WORLD HEALTH ORGANIZATION
ORGAN~SATION MONDIALE DE LA
SANTE
B L G / U N O P / ~ ~ . ~ Rev.1 ENGLISH ONLY
MANUAL FOR THE PRODUCTION AND CONTROL OF VACCINES
PERTUSSIS VACCINE
l h o issue of thds docun1cr.t dues not Constitute CB document ne constitua pas une publication.
!ornlal publication. I t should not be reviewed. II ne duit laire I'objet d'aucvn compte rendu ou abntracled or quoted vrithout the anreement Of rtsurne nl d-aucune Citation Sans I'autorisation dr?
the Worfd Heat!h Organrlation. Authors alone I'Organisahon Mondiale de la Sanre. Les opinions are rosponsibfc for views expressed in signed exprimees dans les art~cles sign65 n'engagenl
articfes. que leurs auteurs.
A number of c o u n t r i e s a r e w i s h i n g to produce vaccines a t t h e n a t i o n a l level f o r which t h e y r e q u i r e t h e n
E
cessary technical i n f o r m a t i o n . WLth t h i s a i m t h e p r e s e n t manual has beenp r e p a r e d .
I t s h o u l d be s t r e s s e d t h a t t h i s manual is i n t e n d e d o n l y t o p r o v i d e g e n e r a l i n f o r m a t i o r . on methods
-
p r e f e r a b l y the l e a s t c o m p l i c a t e d ones-
f o r t h e p r o d u c t i o n of a p e r t u s s ' s v a c c i n et
of a c c e p t a b l e q u a l i t y m e e t i n g t h e WHO Requirements and t o d e s c r i b e t h e n e c e s s a r y tests. There e x i s t o t h e r p r o d u c t i o n methods, whtch may l e a d t o a s i m i l a r r e s u l t . The manual,
t h e r e f o r e , does n o t n e c e s s a r i l y express any p r e f e r e n c e f o r t h e methods chosen and s h o u l d n o t be r e g a r d e d a s a d e s c r i p t i o n of
t h o s e p r o d u c t i o n methods p r e f e r r e d by WHO. This is t r u e a l s o f o r a l l ( b i o ) c h e m i c a l p r e p a r a t i o n s , s p e c i f i e d i n t h e manual, a s w e l l as f o r t h e m a n u f a c t u r e r s and s p e c i a l equtpment mentioned.
I f c l a r i f i c a t i o n i s r e q u i r e d on any p o i n t , r e f e r e n c e s h o u l d be made t o t h e WHO S e c r e t a r i a t ( C h i e f , B i o l o g i c a l s ) .
%he manual was p r e p a r e d by t h e f o l l o w i n g c o n s u l t a n t s and members o f t h e WHO S e c r e t a r i a t :
D r J. Camexon, Connaught L a b o r a t o r i e s , Willowdale, O n t a r i o , Canada ( C o n s u l t a n t )
Dr S. Guptarak, Government P h a r m a c e u t i c a l O r g a n i s a t i o n , Bangkok, T h a i l a n d ( C o n s u l t a n t ) D r Lj. ~ i g y - m n d i E , B i o l o g i c a l s ; WHO, Geneva,
Swf t z e r l a n d
D r P. Knight, The Wellcome Research L a b o r a t o r i e s , Beckenham, Kent, UK ( C o n s u l t a n t )
D r Y. S . Nimbkar, H a f f k i n e Biophalcmaceutical C o r p o r a t i o n , Bombay, I n d i a ( C o n s u l t a n t )
Dr F. T. P e r k i n s , C h i e f , B i o l o g i c a l s , WHO, Geneva, S w i t z e r l a n d
Dr J.
n.
van Ramshorst, B i o l o g i c a l s , WHO, Geneva, S w i t z e r l a n d1
-
continuedDr N. 3 k a r i c a , Immunological I n s t i t u t e , Zagreb, Jugos Lavia (Consultant)
*
WHO Technical Report Series 1979 (to b e p u b l i s h e d ) ; Appendix P . 2 1 of t h i s manual.CONTENTS
P1
.
INTRODUCTION. .
m 7P1.l Description of the vaccine
. . .
7P1.2 Efficacy of vaccine
. . .
7. . .
P1.3 Suggested method of production 7 P1.4 WHO l&quirements. . .
8P1.5 Glossary o f terms
. . .
9P2
.
PREMISES 10 P2.2 Design of premises. . .
l0 P2.2 Qualityofmateriats. . . to
P2.3 Changing facilities. . .
t t P2.4 Premises for breeding and keeping animals. .
