ments were performed i n whi ch (a) fl ui d vel oci ty was vari ed by changi ng homogeni zer pressure and (b) back pressure was appl i ed to the homogeni zer outl et. To appl y 0-60 psi back pressure i n the i mpi ngement area, the homog- eni zer outl et was modi fi ed wi th a pressure gauge and di aphragm val ve. The appl i cati on of back pressure (e2 atm) had no effect on the homogeni zati on pressure (>1600 psi ) as measured on the homogeni zer gauge. CapillaryTubes. Capi l l ary tubes (0.005 and 0.007 i n. i .d.) of pol yethyl ether ketone (Rai ni n, PEEK) were cut to predefi ned l engths and attached to a hi gh-pressure, hi gh-fl ow-rate l i qui d chromatography pump (Rai ni n SD- 1, Woburn, MA) for di screte pass, fl ow through experi - ments. Sonic Horn. A commerci al 20-kHz ul trasoni c cel l di srupter (Branson model W-350, Danbury, CT) wi th a mi croti p probe and an ampl i tude range of 60-250 m was empl oyed. The mi croti p probe of the soni c horn was pl aced i n a 50 mL pol ypropyl ene centri fuge tube that contai ned Ps. The tube was suspended i n a cool i ng bath to mai ntai n constant temperature duri ng extended soni - cati on treatments. Sampl es were removed at peri odi c i nterval s duri ng the experi ment for si ze anal ysi s by HPSEC/MALLS/RI . Experimental Results Free Radical Formation: Effect of Operating PressureandBack Pressure. Two sets of experi ments were performed wi th potassi um i odi de sol uti ons i n recycl e mode, and ti me sampl es were taken for absor- bance measurements. I n the fi rst case, Gaul i n pressures of 0, 1600, and 5000 psi were uti l i zed. As shown i n Fi gure 3, there was no measurabl e generati on of i odi ne at zero or even 1600 psi . However, at 5000 psi i odi ne was conti nual l y generated throughout the course of the experi ment. I n the second experi ment, shown i n Fi gure 4, back pressure was appl i ed to the homogeni zer at 5000 psi for about 30 mi n and then rel i eved. Whereas the l evel of free radi cal s was constant for the 30 mi n phase when back pressure was used, free radi cal s were produced as soon as the back pressure was rel eased. Figure 1. Structure of the repeati ng uni t of Ps 19F. Figure 2. Experi mental homogeni zer confi gurati ons for (a) recycl e, (b) mul ti pl e, si ngl e pass, and (c) use of backpressure wi th ei ther mul ti pl e pass or recycl e modes. Biotechnol. Prog., 2000, Vol. 16, No. 1 81 The basel i ne l evel of free radi cal s i n the l atter experi - ment was hi gher than that of the experi ment shown i n Fi gure 3. Thi s was a resul t of di ffi cul ty i n control l i ng the modest back pressure duri ng the i ni ti al moments when Gaul i n pressure was bei ng i ncreased to 5000 psi . Because of the hi gh fl ow rates (1 L/mi n) and smal l fl ui d vol umes (0.5 L), even a momentary overshoot of pressure duri ng the adjustment at startup coul d expl ai n the i ni ti al burst of free radi cal s i n thi s pl ot. Thi s experi ment provi des di rect evi dence of cavi tati on i n the homogeni zer and shows that such cavi tati on begi ns at a threshol d pressure between 1600 and 5000 psi . Furthermore, the suppressi on of free radi cal s by back pressure i ndi cates that the l ocati on of cavi tati on i s i n the i mpi ngement secti on of the homogeni zer si nce the val ues of back pressure are smal l compared to the pressure drop of 5000 psi whi ch occurs across the val ve gap. The rel ati onshi p between cavi tati on and back pressure i s di scussed i n the model i ng secti on. Ps Breakageby Fluid Shear: Capillary Flow. To characteri ze the breakage pattern of the pol ysacchari de by si mpl e