thesis on vaccine production

8/13/2019 Thesis on Vaccine Production

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VACCINE PRODUCTION AS A UNIT PROCESS

PROEFSCHRIFT

T E R V E R K R I J G I N G V A N D E G R A A D V A N D O C T O R I NDE T E C H N I S C H E W E T E N S C H A P P E N A A N D E T E C H N I S C HE H O G E S C H O O L T E D E L F T , O P G E Z A G V A N D ER E C TO R M A G N I F I C U S I R . H . R . V A N N A U T A L E M K E ,H O O G L E R A A R I N D E A F D E L I N G D ER E L E K T R O T E C H N I E K , V O O R E E N C O M M I S S I E U I T D E S E N A A T T EV E R D E D I G E N O P W O E N S D A G 2 4 N O V E M B E R 1 9 7 1 T E

14 UUR

R / p - 3 2 3,o6PAULUS ALOYSIUS VAN HEMERT

S C H E I K U N D I G I N G E N I E U RG E B O R E N T E V O O R B U R G


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DIT PROEFSCHRIFTISGOEDGEKEURD DOORDE PROMOTORPROF.DR.T.O. WIKE 'N

DIT PROEFSCHRIFT WERD BEWERKT OPHETRIJKS INSTITUUT VOOR DE VOLKSGEZONDHEID


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-et renovabis faciem terrae-

Aan de nagedachtenis van mi jn oudersAan m i j n v rouwAan mi jn k inderen


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drukkerij elinkwijk, utrecht


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C H A P T E R 1

Introduction

This thesis deals w i th the product ion of vaccines. More speci f ica l ly , w i ththe manufactur ing of vaccines for human appl icat ion. I t encompasses morethan a decade of study in the f i e l d . In th is per iod the character of theVacc ine Depar tment o f the R i jks Ins t i t uu t voor de Vo lksgezondhe id inBi l thoven has changed considerably . This has been due, not only to theknowledge gained dur ing the study of the var iables of the processes involved,but especial ly to the better insight gained in the technological aspects ofvaccine product ion. Because of the great f inancia l and technical considerat ions , the work descr ibed here involved the cooperat ion wi th many others.The necessary investments would never have been made if the Board ofD i rec tors o f the R i jks Ins t i t uu t voor de Vo lksgezondhe id had not be l ieved,in advance, in the appl icabi l i ty of the concepts concerned. Dr. H.H.Cohen, inh is responsib i l i ty for the vaccines produced by the Inst i tu te, s t imulated to agreat extent the development of the concepts. A complete laboratory was atmy d isposa l in wh ich par t icu la r ment ion must be g iven to Ir.A.L.van W ezeland Mr. P.Smid who were col laborat ing a lmost dai ly in d iscussing the designof ex per im ents, and a lso in pe r form ing th em . In ad di t io n, a smal l s taf f ofconstruct ion designers was present, a rather unusual asset for a laboratory; inth is team the ac t iv i t y o f Mr.J.van Hooijdonk canno t r ema in unm en t ione d .Th e deta i led develop me nt of the technical o u t f i t to be descr ibed in Chapter3 could never have been real ised without the presence of this staf f .

The d iscuss ions w i th the promotor Prof.Dr. T.O.Wikn, on the preparat ion of the text , have been most usefu l , and were welcomed by the author .Whereas a thesis is co m m on ly the resul t of endogenous st im ula t io n, th isbo ok has also received exog enou s im pe tus in sofar as col leagues in the f ie ld of

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vaccine product ion have f requent ly suggested that they would welcome acomprehensive treat ise of the work done in Bi l thoven on this subject. Forthis reason, digressions, as usual ly enco unte red in a te xt bo ok , have beensom ewha t indulged in by the autho r. W hi le covering a rather wid e f i e l d , i thas on ly appeared feasible to wo rk ou t the subject to the wh ole de pth in acertain l imi ted number of cases. The book not only contains the record of apiece o f scient i f ic w or k, in clud ing a goo d deal of app l ied techn ical science,but also a phi losophy: the concept of Uni t Process.The leading thought of th is thesis is a logical consequence of theobservation that processes used in the preparation of several vaccines havemo re features in co m m on than is general ly recog nized. Th is led, as w i l l beexplained in Chapter 2, to the appl icat ion of the concept of uni t process tovacc ine p ro duc t ion .

Data on general methodology and appl icable to the great major i ty of thespecif ic processes leading to the production of vaccines are treated as "BasicTo ols o f the U ni t P rocess" in a separate chap ter (Chap ter 3). It is on ly af tert his dis pla y o f th e " a r m a m e n t a r i u m " , t ha t a n um b er o f s pe cif icprocesses are detai led (Chapter 4). They serve as examples, and form thejust i f ica t ion for the appl icat ion of the pr incip le o f Uni t Process.Product ion of vaccines in large quant i t ies, requir ing a technologicalapproach, becomes necessary when systematic vaccinat ion of larger parts ofa populat ion is considered. An ef fect ive systemat ic vaccinat ion programmerequires a vaccine o f prope r ef fect iveness, as we l l as the po ss ibi l i ty toestabl ish a vaccinat ion programm e and to ob ta in a suf f ic ient supply o f thevaccine for such a programme. The programming depends upon theadministrat ive accessibi l i ty of a populat ion and the vaccine supply ontechno logy. A par t f rom sma l lpox vacc ina t ion , and the p rogrammesconducted in the armies dur ing both wor ld wars ( typhoid and te tanus; thela t ter on ly in Wor ld War I I ) , and d iphter ia immunizat ion in the la te th i r t ies,system atic va ccina t ions as such were on ly started after W orld War I I . Fieldt r ia ls , as described by Cockburn (1955 ) for pertussis in England, and by

Francis (1955) fo r po l iom ye l i t is in the Un i ted States, have co ntr ibu tedgreat ly to st imulat ing systemat ic vaccinat ion, whereas the in t roduct ion ofcombined vaccines, such as the diphtheria-tetanus-pertussis-pol io-- comb i na t i on (Brand wi/k et al., 1961) have s im pl i f ied the ap pl ica t ion.Stat ist ica l data concern ing coverage of the populat ion by vaccinat ion,even wi th in h igh ly developed countr ies, are not a lways obta inable . An estimated va lue of 50 to 90 per cent for po l io and smal lpox in Europe and N or thAmerica, and eventual ly the same for pertussis, diphtheria, and tetanus maybe assumed. Th is impl ies that on ly 20 per cent o f the to ta l wor ld populat ionof more than 3 mi l l iard people has been reached by the most important

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vaccines, and this only during the last ten years. Smal lpox vaccine forms anexce p t ion . The sma l lpox e rad ica t ion p rogramm e o f the W or ld H ea l thOrg anizat ion has gradual ly broug ht the f igure fo r sm al lpox vac cinat ion on aconsiderably higher level during the last f ive years.A rap id expansion of the product ion of vaccines must therefore beexpected in the next decades, not on ly o f the above ment ioned, but a lsothose fo r use in special disease areas, such as vaccines against ye llo w feverand those that have been developed more recent ly (see review by Edsall,1961) such as measles and rubel la vaccines. This requires that the technologyof vaccine product ion should be considerab ly improved. Th is should takein to accoun t improv em ent no t o n ly in qu an t i t y bu t also in qua l i t y . Th is w i l lbe exp la ined be low in terms of consistency.In order to give an impression of the cul ture volumes concerned in massvaccine pro du ct ion , in Table 1-1 the cu l ture vo lum e for the preparat ion o fone total human dose is given for each of several vaccines. The data areapproximat ions, depending not on ly on the y ie ld o f ce l ls or product per un i tvo lum e, bu t a lso on the dose adm in istere d; i f severa l, st rongly d i f f ere nt ,doses of one vaccine are present, as for instance when oral vaccinat ion of

Table 1-1 Ap pro xim ate cul ture volumes ( in m l) , requi red for the preparat ion of one tota lhum an dose (t.h.d.) of several bacterial and viral vaccines. For ex pl ic atio n, seetex t .type of vaccine

pertussisdiphther iatetanusstaphylococcus acholera, parenteralcholera, oraltyphoid, parentera ltypho id , o ra lB.C.G.pol io, inactivatedpol io, l ivesmal lpoxmeasles, inactivatedmeasles, liverubella, l ive

inoculat ionsper t .h .d .333222221331311

ml cul t .per t .h .d .1.00.30.30.50.220.0220.02-0.160.10.031.00.0030.1

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entero bac ter ia l vaccines is com pared w it h the parenteral rout e, tw o data aregiven. For vi ral vaccines the m ost m ode rn m eth od o f pre para t ion is chose n;so the sm al lpox f igure is derived fr om t issue cu l tur e, as the classical m eth odon ca l f 's skin cannot be eva luated in terms of vo lume. Fur thermore, forsome virus vaccines the f igure is based on a method in homogeneous cul ture,which is under development (see Chapter 4 .3) . The tab le shows that , wi ththe except ion of l ive vi rus vaccines, considerable volumes are required.Larger volumes than the ones mentioned here wi l l be needed, i f pur i f icat ionprocedures, such as envisaged for instance for pertussis vaccine, would bepract ised.

Direct ing our a t tent ion to the technology of vaccine preparat ion, we muststate that in the early years vaccines were being prepared in a laboratory on alaboratory scale and with laboratory methods. This picture seems to persistmore than is desirable because of some features inherent to vaccinep r o d u c t i o n :

1. Mu ch o f the ear ly de velopm ent has been done by governm ent .Preparat ion took p lace in nat ional inst i tu tes or o ther non-prof i torganizat ions. The preparat ion was done on the bench of the medicalbacter io log ist ; e f f ic iency and product iv i ty were on ly o f secondaryconcern.2 . Whereas, for instance in the pro du ct io n of ant ib io t ics , the cu l turevolumes involved are so large that ef f icient technological procedureshave been necessary almost f rom the beginning, in vaccine product ion asomew hat in tensi f ied labora tory procedure could m eet the dem and ,even for a populat ion of , say, 50 mi l l ion people.3. Virus vaccines, prepared rout inely on such substrates as cal f 's skin andchicken eggs, lend themselves poorly to industr ia l preparat ion. Even theuse of monolayer cul tures of t rypsinized t issue cel ls does not provide aso lu t ion in th is respect . Only la te ly, wi th the in t roduct ion ofhomogeneous t issue cul ture techniques, a possible break-through hasbeen made.4. The com plex cu l tura l co ndi t ion s of many pathogenic m icroorganismsand viruses has f req ue nt ly led to the be l ie f that c u l t iva t ion in " t a n k s "was e i ther impossib le or ext remely d i f f icu l t to per form.The ch aracter ist ic o f labora tory cu l t iva t ion of m icroorganisms is, in th isco ntex t , gro wt h on a smal l am oun t o f subst ra te , a t a cons tant tem peratu re.Variables such as pH and real incubat ion t ime (that is, the t ime to reach adef ined physico-chemica l and b io log ica l co nd i t io n, cf . p . ) are se ldomcon t r o l l ed . This method of cul t ivat ion is not ideal for producing vaccines ofgood consistency (for a def in i t ion of consistency, see p. 17). The reason is

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that th e re pro du cib i l i ty o f the process can not be checked adequate ly due tolack of cr i ter ia.Consistency is becom ing m ore and mo re a necessity for the f o l l ow ingreasons:1. Extensive f ie ld tr ia ls such as those p reviou sly m en t ion ed can givedependable resul ts only i f the vaccine used is of constant qual i ty. Onemay expect s imi lar resu l ts in fur ther vaccinat ion programmes on ly i flater suppl ies of vaccine are of the same qual i ty.2 . Mo re e ff ici en t tests of po ten cy and safety, such as the mouseprotect ion test for per tussis vaccine and the t remendous safety contro ltests for vi rus vaccines such as pol io, enable us to dist inguish betweengood and bad vaccines.