11P3
.
EQUIPMENT . . . . . . . . . . . . . . . . a . * . . l 3 P4.
STAFF. . .
13P4.L Qualification a d numbers
. . .
13P4.2 Experience
. . .
14P4.3 Health
. . .
14P4.4 Organization of activity
. . .
15P 5
.
MEDIA. . .
. l 5 P6.
STRAINS OF BORDETELLA PERTUSSIS. . .
16P6.1 Characterization and maintenance
. . .
16P6.2 Preparation o f seed cultures
. . .
17BLG/UNDP/~ 7.3 Rev
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lpage 5 Page P7
.
PRODL'CTION OF CONCEh:TRA'ED SUSPENSIONS OF. . .
5 . P E R T U S S I S . 18
. . .
P7.1 On solid media 18
. . .
Method A 18
Method B
. . .
2 0. . .
P7.2 T e s t s ou b a c t e r i a l harvest 2 1
. . .
P7.3 I n l i q u i d media 22
. . .
Method A in b o t t l e s 2 2
. . .
Method B in fermenters 24
. . .
P7.4 T e s t s in b a c t e r i a l harvest 2 6
. . .
.
p8 POOLING AND STORAGE 27
P7
.
PREPARATION OF FINAL BL'LK VACCINE; PLAIN OR. . .
ADSORBED 2 8
. . .
P9.L Adjuvants 28
. . .
P 9 . 2 Preparation of b u l k vaccine 28
. . .
P9.3 Testing 2 0
. . .
P9.4 F i l l i n g i n t o final containers 3 1
. . .
P 9 . 4 I n s p e c t i o n 33
P10
.
TESTS ON FINAL PRODUCT. . .
33P 1 0 . 1 T e s t s
. . .
33. . .
P10.2 Reporting of data 34
B L G / U ~ W P / ~ ~
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3 Rev.
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%
P11
.
LABELLING AND PACKAGING. . .
35P11.1 Labelling
. . .
35P l L . 2 Packaging
. . .
35. . .
P12
.
RELEASE 36. . .
P 1 3
.
RETAINED SAMPLES 3 6P14
.
SURVEILLANCE OF VACCINES. . .
37. . . .
P14. 1 S a f e t y 37
P14.2 Efficacy
. . .
37. . .
P15
.
WHO RJ3QUIREMENTS 38P16
.
SUMMARY PROTOCOLS. . .
38LIST OF APPENDICES
. . .
+. . .
39B L G / I : N D P , / ~ ~ . ~ Rev. l Page 7
PERTUSSIS VACCINE
(Requirements for Riological S u b s t a n c e s Xo. 8 ( r e v i s e d 1978)) See Appendix P . 2 1
P 1 . INTRODUCTION
P1.l D e s c r i p t i o n o f the vaccine ( P a r t A , S e c t i o n 1 . 2 , page 3 1 ) See A p p e n d i x P . 2 1
Pertussis v a c c i n e is a s u s p e n s i o n o f k i l l e d Rordetella p e r t u s s i s . S u i t a b l e s t r a i n s are grown in or on a s u i t a b l e medium and, a f t e r harvesting and s e p a r a t i n g from the medium, are k i l l e d by a method known t o preserve a n t i g e n i c i t y . p:. 2 Efficacy of vaccine
Pertussis v a c c i n e c o n t a i n i n g a t l e a s t four I n t e r n a t i o n a l U n i t s p e r human dose and g i v e n in three doses suitably
spaced has been shown to prevent whooping cough. The vaccine i s not e a s y t o make and its efficacy has b e e n q u e s t i u n e d when vaccines of low potency have been used or when the vaccine has not been u s e d i n an effective immunization schedule. Under these c i r c u m s t a n c e s vaccines cannot b e expected t o b e
effective and only p o t e n t vaccines c o r r e c t l y u s e d can r e d u c e t h e incidence o f t h e disease.
The vaccine i s rarely given alone. I t i s more mmmon f o r i t to be u s e d combined w i t h d i p h t h e r i a and tetanus toxoids, and an a d j u v a n t ( a l u m i n i u m hydruxide sr p h o s p h a t e ) . P 1 . 3 Suggested method o f production
T h i s manual describing t h e production and control o f p e r t u s s i s v a c c i n e suggests methods of p r u d u c t i . o n t h a t have b e e n shown to give satisfactory products. The methods w i l l not n e c e s s a r i l y u t i l i z e the most r e f i n e d , expensive and modern equipment that may b e in use in some labora- t o r i e s having many years' experience in t h e p r o d u c t i o n o f
BU;/UNDP!~~. 3 Rev. l
the vaccine, but the suggested methods are known to yield vaccine of consistently good q u a l i t y . Only when a new produc- tion unit has had a number of years o f experience in the
production of the vaccine by a relatively simple method should more refined methods be considered.