fl ui d shear, sol uti ons were subjected to re- peated passes through a capi l l ary tube. A range of shear rates was achi eved wi th fl ow rates of 30, 50, and 80 mL/ mi n i n a 0.007 i n. PEEK tube. The data of Fi gure 5 show that greater breakage rates are achi eved as shear rate i s i ncreased. Thi s i s consi stent wi th the di rect rel ati on- shi p between breakage of pol ymers and capi l l ary fl ui d shear descri bed by Ram and Kadi m (14). I n the same experi ment, Fi gure 6 shows that pol ydi s- persi ty decreases sharpl y wi th repeated passes. Thi s nonrandom breakage pattern i s not unusual when pol y- mers are extended by a shear fi el d such that sci ssi on occurs at a l ocati on central to the mol ecul e (14). Breakageby Sonication: Cavitation. To character- i ze the breakage pattern by cavi tati on, the pol ysaccha- ri de was subjected to soni cati on at vari ous horn i nten- si ti es. The data i n Fi gure 7 show cl earl y that the rate of mol ecul ar wei ght reducti on i s i ncreased wi th horn i n- tensi ty, but the termi nal val ues of mol ecul ar wei ghts approach si mi l ar asymptoti c val ues of approxi matel y 100 000. Fi gure 8 shows that, i n contrast to the capi l l ary breakage pattern of Fi gure 6, pol ydi spersi ty i s unchanged duri ng breakage by soni cati on. Mol ecul ar extensi on, whi ch i s necessary for central sci ssi on, i s evi dentl y not possi bl e i n the shorter ti me frames of the more i ntense cavi tati ng shear fi el d. PsBreakageintheHomogenizer. Mol ecul ar wei ght reducti on was measured for 10 di screte passes at several homogeni zer pressures. The resul ts of Fi gure 9 show that the rate and extent of mol ecul ar wei ght reducti on i s di rectl y rel ated to homogeni zer pressure. I ncreasi ng homogeni zer pressure corresponds to smal l er gap si zes and therefore hi gher val ues of shear i n both the gap and i mpi ngement secti ons (6). For the case of 5000 psi homogeni zer pressure, both pol ydi spersi ty and mol ecul ar wei ght changes are pl otted i n Fi gure 10. The reducti on i n pol ydi spersi ty i s si mi l ar to the capi l l ary data, i ndi cati ng that fl ui d shear i s the domi nant mechani sm for pol ysacchari de breakage. Suppression of PolysaccharideBreakageby Ho- mogenizer Back Pressure. Al though i t has been shown that si mpl e shear i s the domi nant mechani sm of pol ysac- chari de breakage at 5000 psi , the potenti al contri buti on Figure 3. Free radi cal s are detected at an homogeni zer pressure of 5000 psi but not at 1600 psi . Figure 4. Use of 60 psi back pressure compl etel y suppresses free radi cal formati on at an homogeni zer operati ng pressure of 5000 psi . Free radi cal s are formed when back pressure i s rel eased. Figure5. Pol ysacchari de mol ecul ar si ze reducti on i s i nversel y rel ated to the shear rate. Figure6. Mul ti pass breakage i n a capi l l ary tube. Pol ysaccha- ri de breakage by si mpl e fl ui d shear i s nonrandom. 82 Biotechnol. Prog., 2000, Vol. 16, No. 1 by cavi tati on was assessed by appl yi ng back pressure. Fi gure 11 shows that the appl i cati on of onl y 30 psi back pressure reduced the rate of mol ecul ar si ze reducti on, affordi ng a nearl y constant val ue of 150 000. The freel y vented case, by contrast, exhi bi ts steady mol ecul ar wei ght decl i ne throughout the 10 pass profi l e and reaches a val ue of nearl y 100 000. The di fference i n the two profi l es becomes apparent when the pol ysacchari de at- tai ns a si ze that can onl y be further reduced by the more i ntense shear of cavi tati on. The di fference between the fai rl y steady val ue of 150 000 and the