The character ist ic o f indust r ia l cu l t ivat ion of microorganisms, on theco nt rar y, is tha t an eff icie nt w ay of scal ing-up is present and tha t enoughvariables can be measured to ensure the exact reproduct ion of the process.O nly in th is way may a consistent prod uct be expe cted. Bo th theserequi rements can on ly be fu l f i l le d by cu l t iva t ion in a homogeneous system ,as w i l l be exp la ined in the in t ro du ct ion of Chap ter 3 .

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C H A P T E R 2

Group Characteristics of Vaccine Production

Turgueniew s tated in 1857 tha t "system s at t ract those w ho do no tsucceed in captur ing t ru th in i t s to ta l i t y . . . " . I am, however , o f the op in ionth at i t is in man y instances advantageous to b r in g al l aspects of a sub ject in toa system. A system may help to f ind the features that are in common in agrou p o f phenom ena. I t may s im pl i f y f ind ing a com m on approach in caseswhere th is d id not seem c lear before. A system should only serve, however ,to guide thoughts; i t may never become autonomous and imperat iveo b li te ra t i n g n u a n c e s t ha t m a y be i m p o rt a n t.In the ear ly t ime o f chemica l techno logy , Lewis (1923) t r ied to createorder in the mu l t i tu de of act iv i t ies leading to a f in ished pro du ct , by thedesignat ion Uni t Process and Uni t Operat ion. Examples of the f i rs t arechemica l reac t ions such as ox ida t ion , hy dro xy la t i on and a lky la t i on ; thesecond is exe m pl i f ied by opera t ions l i ke f i l t ra t io n , d is t i l la t ion and cent r i f u -ga t ion . This system had the advantage that both ent i t ies could be descr ibedindependent ly of the par t icu lar procedure, thus establ ish ing knowledge of

mo re general app l ica t ion . W ork ing o ut a speci f ic procedure is thus b rou gh tback to the cho ice o f mater ia ls and cond i t ions ; w i th o ther words to mak ingvar ia t ions on a kn ow n the me.Al though the procedures used for the preparat ion of vaccines are of qui teanother nature than the chemical processes on which Lewis appl ied hissys tem, w e f e l t t h a t t h e t e r m " U n i t P ro ce ss " w as a p p li ca b le m u t a t i sm u t a n d i s to vaccine p ro d uc tio n .For the app l icat io n o f the conce pt of "U n i t Process" i t is required th atthe procedures concerned have some group character is t ics in common. In thecase of vaccine product ion these are:

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1. A cult ivat ion of bacter ial or t issue cel ls, at temperatures around 37C inmedia that are somet imes considerably complex.2. A cu l t i va t ion vo lume tha t is smal l , whe n compared w i th o ther indus t r ia lcul t ivat ion procedures, volumes being in the order of 25-1000 l i t re.3. The need for str ic t asepsis, as we ll as pr ote ct io n o f the ope rato r againstin fec t ion w i th the mic robe under cu l t i va t ion . The f i r s t cons idera t ion ismade because most organisms involved are cul t ivated in media in whichthey are easi ly overgrown by common contaminat ing bacter ia; thesecond because the laboratory strains used are of ten considered st i l l tobe pathogenic to man.

4 . The pro du c t ion o f a subs tance tha t is no t a main metabo l ic p rod uc tsuch as ethano l f r om yeast or c i t r ic ac id f ro m Aspergillus niger. This ist rue w heth er the end p rod uc t consis ts of cells or is a bacter ia l p rod uc tseparated f rom the cel ls. In both cases i t is the format ion of one ormore ant igens, of a com plex chemical nature, that cou nts.5 . The ob ta inm en t o f an end produ c t w i t h a ma x im um o f sa fe ty fo r ma n,which is an impor tant feature. Vaccines belong to the prevent ivemedicaments. Especial ly when the disease, against which one isimm uniz ed has a lmost com plete ly d isappeared, the s ide-ef fects of theimmuniz ing agent , appl ied to heal thy persons, come into focus.6. Because i t is in most cases impossible to determine the potency of thevaccine dur ing the cult ivat ion process, more than usual emphasis is laidupo n rep rod uc ib i l i t y o f the process and hence on dete rmin at ion o fprocess variables.

In s tudying the l i terature on the procedures publ ished for preparat ion ofthe d i f ferent vaccines one cannot help to be st ruck by the fact that eachvaccine is considered ir respect ive of the data on other vaccines that could beapplicable. The best i l lustrat ion is the fact that apparatus have beendescr ibed for the cul t ivat ion of for instance Corynebacterium diphtheriae o rBrucella abortus, that have no deta i l speci f ic for the microorganismment ioned. Hence, up to the present in several inst i tu tes, the product ion ofd i f ferent vaccines is per formed in separate rooms by d i f ferent personnelus ing d i f fe ren t methods w i thout any jus t i f i ca t ion fo r th is sp l i t t ing in theclosely related basic procedure.

Th e ap plic at io n of the co nce pt o f U ni t Process requires a generalconsiderat ion of a l l mater ia ls and methods that are common to vaccines,leaving only those detai ls that are specif ic for the preparat ion of a certainvaccine to special t reatment.The conce pt is v isual ized in Fig. 2 .1 . The feature most ob viou slybelonging to the Uni t Process is the cul t ivat ion apparatus, inc luding theins t rum enta t ion fo r the measurement and con t ro l o f phys ico-chemica l

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apparatus con tro ls cul ture con di t ions

/ \kinet ics cul ture methods type of med ium selected stra inFig. 2.1 The concep t of Vaccine Pro du ction as a U nit Process.

variables. We have shown that one set of apparatus wi l l g ive an opt imalso lu t ion for the cu l t ivat ion process of a l l bacter ia (and t issue ce l ls )concerned. In addi t ion, a number o f aspects o f the cu l ture methodology isshared by al l organisms studied. This also appl ies to the especial ly di f f icul tposi t ion in regard to the k inet ic approach of the product ion of such complexcom pou nds as ant igens.Th us the aspects lef t fo r ind ivid ua l t re atm en t are the select ion of strainand med ium, and the cho ice o f the op t ima l cu l tu re cond i t i ons fo r thepro du ct io n of a cer ta in vaccine.

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C H A P T E R 3

Basic Tools of the Unit Process

In the foregoing ch apter the cho ice of the " U n i t P rocess" pr inc ip le as anappro ach to th e pr od uc t io n of vaccines has been reasoned. I t has served toguide our thoughts. The material basis, however, which was necessary for thepract ica l study of the cu l t ivat ion processes invo lved, st i l l had to be provided.A cu l t ivat ion process presupposes the appl icat ion of appropr ia tetechniques, and these techniques can on ly be stud ied i f su i tab le equipment isava i lab le . Th is appl ies not on ly to the conta iner in which the microorganismis a l lowed to mul t ip ly and form the desi red product , but a lso to the auxi l ia ryinstruments that enable us to measure, indicate, register and controlphysico-chemica l var iab les. The importance of these var iab les in the cu l tureprocesses stud ied can not be overes t imated. They g ive co nt in uou s in form at ion wi th a lmost no t ime de lay. Knowledge of these parameters provides thebasis for se lect ing opt imal c i rcumstances, thus ensur ing repet i t ion o f theop t im al va lues in subsequent cu l t ivat io ns . I t is on th is last phen om enon tha tthe consistency of the product , i .e . the vaccine, depends. Consistency in th iscontext means the repeated format ion of an end product o f good qual i tyw i th o u t occasional in fer io r batches. One has to depend upo n exactrepet i t ion of the procedure to ensure consistency, because some vaccines ared i f f ic u l t to test fo r e f f icacy and in no cu i ty, as w i l l be stressed in the n extchapter . The contro l o f physico-chemica l var iab les is therefore an ind ispensable requi rement .The re are tw o reasons for consider ing homogen eous cu l ture as the on lyacceptab le way of cu l t ivat ion. The f i rst d i rect ly bears upon the ind ispensa-b i l i ty of phy sico-c hem ical var iables in this U ni t Process. Th e value measuredonly g ives in format ion about the spot where the probe is inser ted; i f the

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physical condi t ions in this locat ion can be considered representat ive for thew ho le cu l tu re, va l id conclusions can be draw n a bout th e process. Th is is on lythe case in a homogeneous cul ture i .e. in a cul ture in which al l const i tuentsare un i fo rmly d is t r i bu ted . In microb io logy the express ion "microenv i ron-m e n t" is used to designate the part of the universe th at is "s e en ", in aphysico-chemical sense, by a microorganism i .e. the part in which a directin teract ion between microbe and surrounding exists: on ly i f a l l microenvi -ronments of a cul ture are ident ical i t can be considered as homogeneous.This homogenei ty is not go ing down to the molecu lar leve l . Themicroorganisms themselves, being part icles of the order of magnitude of 1 IJ.are present. Sometimes smal l clumps of bacter ia are almost impossible toavo id , as w i th M yco bac ter ium tubercu losis (see p.113) . In the cu l t iva t ion o ft issue cel ls, the cel ls are in the order of 10/ i . When cul t ivated on theso-ca l led "microcarr iers" (see p.156) c lumps of 100-200/1 in d iameter areregular . Wi th e f fect ive mixing wi th mechanica l st i r rers there wi l l be an evendis t r ib ut i on of par t ic les, and the use of the term hom ogeneous is jus t i f ied .L ikewise there is no complete homogenei ty a t ta inable o f the concentra t ionof those compounds that are d i f fusing in to and out the ce l l in connect ionwi th i ts metabol ism. A cer ta in concentra t ion grad ient wi l l a lways occur . I fthe gradients around al l cel ls are of the same character, due to suff icientm i x i n g , the term homogeneous is again appl icable. The same holds true forthe undissolved gases present as bubb les in the cu l ture . An even di st r ib ut io n ,and ident ical gradients around each bubble give the cul ture a homogeneouscharacter in the sense of this treatise. It is understood that i t is not easy todef ine how a cul ture should be agi tated in order to achieve sat isfactoryhomogenei ty. One is re ferred to the work o f Hansford and Humphrey(1966) .