The controls applied both during production and on the final product follow closely those included in the WHO
Requirements (see Appendix P.21). A most important factor in vaccine manufacture is consistency of production o f quality vaccine and additional controls to ensure t h i s are suggested.
In attempting to make the manual as widely applicable as p o s s i b l e it has been necessary in some instances to suggest alternative control t e s t s . I f a laboratory has no
experience of any of the suggested t e s t s , therefore, the one of choice should be the one most easily carried out having regard to the availability o f materials and resources.
In general the methods recommended will comply with the requirements of most narional authorities for the production and control of biological s u b s t a n c e s . I f i n any d e t a i l . there is a divergence from national requirements then permission to use the new method f o r the manufacture or control of the vaccine must be obtained from the national control authority.
P1.4 WHO Requirements (Technical Report S e r i e s (to b e published 1979)) See Appendix P.21
The WHO Requirements f o r Pertussis Vaccine were f i r s t formulared in 1964; since then there have been several developments in vaccine p r o d u c t i o n and t h e Requirements were therefore revised i n 1978. The control procedures s u g g e s t e d i n t h i s manual EolLuw them closely. Although t h e methodssuggested in the manual may not b e identical t o t h o s e used by some countries, t h e q u a l i t y of the v a c c i n e produced w i l l satisfy the requirements of a l l countries.
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. .
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I . \ ' . - l y ; t c r n : l t ; i o n s l t:r;j. i.: s l ; f c i f i c s c t i v i ~ y a f n t a t e d on;::unt !;I: tile .:r:Lcrnar.i:.r.al S tar-:dard a s d e f i n e d by t ! l k
kH!1 E x p c r ? Ccm::i. c cee or) ; I i ~ . r i o g i c a l S t a n J a r d l : : a t i o n .
Gpacity I ; r ? i t is the measure o f opaciil):
a s Cc-icrmineci in c t ~ m p a r i s o r : w i t h t h e I!ttcr~:ational Reference P r e p a r a t i o n o f Opaili t y .
= T11c dosri cf vaccine which p r o t e c t s 50.X c,!: t h e irnrir~li zcd mice agains l: a
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p e r t u s s i s 2Li;e:l by t h e i;ltraccrebral r o u t e ,$ ' ...l-
..
I I i f i charvcs t = A siispensicn O S b a c t e r i a I~arvested on t l : ~ saaie day £rum ;:he c u i t u r e s of onf s i r a i n u l j3ordeteLla pertossis.
'-4 P - . . i = l'he i; ,..l..,l,ed 7, ' c-. v a c c i n e prcscllt i : ~ the
concn-iner fro~n w h i c h the final coriLainers are f i l l e d .
E ' i ~ a l 1.ot = A c o l l e c r , i o n o f s e a l e d final containers
3 r t h a t if: homogerlcous r ~ i th respect to, the Filling lul: r i s k o f cor.tami.nation during f i l l i . n g .
21 Einal L o t m u s t , t h e r e f o r e , have been f i l l e d i n one working session.
SI.X:,:~;?;DF/~~.~ Rev. l page LG
F o r more d e r a i l s , see a l s o : Xalnnual f o r t h e D e s i g n , E q u i p p i n g and Staffing of Facilities f o r Production a n d Q u a l i t y C c n t r o l . clf Yacrerial C'accines < B I ; G / U K D P / ~ ~ . I ) P2.1 Design of premises
--
( P a r t A , S e c t i o n 2, page 1 3 ;See Appendix P . 2 3
The m o s t important requirements f o r the premises i n ~ h i c h vaccine i s t o be produced are that they should b e clean,
comfortable to work i n , have c o n t r o l of temperature and
h u m i d i t y , i n c l u d e all t h e essential f a c i l i t i e s , s a d have built- in safety measures against hazards s u c h as c o n t a m i n a t i o n . The l i g h t i n g s h o u l d be such t h a t a l l equipment can b e s e e n a n d o p e r a t e d w i t h o u t d i f f i c u l t y . I t i s advisable t h a t the laboratories should b e s u p p l i e d with f i l t e r e d a i r or t h a t work s h o u l d b e done i n l a m i n a r - f l o w c a b i n e t s t h a t a r e m o n i t o r e d regularly t o e n s u r e that t h e y are o p e r a t h g
correctly. I t s h o u l d b e s t r e s s e d , however, t h a t the u a e of l a m i n a r - f l o w cabinets does not obviate t h e need f o r a h i g h s t a n d a r d of m i c r o b i o l o g i c a l t e c h n i q u e .