steady decl i ne to approxi matel y 100 000 i s wi thi n the preci si on of the assay, whi ch i s 5% (12). Cavitation Model: Submerged J et Cavitation. Fi gure 12 i s a schemati c of the val ve i mpi ngement secti on of the homogeni zer and i l l ustrates the submerged-jet cavi tati on model . The hi gh-speed jet emerges from the val ve gap i nto the i mpi ngement secti on and spreads out i nto the surroundi ng fl ui d. Hi gh stresses and turbul ence exi st i n the mi xi ng regi on i mmedi atel y adjacent to the jet where the surroundi ng fl ui d i s accel erated. Thi s l eads to l ocal pressure l oss and the i ncepti on of cavi tati on by bubbl e formati on. Rouse (15, 16) has studi ed the forma- ti on of bubbl es from submerged jets, defi ned thei r l oca- ti on on the jet surface, and shown that a cavi tati on number of <0.7 i s requi red for thei r formati on. Cal cul ati on of the cavi tati on number for a submerged jet i s based upon the jet vel oci ty, l ocal pressure, and the vapor pressure of the sol vent and i s gi ven by Young (17): P i s the pressure i n the i mpi ngement regi on, P v i s the vapor pressure of the sol vent, and V avg i s the vel oci ty of the ci rcul ar jet as i t exi ts the homogeni zer. Fi gure 13 i s a schemati c of the ci rcul ar val ve gap regi on. The average jet vel oci ty, V avg , i s cal cul ated from Figure7. Pol ysacchari de breakage by cavi tati on wi th the soni c horn. Breakage rate i s affected by the horn ampl i tude but a rel ati vel y constant l i mi t i s evi dent at about 100,000 Da. Figure 8. Pol ysacchari de breakage by soni c horn at an ampl i tude of 224 m. Pol ydi spersi ty i s unchanged duri ng breakage by the soni c horn, i n contrast to the capi l l ary data of Fi gure 6. Figure9. Mol ecul ar si ze as a functi on of homogeni zer passes at vari ous pressures. Lower val ues are achi eved by hi gher homogeni zer pressure correspondi ng to narrower val ve gap wi dth. Figure10. Pol ydi spersi ty i s reduced by homogeni zer breakage, si mi l ar to the trend for capi l l ary breakage i n Fi gure 6. Figure 11. Back pressure of onl y 30 psi reduces the pol ysac- chari de breakage rate compared to a freel y vented homogeni zer. Cavi tati on i n the i mpi ngement secti on i s suppressed by back pressure. C ) (P - P v )/( 1 / 2 V avg 2 ) (1) Biotechnol. Prog., 2000, Vol. 16, No. 1 83 a knowl edge of the homogeni zer fl ow rate Q, the val ve gap wi dth , and the homogeni zer ri ng radi us, R: For the M-15 homogeni zer and the CD-type val ve, the val ve gap wi dth has been measured by Kl ei ni g and Mi ddel berg (6) and i s gi ven by the correl ati on where i s the gap wi dth i n mi crons and P i s the homogeni zer pressure i n uni ts of MPa. From eqs 1-3, the cal cul ated cavi tati on number at 5000 psi i s 0.07. Tabl e 1 shows cal cul ated val ues of the cavi tati on number for the enti re range of homogeni zer pressures. Wi thi n the accuracy of the cal cul ati on, the model shows the potenti al for submerged jet cavi tati on i n the i mpi ngement secti on si nce the cal cul ated cavi ta- ti on number i s l ess than 0.7 for al l pressures. To esti mate the i mpact of back pressure, i t i s i nstruc- ti ve to cal cul ate the l ocal pressure drop created i n the i mpi ngement regi on by the jet, whi ch i s gi ven by the Bernoul l i formul a: Substi tuti ng the cal cul ated val ue of vel oci ty, 54 m/s at 5000 psi , a val ue of 211 psi i s obtai ned. Because the downstream secti on i s vented to the atmosphere, thi s pressure drop resul ts i n the creati on of the vacuum condi ti ons i n the i mpi ngement secti on, whi ch are es- senti al