The second reason to choose a homogeneous cu l ture is as fo l lowing:assuming that there is an opt imal condi t ion for a microorganism to fu l f i l l acer ta in task of reproduct ion or product ion, then a l l members o f a bacter ia lpop ulat ion sh ould be kept in th is con di t ion . An d th is , again , can on ly beachieved in a homogeneous cul ture. The inadequacy, in th is respect, of acu l ture on a so l id m ediu m is so obvious (compa re the co nd i t ion of a ce l l inthe to p of a co lo ny w i th one in the m idd le ) tha t i t is d i f f i cu l t t ounderstand that the use of sol id cul ture is st i l l f i rmly defended in severalcases in vaccine product ion. For per tussis vaccine product ion, the opposingviews appeared c lear ly dur ing a l ive ly d iscussion on the formulat ion ofconclusions and recommendat ions for per tussis vaccine preparat ion at thePertussis Conference Bi l thoven (1969; see Symp.Ser . immunobio l .Standard.1 3 ( 1 9 7 0 ) p . 2 7 3 ) .For the ful f i l lment of our purpose i t was considered necessary to set up asys tem, a l l components o f which were tuned to each other . In the ear ly

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sixt ies when th is work star ted no commercia l ly ava i lab le apparatus wasconsidered sat isfactory . There fore we decided to design the eq uipm entourselves. In the course of ten years much work has been done, i l lustrated bythe fact that more than two thousand drawings have been made by the sta f fo f the Ri jks Inst i tuut voor de Volksgezondheid carry ing out th is pro ject . Thesystem not on ly com prised a set of cu l t iv at ion vessels of increasing cap aci tybut also other vessels for auxil iary purposes, and al l the necessaryinst rumentat ion, brought together in panels.A few basic pr incip les were mainta ined throughout the development :I.The use wh ere possib le , o f c om m erc ia l ly ava i lab le com pon ents ,provided they are wel l s tandard ized and of sat isfactory constantqual i ty. This appl ies to simple parts such as 0-r ings and glass containers,as we l l as to com pl icated com pon ents such as e lect ron ic measur ing and

con t ro l l i ng c i rcu i t s .2. In terchang eabi l i ty o f par ts w i th in a grou p of apparatus, and a lso w i t hcom parab le parts in oth er groups . Insofa r as i t invo lved n ew lydeveloped par ts i t imp l ied a r ig id system of pro du ct i on c on tro l , usingaccurate measuring devices and cal ibrat ing reference parts. Examplesare the inlets for stainless steel tubes, and the l ids for the standard glassvessels.3 . Arrange me nt in the f ina l system o f a l l com pon ents in a com pac t andsurveyable way.In th is chapter the cu l t iv at io n vessels w i l l be deal t w i t h f i rs t , togethe r w i t hsome conta iners that have d i rec t ly been der ived f ro m th em . In a separatesect ion the measur ing pr incip les and the d i f ferent combinat ion panels wi l lbe described.Theory and p ract i ce o f cu l t i va t ion techn iques (ba tch and con t inuous)conclude the chapter .

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3.1 Cultivation Vessels

The f i rst impetus for the development o f fermentors o f laboratory andpi lot plant scale was given, in the years after 1945, by the ant ib iot icsfermentat ion industry. Designs for more special ized purposes eventual lyf o l l ow ed .A cr i t ica l s um ming up of co ndi t ion s for a good ferm en tor is g iven byMalmgren and Heden {1952a) . A survey of the most impor tant l i te ra ture upto 1957 has been wri t ten by Fuld and Dunn (19 58 ), whereas a great deal ofin fo rm at io n a bout apparatus for co nt inu ous cu l tur e has been co l lected byRicica (19 58 , and also later reviews of Maiek and Ricica, e.g. 19 70 a and1970 b) . In addi t ion to the l i tera ture quoted in the fo l lowing, one is re ferredto the art icles of Brown andPeterson { 1950b ) , Callow and Pirt { 1961 ) , Chainet al . (1952 ; 1954) , Dispigno ( 1961 ) , Feustel and Hum feld {^946), Fortuneet al . ( 1950 ) , Heden (1957 ; 1962) ,Heden andMalmbo rg ^58). Heden andHolme (1961 ) , and Slezak and Sikyta (19 61 ) . Cr i t ica l reviews of comm ercial ly avai lable laboratory fermentors have been made by Solomons ( 1967;1971) .

The most common design in these early days was a cyl indr ical glass tubeor vessel , enclosed between two stainless steel plates held together wi th twolong screws; see Bartholomew et al. ( 1950 ) , Brown and Peterson (1950a) ,Rivett e t al . ( 1950 ) , Humfeld (19 47 ) , andMaIek (1961) . A var ia t ion on th isconcept is the type o f ferm ento r descr ibed by Elsworth etal. (195 6 ; 1958) .It consists of glass pipeline f i t ted with stainless steel plates at top andb o t t o m . Free hanging glass vessels f i t t ing against a top l id with a similarf lange construct ion are described by Lumb and Fawcett (1951) and byRicica (19 58) . O ur ow n ferm ento r , to be descr ibed in the fo l l ow ing , is o fthis type. I t most closely resembles the smal l stainless steel fermentors, asdescribed by Friedland et al . ( 1956 ) , Nelson et al . ( 1956 ) , Falini ( 1960 ) ,Malmgren and Heden {1952 b ) , Fuld and Dunn (195 8) , and Jo et al.(1959), and shares with these ease of assembly and handl ing, but has theadvantage of f ree visibi l i ty of the process through the completely accessibleglass walls. Elsworth objects to the use o f a glass bo tto m o n the ground s tha tthe ov er f low canno t be brought throu gh i t . We have overcome th is by usingan overf low consist ing of anU-bent tube in t roduced th rough the top .The earl iest fermentors most ly had packed stuff ing boxes (see Elsworth etal., 1956) as st i rrer shaft seals. Be tter so lut ions to t his pr ob lem are:1. The radial mechanical seal, as desc ribed, for instance, by Elsworth etal.

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Fig. 3.01 Cross section of 10 l i tre ferm en tor.

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Fig. 3.02 Exp loded view of 10 l i tre ferm ent or.I. st i rrer shaft; 2. fel t oi l -seal; 3. screw-cap for adjustment of rol ler bearings;4 . roller bearing; 5. housing for roller bearings; 6. roller bearing; 7. O-ring;8. inlet for stainless steel tube; 9. stainless steel l id; 10. oil-seal (Simmerring);I I . O-ring; 12. housing for Sim me rring; 13. add it ional oi l-seal (Hutm ansc hette);14. r ing for posit ioning Hutmanschette; 15. rubber l ining; 16. f lange, l ightmeta l ; 17. glass vessel; 18. interchangeable impeller; 19. cap nut.

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Fig. 3.03 Lid of ferm en tor, seen fro m above.1. In let for 8 mm o.d. tube.2. In let for 6 mm o.d. tube.3. Inlet for pH- or PO2-electrode.

(1958) and also used by Friedland et al . ( 1 9 5 6 ) , Malmgren and Heden(1952 b), and Wec/n( 1958).2 . Th e o i l seal o f th e ty pe used f re qu en t ly in moto rcars , as appl ied byKroll et al . ( 1956 ) , Lumb and Fawcett (19 51 ) , and mo st prob ably a lsoby/Wa7eA:(1961).3. Instead of seals, a magnetic coupling is suggested recently and alreadyappl ied to some commercia l types of smal l fermentors.

As the most s imple and ef fect ive so lu t ion to the prob lem of shaf t sea l ing,we have adapted the oi l seal, improv ing the s tandard app l i ca t ion .Other features of smal l fermentors which have been subject to gradualdevelopment are heat ing and ster i l i za t ion methods. Almost a lways the ear l ierfermentors were kept in a waterbath, and severa l commercia l ly ava i lab lefermentors are st i l l of th is type. The disadvantages are the r isk oftemperature d i f ferences between waterbath and cu l ture f l u i d , decreasedvis ib i l i ty o f the vessel contents, and an ext ra contaminat ion hazard.Stainless steel fermentors, even relat ively smal l ones of 100 l i t res caneasily be equipped wi th a jack et , bo th fo r ster i l i za t ion and for the rm osta t icregu la t ion . For the smal l g lass vessels, good solut ions have been found byElsworth et al. ( 1956 ) , Ricica ( 1 9 5 8 ) , Malek (196 1) , and Fiechter (1962) .The heat ing uni t consists of water coi ls or electr ical heat ing elements, or acomb i na t i on o f b o th .Most invest igators, however, ster i l ize their smal l g lass fermentors in the

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autoclave, accept ing thereby the compl icat ions of reat tachment o f dr iv inguni t and connect ions af ter ster i l i za t ion. Elsworth etal. (1958 ) ster i l ize the i rimproved fermentor by b lowing l ive steam through the in ter ior , f i l l ing in them ed ium asep t ical ly af te rwa rds. As far as we are aware, no au tho r hasdescribed a smal l g lass fermentor to be ster i l ized in si tu wi th the medium. I tis the so lu t io n we have chosen for ou r ferm ento r .Most fermentors, glass as well as stainless steel ones, have relativelypr im i t ive tube conne ct ions. In some cases they are s im ply welded in the l id ,making them completely inexchangeable. In other cases they can be fastenedby some type of un ion jo in t making them exchangeable, a l though i t is st i l ld i f f icu l t to ad just them in every possib le posi t ion. Improvements have beenmade by Elsworth et al. (1956) , using 0-r ings, and by Heden ( 1958 ) , whodeveloped specia l "Ste r ico nn ec tors " . T he la t ter devices, per fec t ly designed asthey are, are comp l icated and d i f f ic u l t to apply to smal l vessels w i t h l im i tedl id d im e n sio n s. T h e a rg u m en t o f c o m plic a te dn e ss a pp lie s a f o r t i o r i f o rthe inlets designed by Fiechter (1962) and now adapted by tw o Swiss ma nufacturers. The design of our tube connect ions is described on p. 27. I t hasbeen used on a l l equ ipm ent , and is, w i th o ut any ada ptat io n, ab le to f i tmanometers, safety valves, etc.