P2.2 Quality of m a t e r i a l s
The main o b j e c t i v e i n t h e choice o f b u i l d i n g materials f o r a l a b o r a t o r y i s that the ahedding or c o l l e c t i o n of d u s t s h a l l b e avoided. T h i s a p p l i e s t o all s u r f a c e s i n c l u d i n g t h e f l o o r which s h o u l d be covered w i t h a n i m p e r v i o u s epoxy r e s i n s u r f a c e . Where t h e r e i s heavy t r a f f i c quarry t i l e s o r t e r r a s s o floors are p r e f e r a b l e . The walls and c e i l i n g s h o u l d b e of a high q u a l i e y h a r d p l a s t e r s e a l e d by p a i n t i n g w i t h a w a s h a b l e material that d o e s n o t flake. Ledges,
t h a t i n e v i t a b l y c o l l e c t d u s t , should be avoided, and benches s h o u l d b e i m p e r v i o u s a n d wirhout c r a c k a o r c r e v i c e s . S u i t a b l e c o n s t r u c t i o n m a t e r i a l s f o r benches i n c l u d e l a m i n a t e d p l a s t i c s , m e t a l and wood c o v e r e d w i t h an epoxy r e s i n . They should b e a b l e t o w i t h a t a n d f r e q u e n t swabbing and d i s i n f e c t i o n without d e t e r i o r a t i o n . I t must b e p o s s t b l e t o spray t h e whole area w i t h a d i s i n f e c t a n t w i t h o u t damage t o any of t h e m a t e r i a l s .
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. . . .
I. s i:~cau:: e :F,€.:.:. art.: r-lrI i,ur., ;. j.,Lel:icr: I:T j.IIBt; L; L7&ti C:: 6 L E > < : E .-
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i nvclvrd i r i t i i o :,rc.ci:lctj..Jr: I:r-cci;!;s . i;J.!-;?Gilgh sLI=].: 12 ~t:il~~f:li:
precari l-i.o~ls 1 . e ~ ; i::;>i>rtaot: ttke Frsd:jtlr. <:I' a tr?xcld or bac teri.ai. va(:clne i n w!l-;.c& rile cr.l;al--sac are iij.LLcc by a c1:en:ica;.
u r i n w h i c h p u r i fi.caii;:n. in-\:..,ir;ed, j i s EsserlliaL to have some barrier between t h e o u t s i d e d u s t y cDn<at;;ineted atmosphere a:l& the p r - ~ d u c t j . o n area. The ari?as a s i . j e f o r tlte c l i l ~ u r e 3 5 organisms, processing, bl.ca<!;r,g a n d f i l l l n g cf f i n a l
vaccj. r,e mus L i;e approachah le o n l y thruueb a n a c t e r a o n
e q u i p p e d f o r washink, change of E ~ o t u e a r , a:~d t h e d o x n i ~ g o f p r o t e c t i v e clothing s n c h as c l f a n go?:ns t h a t d o n o t shed d u s t or iihres. Where open operaCiil~is arc c a r r i c d o a t , ei.ther z cap ar.d mask o r a t c t a l hcaci hood s h o c ~ l d i;e v D r r j . ~ ? , c c e s s C O
t h i s antcroorn T T ~ L I S ~ he from other parts of tl?e producti.on area acd n o r d i r e c t l y from tbc exterFor.
A p r o d u - c i o : ~ ur.ir 2nd i t s s t a f f czn !Ic 1.1sed f o r t h e production of ntcre t h a n one vaccirle i n a s e q u e n t i a l . produc- t i o n p r o g r a m e . Some vacc ir.es (c. E;. :Icl:..::.lu:;) reqaire e s e p a r a r e facility.