for cavi tati on. Wi thi n an order of magni tude, thi s cal cul ati on i s al so consi stent wi th the observed suppres- si on of free radi cal formati on that resul ted from the i mposi ti on of ca. 60 psi or even 30 psi back pressure (data from Fi gures 3 and 10). Natural back pressure due to the fl ow through the i mpi ngement regi on wi l l al so contri bute to the suppressi ve effect. Al so, i t i s recogni zed that cavi tati on mi ght be suppressed to an extent that does not necessari l y produce free radi cal s but may nonethel ess i mpart suffi ci ent shear to break pol ysaccha- ri des. Conclusions The occurrence of cavi tati on wi thi n the Gaul i n homog- eni zer has been demonstrated by the detecti on of free radi cal s. Free radi cal formati on begi ns i n a pressure range from 1600 to 5000 psi . Moreover, free radi cal formati on i s compl etel y suppressed by the appl i cati on of 60 psi back pressure at an homogeni zer pressure of 5000 psi . Thi s behavi or i s consi stent wi th a submerged jet descri pti on of cavi tati on occurri ng i n the i mpi ngement secti on. The hi gh-speed jet exi ti ng from the homogeni zer val ve entrai ns stagnant fl ui d, thereby creati ng the neces- sary vacuum for bubbl e formati on. The jet suddenl y stri kes the i mpi ngement pl ate, whereupon the bubbl es col l apse as a resul t of the repressuri zati on of thi s i mpact. Usi ng correl ati ons devel oped for the val ve gap si ze, jet vel oci ti es have been cal cul ated. The cal cul ated cavi tati on number for the homogeni zer i mpi ngement regi on i s <0.1 at 5000 psi and 0.15 at 1600 psi . Cavi tati on i n the i mpi ngement regi on i s therefore consi stent wi th i nves- ti gati ons i n model systems (15, 16) wherei n submerged jet cavi tati on occurs when the cavi tati on number fal l s bel ow 0.5. The marked reducti on i n pol ysacchari de pol ydi spersi ty duri ng si ze reducti on i n the homogeni zer i s i denti cal to the breakage resul ts i n the model capi l l ary system and contrasts to the pattern caused by soni cati on, where pol ydi spersi ty i s unchanged by breakage. Thi s compari - son between homogeni zer and model systems i ndi cates that si mpl e fl ui d shear i s responsi bl e for most of the breakage i n the homogeni zer. However, a contri buti on of cavi tati on to breakage can be observed through the i mposi ti on of 60 psi back pressure, but onl y after the pol ymer si ze has been reduced to 150 000. At the l ower mol ecul ar wei ght range, the breakage rate by shear i s di mi ni shed and the contri buti on of cavi tati on becomes apparent. Wi th thi s evi dence of cavi tati on i n the i mpi ngement regi on and i ts rol e i n the breakage of suffi ci entl y smal l pol ymer mol ecul es, i t becomes possi bl e to specul ate and attempt to reconci l e data of other i nvesti gators. I mpi nge- ment shear was i nvoked to expl ai n the effect of vari abl e i mpi ngement di stances upon the breakage of S. cerevisiae (1). However, shorter i mpi ngement di stances woul d al so ampl i fy the energy rel ease from i mpl odi ng cavi tati on bubbl es. Thi s vi ewpoi nt provi des an expl anati on for the pressure exponent of 2.9 i n the breakage of S. cerevisiae (5). A si mi l ar exponent was found i n the earl y work of Gooberman (18), who showed from both theoreti cal and experi mental grounds that cavi tati on shear force i s rel ated to the cavi tati on pressure by an exponent of 2.33. I n fact, cavi tati on may be the domi nant mechani sm i f the cel l wal l s of S. cerevisiaeare stronger than the ether Figure12. Schemati c of the homogeni zer val ve and i mpi nge- ment