The advantages of glass vessels are obvious: the contents can be inspected,wh ich is o f pa ramoun t imp or tance in deve lopmenta l w or k .

Fig. 3 .04. 50 l i t re ferme ntor wi t ha culture of B.pertussis

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The fo l lowing considerat ions apply to the vo lume to be chosen:1. The minimal size of the cul t ur e vessel is de term ine d by con side rat ionsof exper imenta l technique. Not on ly is i t d i f f icu l t to p lace p ip ing andelectrodes in a smal l volume, but there wi l l be also restr ict ions to sizeand number o f samples. Bio log ica l tests f requent ly requi re considerab lesample vo lumes, and no microde te rmina t ions ex is t fo r them. Amin imum of 2-3 l i t res was considered pract ica l .2. 7776maxim al size is determ ined b y ease of h andl ing and by the am oun tof cu l ture f iu id needed for a par t icu lar programme. Since the year lyp ro duc t ion o f most vacc ines in a co un t ry w i th a pop u la t ion up to 50mi l l ion people, is in the order o f thousands of l i t res, batch cu l tures of25-50 l i t re and cont inuous cu l tures wi th vo lumes of 3-8 l i t re are them ax im um needed and a set of glass vessels ranging fr o m 1 to 5 0 l i t rehas been found most pract ica l .

In respect to s ize and mater ia l , te tanus toxin product ion forms anexcept ion, and in sect ion 4.2.2 stainless steel vessels of a size of 200-400l i t re wi l l be described.In Fig. 3.01 and 3.02 a cross sec t ion and an ex plod ed view of t hefermentor developed by us [Van Hemert, 19 64 b) has been given and in Fig .3.03 the disposi t ion on the l id is shown. Fig. 3.04 gives an impression of apract ical set up with the 50 l i t re vessel . Fig. 3.25 gives a picture of the 10l i t re fe rmento r .

0.6D{^My/ > ^v////} ' 7' ^^^ '

^ ' - '' / / / / / / / y / / y y / / , /

H h-

5o i0.6 D

Fig. 3.05 Recom mended dimen sions for O-ring grooves in glass/metal joint s.Left: f lange joint. Right: screw-cap joint. D = diameter of O-ring.

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The fermentor consis ts of a s ta in less steel l id , in which are located thest ir rer shaft with seals and rol ler bear ings, the pH-electrodes and the pipesfor t ranspor t of f lu ids and gases and for temperature cont ro l . To th is l id , theglass vessel is attached by an O-ring seal.Th e glass part is com pos ed o f in du str ial Jena glass w it h a f lat f lange o f th eso-cal led KF type. Sizes of 5, 10 and 50 l i t re are avai lable with the samef lange s ize of 200 mm.An hermet ical seal between f lange and glass is achieved by an O-r ingplaced in a groove in such a wa y t ha t , even af ter com press ion th at causes theO-r ing to f i l l the groove, no contact between metal and glass is caused. Forth is purpose the re lative d ime nsions as sho wn in F ig. 3.05 have been fou ndop t ima l .Th e s t i r rer consis ts of a s t i r rer shaf t rota t ing on tw o conical ro l ler bear ingsenclosed in a cy l indr ical housing screwed on top of the l id . Asept ic seal ing isacco mp lished by a shaft seal (Sim me rr ing) and a hatshaped seal(Hutmanschet te) . Both are easi ly replaced, but at least 3000 hoursoperat ion, inc luding several s ter i l izat ions, can be ef fected wi thout renewal .As both seals exert a high surface pressure on the shaft , hardening of the partin contact with the seals is needed to avoid rapid wear. The stainless steeltypes 304 or 316 that are in f requent use where h igh cor ros ion res is tance is

Fig. 3.06 Stand ard inlet fo r stainless steeltube (shown is 8 mm tube; inlet for6 mm tube is simi lar).1. Stainless steel sleeve, welded instainless steel l id. 2. Saturnus ring(nylon). 3. Gland nut (brass).4. Tube.

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of which e ight are 6 mm o.d. p ipe and two are 8 mm o.d. p ipe. In a typ ica lprocess they are used as fol lows:1. in let heat ing co i l ,2 . ou t le t heat ing co i l ,3. inle t air,4 . ou t let ai r ,5 . the rmocoup le ,6 . i nocu la t ion ,7. sampl ing,8. in le t m ed ium ,9. ou t le t cu l tu re f l u i d ,10. spare.

The ferm en tor l id is a t tached to a movab le sup por t . A n e lect r ic m oto r wi th acont inuously var iable speed dr ive is placed on top of the st i rrer shaft andat tached independ ent ly to the same supp or t , as show n in F ig . 3 .08. Fro mth is un i t the fo l lowing connect ions, wi th standard f i t t ings, lead to theins t rum ent pane l :1.2 .3.4.5.6 .7 .8 .

tube to heat ing co i l ,tube f rom hea t ing co i l ,the rmo coup le cab le,motor cab le ,pH-e lect rode cable ,oxygen electrode cable,gas inlet tube,gas out let tube.Al l cables and tubes are f lexible and easi ly detachable: a new fermentor maybe at tached to the same panel connect ion wi th in a few minutes.Th e coi l in the f er m en tor is used for ste r i l iza t ion as we l l as fo rtherm osta t ic regula t ion. In the f i rst p lace steam (of about 2 .5 kg f /c m ^) ispassed through the c o i l , heat ing the water or the cu l ture medium in thevessel to bo i l ing at a tem per atur e of 110C (0.5 kg f/cm ^ overpressure). Byle t t ing the steam out o f a l l connect ions on the fermentor , one by one, theyare all ster i l ize d. The f i l t er in the ingo ing air l ine is steam ed as the last on e;imm ediate ly thereaf ter a i r (or another gas m ixture ) is a l lowed throu gh thef i l te r in to th e vessel. R egu lat ion of gas f lo w rate and gas co m po si t ion w i l l bedescribed in sect ion 3.2.3.For therm ostat ic regula t ion wa ter is passed throu gh th e c o i l . The p r inc ip leof the control i tsel f wi l l be described in sect ion 3 .2 .1 .

con t inuous cu l tu re

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Fig. 3 .08 50 l i t re ferme ntor wi t h mot or and supp ort . Vert ica l cross sect ion.

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Fig. 3.09 Im pro ved aseptic seal for smallVibromixer shafts, as used withB-containers. Ve rtica l cross sect ion.1. Lid of B-container. 2. Glandwith three screws. 3. Bel lows(perbunan). 4. Mixershaft .5. Horizontal cross section.

From the fermentor several other containers have been developed forpurposes of vaccine product ion. They fa l l in to three groups and wi l l bedescribed subsequent ly:S O - C A L L E D B - C O N T A I N E R STh ey consist o f a standa rdized Jena f lask of 10 or 20 l i t re vo lum e,pro vide d w it h t he same f lat K F f lange as the fer m en tor vessel, bu t of o nly80 mm diameter. The l id has six standard in lets for 6 mm stainless steel tube,one of which may be replaced by an electrode in let . The l id can be providedw i t h a centra l opening fo r a V ibr om ixe r sh af t . Because the design of th eor ig ina l stu f f ing box was thought to be unsat isfactory in prevent ingco nt am ina t io n, a new design had to be made, as sho wn in Fig. 3.09 . In th is,the v ibrator shaf t is provided wi th a r ing welded to the shaf t . A be l lows ofperbunan is pressed against th is r ing with another r ing. In the same way theother end of the bellows is pressed between a depression in the stainless steell id and a r ing screwed onto i t wi th three screws. A general plan of thecontainers is given in Fig. 3.10 and 3.11.

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Fig. 3 .10 B-conta iner (20 l i tre) w i th Vibr om ixer . Vert ica l cross sect ion.1. Vib rom ixer ; 2. l id ; 3. g lass f lask wi t h standardized neck; 4. mixershaft w i t htwo impel ler b lades; 5. points of at tachment of Vibromixer.The three l id type s are show n, as seen fro m above:2a. with six inlets for 6 mm o.d. tube (type H-1),2b. the same as a, bu t wi th ce ntral opening fo r mixe rsha ft (ty pe H-3),2c. the same as b, but one inlet changed into electrode-inlet (type H-12).

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Fig. 3 .1 1 . B-20 bo t t le w i th V ib rom ixer

B-containers wi th st i rrers are in general use now in our Inst i tute (andseveral others) fo r storage of p art ic ulate vaccines, an d, w i t h o u t st i rrer, fo rstorage o f ster ile f lu ids such as cu l tu re m ediu m . Provided wi th a heat ing co i l ,thermocouple and st i rrer they are used for accurate heat t reatments as indetoxi f icat ion and ki l l ing of vaccines. Smal l scale cul t ivat ions are alsosometimes performed in these vessels, namely for research on tetanus toxinproduct ion. F ina l ly , smal l sca le vaccine mixing procedures have beenper fo rm ed in these conta iners, as for the deve lopme nt o f qu in tup le vaccine(d iphther ia-te tanus-per tussis-po l io-measles) . For th is purpose a com bine dpH-e lect rode was mounted, together wi th a st i r rer .S O - C A L L E D C - C O N T A I N E R S O R T R A N S P O R T V E S S E L SThey consist of a cyl indr ical Jena glass vessel of 400 mm diameter wi th a

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Fig. 3.12 Su ppo rting device for C-container. Ve rtica l cross sectio n.1. Glass vessel. 2. Su pp ortin g c up. 3. S pring-loaded su pports (4 pieces).

30 0 m m f la t KF f lange, at tache d t o a square stainless steel l id , hun g in amovable support . Because of the heavy load the vessel is supported by aspecial d isc suspended on fo ur loaded spiral spr ings as sh ow n in Fig. 3.1 2.Again the l id is provided with standardized in lets. I t can also be f i t tedw i t h a V i b romi xe r .St er i l iz at io n is achieved by int ro du cin g l ive steam in to the vessel thr ou ghan asbestos pad f i l te r, p laced o n the l id ; the same f i l t er fu nc t io ns later as anair f i l ter to equal ize overpressure or to apply pressure for removing thecon tents. The maxim al a l lowa ble overpressure of 0 .55 kgf /cm ^ is co nt ro l ledby a valve, which also had to be developed because no sui table type existedfor this purpose. All internal parts are of stainless steel, and asepsis is ensuredby an O-r ing const ruct ion: cf . F ig . 3 .13.The C-containers are current ly used for storage of mixed vaccine lots andfo r de tox i f i ca t ion and s to rage o f te tanus and d iph ther ia tox in / toxo id . Theyare even used in some other inst i tutes for mixing vaccines (StatensSeruminst i tu t , Copenhagen ; Kn.Nielsen pers .com m.). Fig. 3.14 gives agenera l impression of a C-conta iner wi th V ibr om ixe r .

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o I 2 tm tu be .

B- as we ll as C-co ntaine rs have been used fo r m ed iu m storage and also as areceptac le in cont inuous cul ture.S O - C A L L E D D - C O N T A I N E R S ( M I X I N G V E S S E L S )

For the special purpose of vaccine mixing a range of three double walledstainless steel vessels, with capacit ies of 100, 200 and 400 l i t re have beendesigned.They al l have the same l id, the design of which is shown in Fig. 3.15.Aga in s tandardized in lets for 8 mm tube and for a pH-elect rode were f i t te d ,t oge the r w i t h a V ib r om ixe r sha f t seal. In this case ster i l izat ion is achieved bylet t ing steam into the jacket .Fig. 3.15 gives a schematic representat ion of the 200 l i t re vessel and Fig.3.16 the arrangement in the mixing room. The vessels are al l placed onelect ronic weighing e lements fac i l i ta t ing the mix ing procedure.34


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Fig. 3.14. C-container

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Fig. 3.15 200 li tre D-container, vert ical cross section.1. V ibromixer ; 2 . head p la te ; 3 . connect ion to double w a l l ; 4. double wal ledstainless steel vessel; 5. Vi bro m ixe r shaft w i t h impe l ler; 6. 8 mm tu bin g;7. connection to double wal l .Head plate seen from above:2a . standard pH-inlet (see Fig. 3.07),2b. standard tube-in let (see Fig. 3.06),2c. centra l opening for V ibromixer shaf t wi th membrane seal,2d. openings for acid- and alkal i -containers. In modif ied head plate two bigfi l l ing openings are present instead.

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Fig.3.16 Arrangem ent of m ixin g vessels and instr um ent atio n in the vaccine m ixin gdepartment of the Ri jks Inst i tuut voor de Volksgezondheid.T o p : looking tow ards a s ide wa l l . Bottom: seen from above.1. Indicator and selector switch of electronic weighing apparatus.2. Indicator/recorder for temperature and pH. 3. Panel with valves for steam,cooled water etc. to doub le wal l o f m ixin g vessel. 4. D-100 vessel. 5. D-200vessel. 6. C vessels (transport vessels) to take up the mixed vaccine. 7. D-200vessel.8. D-400 vessel.

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3.2 Measurement and Control

In the beginning of th is chapter i t has been st ressed that much at tent ionshould be g iven to the measurement of physico-chemical var iables. Vir tual lyno cu l t i va t ion process is per fo rmed w i thout tempera ture cont ro l . pH hasproven i ts impor tance f rom the moment good steam ster i l izable e lect rodesbecame available; the same applies to the measurement of dissolved oxygen,fo r wh ich we have con t r ibu ted to the deve lop men t , as w i l l be shown be low .Some other var iables that could be of impor tance and for which thepr inc ip le o f measurement is kn ow n, w i l l be dea l t w i th b r ie f ly , a l though nospeci f ic exper ience has been gained in our own exper iments.Measurement d i rect ly leads to the need for cont ro l a l though th is has notbeen proven of advantage in al l cases. For instance, the shif t in pH dur ingcu l t i va t i on o f B.pertussis gives a clear ind ica t io n o f the stage of gr ow th (seeCha pter 4, page 8 0 and Fig. 4.0 4 and 4.0 6) . In this case pH- co ntr ol has noadvantage because i t gives neither higher cel l y ield nor bet ter protect iveact iv i ty and depr ives us of the ins ight gained by fo l lowing the pH-course.The cont ro l systems wi l l only be t reated insofar they have determined thetechnical lay out of the systems, which have been const ructed.

Th e s implest system of regula t ion is on -of f co nt ro l . I t means tha t thecont ro l l ing act ion is fu l ly appl ied at a l l va lues below setpoint , and is fu l lystopped at al l values above setpoint . Setpoint is the desired cr i t ical value ofthe var iable. Op ening or c los ing of a con tact by m echa nical , magne t ic oropt ica l coup l ing o f a ga lvanometer needle to a sw i t ch is a co m m on exam ple .Accuracy o f c on t ro l is no t a lways achieved w i t h the on-o f f m eth od ,especial ly in those cases wh ere the e f fect of the co nt ro l l i ng ac t ion isretarded. Retardat ion in the onset of ef fect ive cont ro l a lso means prolongat i o n of the e f fect a f ter the setp oin t has again been reache d. Th is results inso-cal led overshoot.pH -co nt ro l is an example whe re o n-of f co nt ro l in a lmost a l l cases g ivessat is factory resul ts : the e lect rode fo l lows the pH wi th negl ig ib le t ime delay,and the ac id or a lkal i pump (or valve) immediate ly g ives fu l l cont ro l act ionaf te r wh ich pH sh i f t f o l lows, aga in w i thout subs tant ia l t ime de lay . The sh i f ti tself is restr icted because of the good buffer ing capacity of most culturemedia. Only in the case of pH-cont ro l by vary ing the carbon diox ideco nc en t rat io n in the inco min g gas is a delay to be exp ected , andpro po r t io na l co nt r o l w i l l im prove the result in mo st cases (c f . Sect ion 3.2.2) .F o r t em p e ra tu re c o n t ro l a nd a f o r t i o r i f o r t he c o n t ro l o f d is so lv edo x y g e n , the s i tuat io n is more co m ple x. Heat ing e lements a l l have the ir ow n

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hea t capac i ty caus ing de lay . The t ime taken fo r the wa te r f l ow, coming f romthe heat exchanger (see Sect ion 3.2.1), to reach the fermentor coi l in ourheat ing system is another reason for delay.Th e si tuat ion is most com plex in the con tro l o f d isso lved ox yg en : f irstthere is the delay caused by the f l ow distance betw een the gas m ix in gchamber (see Sect ion 3.2.3) and the fermentor; then there is the t ime neededto change the oxygen concentra t ion of the headspace of the fermentor , andthe t ime for oxygen to d i f fuse through the gas/ f lu id in ter face. Moreover , thebuf fe r ing capaci ty o f the f lu id fo r oxyg en is very lo w , dem onstra ted by thefact that a qu ick growing microorganism causes oxygen deplet ion in a fewseconds, as the f o l lo w ing ca lc u la t ion shows:The concentra t ion of d isso lved oxygen in water saturated wi th a i r a t 37Cis 0 .22 mM. Taking as a pract ica l va lue for the oxygen consumpt ion ra teof a g iven bacter ia l cu l ture 100 mi l l imoles Oj / lh (cf . F ig . 3 .21) , th iscu l tu re w i l l consume a l l oxygen , con ta ined in the f l u i d , in less than 8seconds.Where on-of f c on t ro l is uns at isfactory , pro po r t ion a l con t ro l is ind icated .I t means tha t the degree of c on tro l act io n of the variable is depe nden t u po nthe devia t ion f rom the setpo in t . Wi th st i l l more sophist icated contro l thedegree of control act ion is also dependent upon the rate at which thedevia t ion is changing (der ivat ive (D) contro l ) and the t ime the devia t ion

exists, ( in tegrat ing ( I ) ac t ion) . On ly inst rum ents exe r t ing the last m ent ion edact ion have no propor t iona l o f f -set ; o f f -set means that the f ina l va lue of thevar iab le d i f fers f ro m the se tpo in t . Th is d i f ferenc e depends upon the exper im ental ci rcumstan ces; on ly in cases whe re these circum stances are not v ary ing to agreat extent, and no great accuracy is needed, can proport ional of f-set beaccepted.The var ious inst ruments chosen in the panels developed in our laboratorywi l l be d iscussed la ter on in th is chapter . The fo l lowing considerat ionshelped us to make a choice in the jungle o f measur ing and contro linst ruments ava i lab le on the market :1. Transm ittance of mea suring c.q. control signalThe comb i na t i on :sensor- ind icator-contro l ler , orsensor- recorder-contro l leris often needed.The normal arrangement has been, for years, to couple the contro l lerme chanica l ly to the ind ica tor c.q . recorder , wh ethe r th is is a ga lvano-metr ic or a potent iometr ic inst rument . The obvious d isadvantage of

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Fig.3 .17 Tw o pr incip les of e lectronic con tro l . To p: dependent upon indica tor /recorder. Bottom: independent of ind icator /recorder.1. Measuring device (sensor). 2a. Ampli f ier. 2b. Converter. 3. Indicator/recorder. 4. Control ler. 5. Control signal.In the top scheme, 2a and 3 are usual ly bui l t together. Dotted l ine: constantsignal l ing current.

cou pl ing to the m oving ind ica tor o f the inst ru m ent is that i t is l iab le tohysteresis and wear, and needs readjustment from t ime to t ime. Inother w ords , con t ro l accuracy is a lso dependent u pon the e lect r ica l andmechanical accuracy of the indicator or recorder. See schematici l lust ra t ion in F ig . 3 .17 ( top) .Systems have recent ly been in t roduced which e l iminate th is drawback.Here the signal of the probe is converted to a current (cal led"standard ized s ignal l ing current" ; German: "e ingepragterG le ic hs t ro m ") . Th is curren t goes throu gh a f ixe d range (o f e .g . 0-20 m Ain one system, and 4-20 mA in another) , i r respect ive o f the measur ingrange. In the loop formed by the output current ind icators, cont ro l lers,recorders, and eventual ly switches, are taken up. In this way thefunct ions are independent o f one another , and no mechanica l par tspart icipate in the transmission of the signals. See Fig. 3.17 (bottom).This new pr incip le has been appl ied to the "Mentor" panel and in the" O x y - T r o H " panel , and also in the new series of "Pal jas" panels.2 . Control actionDepending upon the variable to be measured and the accuracy desired,s imple on-of f c on t ro l act ion or pro po r t ion a l c on t ro l have been chosen;in the lat ter the necessi ty of added D (derivat ive) and/or I ( integrat ing)funct ions was considered.

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3. W ay of effecting proportional controlIn a ll cases bu t one (oxy gen co ntr o l in indu st r ia l ferm ento rs w i t hpos i t ion ing va lve) the pro po r t ion a l c on t ro l has been ef fected by v aryingthe on to o f f ra t io o f the fu l l s t rength con tro l s ignal, kn ow n as t im epro por t io n ing co n t r o l . A n exam ple is the tempera tu re con t ro l . In acycle of e.g. 10 seconds the ful l capaci ty of 750 or 1500 Watts of thee lement is sw i tched on f or a par t o f th is cycle , and com plete ly o f f forth e rest of the c yc le. Fo r sma ll scale w o rk it appeared to be the easiests o l u t i o n , especia l ly f rom the po in t o f v iew of insta l la t ion.In the fo l lowing sect ions the var iab les used in our work wi l l be t reatedone by one . The con t ro l pane ls , con ta in ing the comp le te ins t rumenta t ionw i l l be sh or t ly descr ibed subs equent ly.