assembl y. The proposed cavi tati on occurs as the hi gh- vel oci ty fl ui d exi ti ng from the val ve gap entrai ns and accel erates fl ui d i n the i mpi ngement secti on, thereby creati ng the l ocal vacuum requi red for cavi tati on. There are al so two sources of fl ui d shear: (a) the narrow val ve gap and (b) i mpact upon the i mpi ngement ri ng. Figure 13. Vel oci ty from the val ve gap i s cal cul ated wi th ci rcul ar fl at pl ate geometry. Table 1. Cavitation Number vs Homogenizer Pressure pressure (psi ) val ve gap (m) jet vel oci ty (m/s) cavi tati on number 400 25.94 22.13 0.40 600 22.51 25.50 0.30 800 20.35 28.21 0.25 1200 17.66 32.51 0.19 2000 14.77 38.87 0.13 3000 12.82 44.80 0.10 5000 10.72 53.57 0.07 8000 9.09 63.14 0.05 V avg ) Q/(2R) (2) ) 37P -0.35 (3) P ) F water V avg 2 /2 (4) 84 Biotechnol. Prog., 2000, Vol. 16, No. 1 l i nkage of the pol ysacchari de used i n thi s i nvesti gati on. I t i s recogni zed that, among di fferent cel l l i nes, the pressure exponent di ffers wi del y, from 1.8 for Bacillus brevis to 2.9 for bakers yeast (2). The presence of free radi cal s i n the homogeni zer i s another i mportant fi ndi ng i n thi s i nvesti gati on and seems to confl i ct wi th the resul ts by other i nvesti gators, al - though they uti l i zed a l ess sensi ti ve techni que (7). The resul t i s not i nconsi stent wi th another report (6) that shows that cavi tati on cannot occur i n the homogeni zer val ve uni t because that report di d not study the i mpi nge- ment secti on. Fi nal l y, because degradati on of bi ol ogi cal mol ecul es by free radi cal s i s wel l documented (19), thi s i nvesti gati on provi des some urgency to see whether such reacti ons can occur i n the homogeni zer and whether suppressi on of unwanted reacti ons i s possi bl e by ma- ni pul ati on of i ts operati ng parameters. At the ti me that thi s document was rel eased for cl earance at Merck and Co., I nc., an i denti cal concl usi on regardi ng the exi stence of cavi tati on i n the homogeni zer was reported by Shi rgaonkar et al . (20). Usi ng potassi um i odi de and a Gaul i n homogeni zer, they showed that cavi tati on i s the sol e mechani sm of yeast breakage. They di d not recogni ze, however, that cavi tati on occurs i n the i mpi ngement regi on, as shown i n thi s paper by the effect of backpressure and by cal cul ati on of the cavi tati on number. By more careful l y model i ng cavi tati on, thi s paper reconci l es the negati ve fi ndi ngs of other i nvesti ga- tors (2, 5, 6) who di d not consi der the i mpi ngement secti on as a source of cavi tati on. Notation Greek Symbol s wi dth of val ve gap (cm unl ess i ndi cated as mi crons) F water densi ty of water (gm cm -3 ) Symbol s C cavi tati on number (di mensi onl ess) P pressure (l b i n 2 , gauge pressure) Q vol umetri c fl ow rate (mL mi n -1 ) R radi us of val ve gap (cm) V average vel oci ty of jet from val ve gap (cm s -1 ) References and Notes (1) Keshavarz Moore, E.; Hoare, M.; Dunni l l , P. Di srupti on of bakers yeast i n a hi gh-pressure homogeni zer: New evi dence on mechani sm. EnzymeMicrob. Technol. 1990, 12, 764-770. (2) Engl er, C. R. Cel l Breakage. I n Protein Purification Process Engineering; Harri s, R. G., Ed.; Marcel Dekker: New York, 1994; Chapter 3. (3) Brookman, J. S. G. Further studi es on the mechani sm of cel l di srupti on by extreme pressure extrusi on. Biotechnol. Bioeng. 1975, 17, 465-479. (4) Knudson, M. I ., Jr.; Jones, T. R. U.S. process for manufac- turi ng organocl ays havi ng enhanced gel l i ng properti es. U.S. 4664842, 1987. 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