3 . 2 . 1 T E M P E R A T U R E

Th e opt im al tem pera ture fo r a cu l t iva t ion process is not easy to d ef ine n orto determine accurate ly. Maximal y ie ld o f the desi red ant igen(s) , whetherthis goes in paral le l wi th cel l yie ld or not, must be placed under pr imarycons idera t ion . The tempera tu re o f max ima l y ie ld may d i f fe r f rom thetemperature o f qu ickest growth i .e . shor test doubl ing t ime. Because yie ldscannot be determined wi th great accuracy for most processes under study,opt imal temperatures are not known wi th greater accuracy than 1C. Mostprocesses in vaccine product ion are per formed, not complete ly wi thoutarbi trar iness, at 35C. The fact that most data in the l i terature are obtainedfro m cu l tures p laced in an incuba tor (e i ther cupbo ard o r room) p lays a ro lein th is considerat ion. R. Brouwer (pers.comm .) has observed tem peraturedi f ferences in the order of 2-3C between si tes in commercial incubators.

On ly in one instance is som eth ing kn ow n abo ut tem peratu re dependenceo f y i e l d : i f Cl . te tan i is cu l t iva ted at 33.5 C instead of 35C the yiel d o ftoxin is about 30 % lower.The estab l ishment o f the process temperature is in f luenced by twofeatures:1. The reproducib i l i ty o f the inst rument i .e . the ab i l i ty to repeat the sameabsolute value of the setpoint.2 . The accuracy of the co ntr o l i.e. the devia t ion f ro m the setpo in t va lueon the meter.

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Both va lues together should g ive a devia t ion f rom the desi red absolu te va luethat is less than 0.5C. No process under our considerat ion shows a need fora higher accuracy.In the choice of a heat ing system for our fermentors the maximaltemperature at the surface of the heat exchanger ( i .e. the coi l ) should not beso h igh that decomposi t ion of ce l ls or medium components a t the sur face isto be expected. Therefore a co i l conta in ing water was preferred above anelect r ic e lement in the fermentor . A concomi tant advantage of the system isthat coo l ing in exoth erm ic processes is ach ieved w i th ou t com pl ic at io n, cf.the schematic representat ion in Fig. 3.18. In the system chosen i t isnecessary to use at least PD action control (cf. p. 39).A the rm oc ou ple (Cu -cons tantan o r F e-constantan) has been chosen astemperature sensor. Thermocouples have the advantage over e.g. resistance

thermometers o f smal l d imension ( in fact not th icker than the two wi resto ge th er ) so tha t the y are easi ly soldered in the t i p of a closed 6 m m o.d.sta in less stee l tube. Both thermocouple combinat ions ment ioned have a h ighV/ tem pera tu re ra t io wh ich is im por tan t fo r accuracy. A l tho ugh the ou tp u tcan be ca lcu la ted theoret ica l ly , for maximal accuracy (bet ter than 0.5C)d i rect ca l ibr a t ion against a standard therm om eter has proven recom me n-dable.

Fig.3.18 Schem atic representation of arrangement for ste ri l iza tion , heating and co ol ingin fermentor .A. Connect ion to steam mains.B. Connect ion to water mains.1. Diffe rentia l pressure regulator. 2. Valves. 3. Flo w m eter. 4. Heat exchangerwi th e lectr ic e lement. 5. Backf low valve. 6. Temperature contro l ler . 7.Converter (see Fig. 3.17). 8. Recorder. 9. Thermocouple. 10. Fermentor withco i l . 11 . Out le t to d ra in .

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3.2.2 pH

pH-measurement in i tse l f is no longer a prob lem wi th modern ster i l i zab lecom bine d e lectrodes. They have become avai lab le w i t h increasing dependabi l i ty over the last decade. Whereas i t was formerly necessary to separate atleast the re ference e lect rode f rom the cu l ture by an e lect ro lyte br idge toprevent co nta m ina t ion and leakage of K CI in t o the cu l ture , the mo dernelectrodes are f i t te d w it h a sm al l porou s stone giving very l i t t le leakage andno contaminat ion. Backf low of cu l ture in to the re ference e lect rode reservo i r ,especial ly during ster i l izat ion at overpressure, can be prevented i f the same(or a l i t t l e higher) pressure is app l ied on the KC I reservoir . Th is requires are la t ive ly compl icated arrangement and we have found that wi th the Ingoldcombined e lect rodes c losing of the KCI reservo i r dur ing heat ster i l i za t ion andopening thereafter, and during cul t ivat ion, is an easier and st i l l safeprocedure.

As has already been concluded on p. 38, in pH-control accuracy is easi lyreached. The use of pumps with adjustable speed (e.g. of the peristal t ic type)is prefe rred t o valves, because we f ou nd the lat ter less depe ndab le. M oreov er,va lves do not o f fer the possib i l i ty o f vary ing the f low ra te . I f bothpH-increase and -decrease are to be expected, two point control is necessary.A special case in w hi ch such a tw o p oi nt c on tro l is app l ied is described und erSect ion 4 . 1 . 1 . Schem at ic i l lus t ra t io n of tw o po in t pH-c on tro l is g iven in F ig .3 .19.

Fig. 3 .19 Schematic representat ion of tw o poin t pH -co ntro l .1. Recorder. 2. Converter (see Fig. 3.17) 3. Control lers. 4. Acid l ine fromreservoir (not indicated) through peristal t ic pump to fermentor. 5. Idem, foralkal i . 6 . Fermentor . 7. pH-electrode.

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l/>[n 9 '6r:

^ 3 ^ l

EDC

>

1 2 3 4 5 6 7 8Fig. 3 .20 Schematic representat ion of pH -contro l wi t h carbon d ioxide as the con tro l l ing agent.

1. Recorder. 2. Converter (see Fig. 3.17). 3. Control ler. 4. CO2-inlet. 5. Airinlet. 6. Com bined gases. 7. Ferm entor. 8. pH-electrod e. A. Red uction valves.B. Manometers. C. Needle valves. D. Solenoid valve. E. Flow meters.F. Differential pressure regulator.

A more compl icated pH-cont ro l consis ts of the addi t ion of gaseous carbondiox ide to b icarbonate buf fered systems (cf . Telling and Stone, 1964 andTelling and Elsworth, 19 65) . These systems are f req ue nt ly used incul t ivat ion of animal t issue cel ls . I f such a system is present in ahomogeneous cu l tu re o f t i ssue ce l ls the carbon d iox ide concent ra t ion in theove r ly ing gas phase w i l l in f luen ce th e pH . Th e con t ro l scheme is show n inFig. 3.20. Because, in this case, carbon dioxide gas is added in the gasstream,a reac t ion lag occurs , comparab le w i th the one in oxygen cont ro l . There forean on-o f f cont ro l w i l l no t a lways be accura te . Propor t iona l cont ro l w i l l t henimp rove the resul ts . A co nc om itan t advantage of th is system is th at no e xt raelect ro ly te has to be added to the cul ture.

3.2.3 O X Y G E N T E N S I O N

Unt i l the t ime that accurate measurement of oxygen tension in bacter ia lcul tures became possib le, a c lear ins ight in the ro le of oxygen in bacter ia lcu l t i va t i on w as d i f f i c u l t t o o b t a in .4 4


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Dependence of growth and other metabol ic processes on the supply o foxygen has been known fo r long. The classical division into aerobic andanaerobic microorganisms in fact on ly ind icated that there are microorganisms on which the presence of oxygen has a poisoning effect. For anaerobicmicroorganisms the PO2 wi l l be zero. For aerob ic microorganisms there wi l lexist an opt imal PO2 for product ion of ce l l mater ia l and one for product ionof cer ta in metabol i tes, and these two opt ima do not necessar i ly co incide.In indust r ia l fermentat ions a i r supply is an important factor in considerat io n o f p rod uc t io n costs. The aerat ion ef f ic ien cy is the gu id in g parameter : i tind icates the amount o f oxygen taken up per un i t o f cu l ture vo lume per un i to f t ime under def ined condi t ions of vessel d imensions, f i l l ing vo lume, a i rf low rate, st i rrer speed and method of aerat ion (vortex or sparger and, in thelatter case, type of sparger). Fig. 3.21 shows the resul ts of the determinat iono f the aera t ion e f f i c iency, de te rmined by the method o f Cooper e t al.(1944), as a funct ion of st i rrer speed and air f low rate, in a 5 l i t re vessel ofthe B i l thoven Un i t . In th i s me thod the fe rm ento r is f i l l ed to the requ i red

m m o l OT/ I h3 0 0 - 1

2 0 0 -

1 0 0 -

0 750 1500 2250 3000r .p.m.Fig. 3.21 Ae ratio n eff iciency as a fu nc tio n of impe l ler speed and air f lo w rate. Cu lturevolume 3 l i tres.a. a i r f low rate 10 l /min. b. 3 l /min.

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vo lum e w i th a so lu t ion o f Na 2S 03 , w i th Cu ' ions as a ca ta lys t. The oxy genconcent rat ion in the f lu id is thus a lways kept at zero, because the inter facebetwee n f lu id and m icroorg anism is absent . Hence the me tho d has beenseverely cr i t ic ized on theoret ica l grounds (Herbert, 196 1) . I t w i l l howe vergive at least an impression of the inf luence of the parameters ment ioned andis st i l l in general use for that purpose.I t is obvious that a cul ture grown under condi t ions g iv ing a cer ta inaera t ion e f f i c iency (as de termined w i th the method o f Cooper e t a / . ) , w i l lg ive no in fo rmat ion about the ac tua l concent ra t ion o f oxygen in the cu l tu re .

In the theory of growth k inet ics of a bacter ia l cu l ture the growth rate isassumed to be dependent upon one nut r ient , designated the l imi t ing factor .Maximal growth rate is only achieved i f al l nutr ients are supplied in excess.Oxy gen is in ma ny cases the l i m i t in g factor . O xyge n is no t , as othe r nu t r ien tsnormal ly are, added at the beginning of the cul ture but suppl ied cont inuously d ur in g cul t iva t io n. I t may rest r ic t gro w th in a specia l w ay : the ad di t io n o fequal a mo un ts per u n it o f t im e gives r ise to a l inear instead of a log ar i thm icgro wth pat te rn . I f g row th ra te wo u ld be cons tant as wo u ld happen w i thexcess oxygen, growth is expected to proceed logar i thmical ly , as fo l lowsd i rec t ly f rom the equat ion :^ 1 dN'^ N dt

where /n is the exponent ia l growth rate, N the number of bacter ia per uni t ofvo lum e, and t t he t ime . I f g row th is logar i thm ic , the oxyge n con sum pt ion isincreasing log ar i th m ica l ly . As soon as the oxygen co ns um pt io n reaches themaximal t ransfer rate, under the c i rcumstances of the cul ture, the oxygenconsumpt ion rate becomes constant .In pract ice l inear growth is indeed observed, thus indicat ing scarc i ty ofoxygen . The actual oxyg en c on ce nt ra t ion in the cul tu re is, in th is case,a lmost zero . Accord ing to Herbert ( loc . c i t . ) , t he l im i t in g con cen t ra t ion o foxy gen fo r mo st (aerobic) organisms is in the order of 1 0" ^ m M ; th is imp l ies

th at o xyge n l im i ta t io n is no rm al ly no t to be expe cted above 1 % airsaturat ion in cul tures at 37C.On ly i f the supp ly of ox yge n is such tha t a cer ta in con cen t rat ion ofd issolved oxygen is bui l t up in the cul ture, and mainta ined, by means ofPO 2-cont ro l (see p. 5 4 sqq. ) , is a new s i tua t ion created in whic h dependenceof metabo l ism upon the oxygen concent ra t ion can be s tud ied .The determinat ion o f the oxygen concent ra t ion (ca lcu la ted commonly asoxygen par t ia l pressure, or oxygen tension, and expressed in mm Hg,saturat ion wi th a i r being around 150) has been made possib le by the

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development o f probes, that can be steam ster i l i zed together wi th thecon ten ts o f the fe rmento r . The deve lopment by Van Hem ert, Kilburn,Righelato an d Van Wezel (1969) of such an electrode that has been acceptedin several other laborator ies wi l l be described here in detai l .A l though measurement o f d isso lved oxygen in bacter ia l cu l tures is noproblem theoret ica l ly , there was unt i l recent ly no pract ica l technica lso lu t ion which would make the measurement o f oxygen pressure a rout ineprocedure. The s i tuat ion was comparable wi th the sta te o f pH measurementin bacter ia l cul tures a few decades ago.There was a need for a sturdy, steam ster i l i zab le probe, which onceassembled and put into place, could be easi ly cal ibrated and handled by thelabora to ry techn ic ian .The oxygen e lect rode to be descr ibed here has been developed wi th thesecon d i t i ons in min d . I ts compo nents can be easi ly made in a wo rksh op f r o mnormal ly ava i lab le mater ia ls, and are o f standard design permi t t ing completein terchangeabi l i ty .I t was fe l t that the s i lver / lead ga lvanic e lement separated f rom the cu l tureby a thin membrane, permeable to gases (Mancy et al., 1962 ; Mackereth,1964 ) w as the mos t pr om ising p r inc ip le t o use as a star t ing po in t . Th issystem has a co nsta nt very low zero cu rre nt and gives a high cu rre nt responseto oxygen provided i ts d imensions are proper ly chosen. Since no externa lvo l tage need be app l ied, the e lect r ica l c i rc u i t ry is s im ple.

T h e Mackereth e lect rode ( typ e A 15 A , m arke ted by Elect ro n icInst ruments L td . ) was used by MacLennan and Pirt (1965) . They suggestedthat the e lect rode could be steam ster i l i zed i f a FEP-Tef lon membrane wasused instead of a po lythe ne one, but th is prove d to be impos sib le(MacLennan; pe rs . co m m .) . We fou nd tha t e thy lene ox ide s te r i li za t ion is theonly method pract icab le ; i t was the method used by MacLennan and Pirt(1966) and by Flynn , K ilburn, Lilly and Webb (1967) . These last authorsalso recommended several changes in the design in order to improve thestab i l i ty and the e lect r ica l connect ions.

A n oth er design using the same pri nc iple is tha t o f Johnson, Borkow skiand Engblom (1 96 4; see also Borkowski and Johnson, 196 7) , w ho used anopen e lect ro lyte reservo i r , which is a prerequis i te for dependable heatste r i l i za t io n. Because of i ts sma l l d ime nsions the ou tp ut c urren t is on ly inthe orde r o f 0 .0 5/ zA /m m Hg. The refore an am pl i f ie r is needed, or a h ighresistance has to be put in the output c i rcu i t , which can reduce the potent ia lin the galvanic element impair ing l inear response. Moreover, since the bodyconsists of an open glass tube i t is a rather vulnerable instrument, onlysu i tab le for laboratory exper iments.A n oth er way to ach ieve ste r i l i zab i l i ty is to b r ing the probe, a f terster i l i za t ion, in to a tube c losed wi th an ext ra g lassf ibre-re in forced s i l i cone

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membr ane (Ring e t al., 196 9) . Th e pr inc ip le o f th is e lect rode is of theso-cal led Clark ce l l typ e (polarog raphic ty pe ; see Gleichmann et al., 1960) .This pr inc ip le has cer ta in d isadvantages, i f compared wi th the galvanic type(h igh zero cur ren t ; need ing a more compl ica ted e lec t ron ic ins t rument ) .Moreover , the use of a double membrane (one on the e lect rode i tse l f , asecond one on the ster i l izable tube) , w i l l undoubt ly impair the rate ofresponse to step changes in PO2.In the fo l lowing descr ipt ion of the e lect rode, numbers refer to par ts inFig. 3.22, unless otherwise stated.

S T E R I L I Z A B I L I T YIn order to make the s i lver / lead cel l s team ster i l izable i t was found that aconst ruct ion was needed in which the e lect ro ly te is able to expand. Howeverd i f f i cu l t ies s t i l l occur red when the ce l l w i th e lec t ro ly te was brought to thehigh temperature required for s ter i l izat ion. Therefore i t was decided toster i l ize the assembly f i l led wi th d is t i l led water and to replace the water w i thele ct ro ly te af ter s ter i l izat io n. Th e elec t ro ly te fo un d most sat is factory is hal fsaturated potassium bicarbonate. In the design, measures have been taken foreasy replacem ent o f the f lu ids . Because the ele ctro lyte has the highe rdensi ty , and the probe is normal ly used face down, the ingoing tube (1) isused to br ing the f lu id to just above the s i lver cathode (9) . The outgoingtub e (Fig . 3.23) is conne cted wi th the h ighest poin t of the reservoir.

I N T E R I O R O F T H E C E L LTh e reservoir i tse lf (3) is made of ny lo n . Because th is ma ter ial is easi lydeformed, especia l ly by changes in temperature and humidi ty , i t is encasedin stainless steel (4). The lead anode (11) consists of a str ip of0 .1 X 16 X 400 m m , w ou nd to f i t in to the ny lon . Spacing between thewindings is secured by l i t t le protuberances on the sur face of the lead. Theanode is soldered w i t h in the e lec t ro ly te space di re ct ly to a lead w ire ; th isarrangem ent ensures electr ica l re l ia bi l i ty , wh ile the presence of a bare soldercon nec t ion is app arent ly no t de t r ime ntal to the op erat io n of the e lect rode.Th e s i lver cathod e (9 ) , of ab out 4 00 mm ^ sur face, is separated f ro m thelead by a d isc (7) of porous PVC (Vyon; ' /16 in. th ick) . There are holes of1 mm diameter to br ing the e lect ro ly te to the s i lver sur face adjacent to themem brane (8) . Th is surface should be rough ened , g iv ing just v is ib le scratches(cf . Per formance) .

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Fig. 3.22

Fig. 3.23

Cross section ofoxygen electrode, labora to ry mode l . Connection of the silvercathode is shown.Numbers are referredto in the text, except12 .screws;14. s tandard f i t t ing.

Cross section of oxygenprobe, turned 90 compared with Fig. 3.22. Connection of the lead anode isshown.


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P L A C E M E N T O F T H E M E M B R A N ECyl indr ical membranes have the d isadvantage that they are d i f f icu l t to sealto the electrode; moreover, the required size of plast ic tube mater ial is nota lways avai lable. On the other hand a f la t membrane, i f appl ied over the endof a tube, gives r ise to folds and hence leakage. A special system to apply af lat p iece of me mb rane, a bout 50 x 50 mm was deve loped; i t is f i rs t securedby a stainless steel r ing (6) and then stretched over the cathode, when thisr ing is screwed do w n by fo ur b ol ts . An O-r ing (10) forces the mem branedown onto the outer r im of the s i lver cathode, complete ly seal ing theelect rode, and f ix ing the membrane f i rmly over the lens-shaped sur face ofthe s i lver . This guarantees a con stant m in im al d is tance between mem braneand si lver, necessary for a rapid and reproducible response to the oxygentension outs ide the membrane.I N T E R N A L C O N N E C T I O N SThere are two elect r ica l and two tubing connect ions. The former consis tso f Te f lon insu la ted copper w i re (15) , the la t te r o f Te f lon tub ing (1)connected to the channels in the ny lon body by standard f i t t ings (2) . Oneelect r ica l w ire is soldered at a p lace outs ide the e lect ro ly te into the s i lvercathode; the other is soldered against the end of the lead wire of the anodeat a p lace outs ide the e lect ro ly te and occ luded between two smal l 0- r ings(F ig . 3 .23) .H O U S I N GThe main par t o f the hous ing is cy l indr ica l , approx imate ly 46 mm outerdiameter , and made of s ta in less steel (13) . There are two models: for thelabora tory t yp e the cy l indr ica l p ar t tapers o f f in to a we lded conn ec t ion w i tha stainless steel tub e of 12 m m o ute r diam ete r. This tube f i t s snugly in one o fthe e lec t rode in le ts o f the fe rmentor descr ibed, nex t to the combinedpH -elec trode as sho wn in Fig. 3.25 . Mo reove r, as 12 m m is a stand ard sizefor several marks of pH-elect rodes, the oxygen probe wi l l a lso f i t in manyvessels of other or igin.

In the second typ e (Fig . 3.24) the cy l ind r ica l par t is , w i t h only a s l ightchange in d iameter , welded to a s ta in less steel tube, which in turn is weldedto a s tandard f lange. Th is prov ides an easy ar rangement for insta l la t ionthrough the top or the s ide wal l o f an indust r ia l fermentor .Th e tw o elect r ica l and tw o f lu id leads are bro ugh t throug h the stain lesssteel s tem of e i ther 10 or 32 mm inner d iameter to the exter ior .A S S E M B L YTh e lead anode (11) is soldered int o posi t io n in the ny l on bod y (3) wh ichhas already been screwed in its stainless steel casing (4). The Vyon disc (7) is

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Fig.3.24 Oxygen probe, industrial model w ith flange.

laid over the anode, and the si lver cathode (9) is l ight ly pressed down on thedeepest O-r ing (5). Then electr ical and tubing connect ions are fastenedt ight ly in the connectors. Th is assembly is bo l ted to the main body (13) o fthe probe. For the p lacing of the membrane (8) the e lect rode is kept ver t ica l ,e.g. in a labora tory c lam p, face upwards. Th e m em brane is la id f la t over thesi lver surface. Th e stainless steel r ing (6) is pressed caut iou sly over the o ut err idge (4a) of the reservoir casing, but is not forced down. The O-r ing (10) isla id on the membrane, just under the inner r idge of the stainless steel r ing.Fina l ly the r ing is screwed do w n evenly, st re tch ing the mem brane over thesilver surface.O P E R A T I O NDis t i l led wa ter is in jected thr ou gh the centra l channel (by p lacing asyr inge in the appropr ia te tube) , unt i l i t f lows over f rom the other tube; thereservoir is subsequent ly r insed with several volumes of dist i l led water.The probe is steam ster i l i zed wi th the fermentor , on ly the tube connectedw i t h the centra l channel be ing c losed. A f t er co o l ing , ha l f saturatedpotassium b icarbonate is brought through the centra l channel , unt i l about20 ml o f f lu id have escaped f rom the other channel . Then both tube ends areclosed w i t h a tube c lam p.

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IM . it^

Fig. 3.25. 10 l i tre fermentor wi thO2-electrode

For equi l ibrat ion the e lect rode is le f t in the fermentor whi le s t i r r ing, ineq ui l ib r iu m w i t h a i r un t i l the reading is s table. A l th ou gh th is norm al ly takesabout two hours, in pract ice we always leave the e lect rode overnight for th ispurpose.Ele ctr ical co nn ect io ns are made across a var iable resistor of 0-25 SI. Thepo ten t ia l dro p across the res is tor is measured prefe rably on a 0-1 m Vrecorder, because a low span al lows the use of a low external resistance,which speeds the response to changing oxygen concent rat ions (Mackereth,1 9 6 4 ;M acLennan andPirt, 1966) .Cal ibrat ion is normal ly per formed by feeding mixtures of n i t rogen and ai rth rou gh the fe rm en tor w h ic h is f i l led w i th wa ter o r me d ium . These mix turesare made by mixing streams of known f low rate of both gases.P E R F O R M A N C EThis oxygen electrode has been used extensively in bacter ial and animalce l l cu l t i va t ions inc lud ing cont inuous cu l tu res o f over 4 weeks ' dura t ion ,wi thout appreciable change in cal ibrat ion values. In addi t ion, exper iments tocheck the performance have been carr ied out and are recorded here.

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300 n

O 50 100 150mm HgFig.3.26 Response of O^-electrode at diffe ren t oxygen partial pressures. Externalresistance 1.21 i2. Temperature 33.7C.Dotted curve: insufficiently roughened cathode. Thickness of FEP-Teflon

membrane mentioned in the figure.

Th e response depends upo n m em brane thickn ess , and is of the ord er of2 juA per m m Hg fo r the 0.001 in . (= 0 .025 m m) me mb rane, and 1 juA fo rthe 0 .002 in . (= 0 .05 0 mm ) m em brane, bot h o f FE P-T ef lon. In F ig . 3 .26typ ica l ca l ibra t ion curves for these two th icknesses are g iven. The o xyg enpart ia l pressure of the mixtures appl ied are determined by reading theoxygen percentage on a paramagnet ic analyzer (Magnos, Har tmann andBraun) ; the reading is conver ted to par t ia l pressure, taking in to account theoverpressure and the water vapor pressure in the vessel and the barometr icpressure.For the th innest membrane two curves are shown: the on ly d i f ference isthe roughness of the si lver surface, the rougher surface showing betterl ineari ty. This suggests that the membrane is stretched so t ight ly over thesurface that to o t h in a layer of ele ctr oly te is present at the interfa ce ,resul t ing in a lower relat ive response at higher oxygen tensions. Smal lchannels should be formed by proper roughening of the cathode sur face.

Fig. 3 .27 shows the response t ime using the 0.001 in . membrane when the

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1 ' r20 30seconds O~r10 I ' r^20 30seconds

Fig.3.27 Rate of response of electrode to sudden change in oxygen tension , effected att ime 0 . 100 % = equ i l ibr ium current wi t h a ir c.q. oxygen (approxim ately 250c.q. 900 M A ) . 0 % = equ i l ibr ium current w i th n i t rogen. External resistance1.21 n . Temperature 25C.

e lect rode is brought suddenly f rom n i t rogen to a i r , and f rom n i t rogen tooxygen , and vice versa. The 90 % response time is less than 15 seconds in al lcases, w hic h is cons istent w i t h the response rate required in ba cter ia lcul tures.The electrode has proven to survive at least 25 cycles involvingster i l i za t ion, f i l l ing wi th e lect ro lyte , cu l t ivat ion of 1-7 days, re f i l l ing wi thwater, ster i l izat ion, etc. The decrease in cal ibrat ion value during one cul turedepends a.o. upon the electr ic current during the cul ture. This decrease is inmany cases negligible, and always less than 1 %.O X Y G E N C O N T R O LAs has been men tione d already in sect ion 3.2 (p . 39) the c on tro l ofoxy gen pa rt ia l pressure in a bacter ia l cu l tur e is mo re d i f f ic u l t th an ei thertemperature or pH-contro l in the same envi ronment . The main reason is thatthe b uffe r ing capa ci ty of a cu l ture in respect to oxy gen is very low : a quic kgrow ing c u l ture w i l l be deple ted of oxyg en a few seconds af ter cut t in g of fthe supply.

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This behaviour poses special problems for the device intended to keep theoxy gen p ar t ia l pressure in a cu l ture c onstan t . On-o f f c on t ro l w ou ld causein to lerab le overshoot , so propor t iona l cont ro l is a necessi ty.There are three pr incipa l ly d i f ferent methods to ad just oxygen uptake to acu l ture. In order to i l lust ra te these methods the fo l lowing genera l equat ion isgiven: N v = K | . a ( C * - C | )in which Ny is the oxygen so lu t ion ra te , C* the oxygen concentra t ion of thecu l ture in eq ui l ib r iu m w i t h the gas phase, C| the con ce ntra t ion of oxyg enin so lu t io n in the cu l ture and K| -a the vo lum etr ic mass t ransfer c oe f f ic ien t ,depende nt upon the c on di t ion s o f c u l t iva t io n. I t is c lear tha t , if C| has to bemainta ined in a s i tuat ion of increasing oxygen demand by the cu l ture f l u i d ,

K| . or C* has to be increased; hence the th ree m etho ds :1. Increase of ag i tat io n (st i rre r spe ed), w hi ch increases the tran sfe rcoef f ic ient K| .a .2 . Increase of air f l o w rate. I f a ir is sparged int o the l iq u id , the m ain e ffec tw i l l com e f ro m increase of K| . a, a l th ou gh a higher gas pressure at themoment o f escape wi l l cont r ibute to a h igher C*. When vor tex aerat ionis appl ied, the dependence of the aerat ion ef f ic iency on the f low ra te ,as sho wn in Fig. 3 .2 1 , is clearly presen t; here change of K| .a wi l l a lm ostexclusively be responsible.3 . Increase of the oxy gen c on tent o f the gas of fere d to th e cu l ture.The la t ter method, a l though be ing the most sophist icated and accurateone, i s on ly appl icab le on smal l cu l ture vo lumes. In indust r ia l fermentat ionsthe cost o f oxygen gas is proh ib i tory.Th e pr incipa l ar rangemen t fo r the m ethod s 1 and 2 is show n in theFigures 3.28 and 3.29. In both cases a posi t ioning motor, receiving theco ntr o l l in g s ignal changes the p os i t ion of a speed con tro l on the st i r rermotor c.q . a posi t ion ing gas va lve. A specia l cont ro l inst rument for th isindustr ia l appl icat ion has been developed (Oxy-Trol l 19 I) . For the processes

described in the next chapter, however, whether on 7 or on 40 l i t re scale, thecontro l ar rangement wi th var ia t ion of the oxygen concentra t ion in the gas(3) was preferred.Fig. 3 .30 shows the arrangement for th is cont ro l method. The pressures ofthe three gases are reduced unt i l the three manometers show the samepressure (e.g. 1.5 kg f/c m ^) w he n the re is no f lo w . T he valves C are adjuste dtherea f ter so that the three f lo w meters show the same f lo w ra te . The va lvein the total f low l ine serves to set the f inal rate which is, by vi r tue of thedi f ferent ia l pressure regula tor , independent o f the fact that one, two or threegases are tr an sm it te d.

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r P--'

1 2 3 4 5 6Fig. 3.28 Schematic representation ofPO2-control by means of

varying the stirrer speed.1. Recorder. 2. Converter (seeFig. 3.17). 3. Control ler.4. Sti rrer motor wi th speedvariator, connected with posit ion ing motor . 5 . Fermentor .6. Oxygen probe.

1 2 3 4 5 6 7Fig. 3.29 Schema tic representation ofPO2-control by means of

varying the air f low rate1. Recorder. 2. Converter (seeFig. 3.17). 3. Control ler.4. Posi t ioning valve. 5. Flowmeter. 6. Fermentor.7. Oxygen probe.

T ^ 6*; 0*: -r:

^^ ^ ^ 3f t f1 2Fig. 3.30 4 8 9Schematic representation of pOi-control by means of varying the gascompos i t i on .1. Recorder. 2. Converter (see Fig. 3.17). 3. Control ler wi th selector switchesfor combination of gases (see Table 3-01). 4. Oxygen l ine. 5. Air l ine.6. N i trogen l ine. 7. Com bined gases. 8. Ferm entor. 9. Oxygen p robe.A. Reduction valves. B. Manometers. C. Needle valves. D. Solenoid valve.E. Flow meters. F. Differential pressure regulator.

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Besides the fact t ha t th is co nt r o l system gives a con stan t f lo w rate,pe rm i t t ing ca lcu la t ion o f gas con sum pt ion and ev o lu t ion by means o foxyg en and carbo n diox ide analyzers, i t is the m ost accurate one. Ta ble 3-01shows how the interval o f oxy gen con ce nt ra t ion can be chosen. Th e moreadapted the interval is to the g iven c i rcumstances, the bet ter w i l l the cont ro lbe at a cer ta in value of th e PO2 in the cu l tu re.Fig. 3 .31 shows the ins t

thesis on vaccine production

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