F 2 . !t L e m i s e s f a r b r e e d i n g a n d k e e . p : i - 2 n i . m 1 2
I t i s ct corrn~only n ~ i s t a k e n v i e v thar aoi:nals can be k e p t anywhere and under any c ~ ~ d i t i o n s . A s reliance is tc: b e p l a c e d upon the r e s u 1 . t ~ u f a n i m a l e x p e r i i n e t ~ ~ , the ariiinals must be tiuused under c o d i t i o n s i r : which t h e y will rewair!
healthy and will not be s u b j e c t e d t o c r u s s i n f e c t i u n . They r e q u i r e :
( a ) clean l i ~ i n g conditiocs aith p l e n t y o f iight and good v e n t i 2aticn. Ani.lllaIs generate a great deal of body h e a t a s well as hvmidily and over- crowding can he d i s a s t r o u s ;
ii~C/lt!NUt;.:i'.
3 + ';page 12
( 5 a good balanced d i e t v i c h some v a r i e t y . It i s important t h a t contamination o f f o o d and h e d d i n g , xhic:h i s cailsed u s u a l l y by w i l d r o d e n t s , s h o u l d be p r e v e ~ t e d ;
( c ! l i v i n g quarters t h a t can b e cleaned d a i l y and d i s i n f e c t e d between b a t c h e s of a n i m a l s .
It is important t u c o n t r o l the b a c t e r i a l , fungal and helrriitlth p o p u l a t i o n ir: the breeding s t o c k . Many intercurrent inEeci:ions are a b l e to a f f e c t the r e s u l t s of a n i m a l experi- ments, Such i n f e c t i o n s s h o u l d be k e p t t o a minimm by
segregation o f animals Erom different sources, s e l e c t i v e b r e e d i n g from h e a l t h y stock and a s a l a s t resort by s u i t a b l e treatment w i t h therapeutic agents.
The q u a l i t y control of p p r t u s s i s vaccine requires a large s u p p l y of h e a l t h y mice, and such control should not be attempted u n l e s s good animals are available from one's own facility or can b e obtained on a r e g u l a r b a s i s .
For t h e manufacture and c o n t r o l o f bacterial vaccines such a f a c i l i t y s h o u l d b e capable o f housing and, i f n e c e s s a r y , breeding the animals r e q u i r e d . For the
t e s t i n g of p e r t u s s i s , cholera and typhoid vaccines as well as diphtheria and tetanus toxoids only mice and guinea pigs are needed.
he
UFAW Handbook on t h e Care and Management o f Animals (Ed. Churchill L i v i n g s t o n e , Edinburgh, London, New York, 5 t h E d i t i o n , 1 9 7 6 ) i s a m o s t u s e f u l referencefor a l l aspects involved i n the care o f animals.
'-hc r2qu.i pn:cnt r-cr;::i r e d T o r the productinn of percussis v;?cci.:le r d i i i d e p e n d upcn Lhe rnethod s e l e c t e d f u r the grccrth o f t h e erpst!i.stn.
eactcria groxn c n s o l i d m e d i u m have g i v c n r i s f t c v a c c i n e s
5;j r n c h e Iugl:esc p o t c n c y . T f 1ir;li.red q u a n t i t i e s of vacci.rw a r e r e q u i r e d , t h e r e f c r e , s u c h a ae:hod is t h e onc af c h o i c e . The number c [ bcttlcs r:f solid rricdiuni that cat) b e h a n d l e d at any orrc time v i . t h u u t rur!i~i.-g into a serious problem o f contam- i n a t i o n i s a b o u t 2 0 0 , %hen ths q u a n t i t y of vaccine required
i s l a r g e , therefore, (grcacer than one t c f i v e : n i l l i o n d o s e s pcr y e a r , d e p e n d e n t cn local. circumstances) t h e u s e of a
l a r g e v e s s e l
<
f c r m e ~ ~ ~ e r ) r<i.t;i the g r o w t h o f t h e organisins i n l i q u i d medium s h o u l d be c u n s i d e r e d , e s p e c i a l l y if t h e r e i s a s h o r L n ~ ; e r,f : ; u i t a b : ~ s t a f f .The equipc;cnE r e q u i r e d F3r Ific p r o d u c t i o n of ~ E ~ ~ u s s i . s vaccine: i n f e r m e n t c r s i s s i n i l a r to t h e eqcipment f o r r h e n r o d u c t l . u n of J i p h t n e r i a t0xi.n. Some p i e c e s c f equipr:ent
,
; < h i c k c o u l d !:c constrticted ~ x a l ly, a r e merr:i:)ned i n Appe1:dices i'.2 and F . 3 .
TSp nl;m:,er or staff ir?i.-,:.::.red ir: t h e p t i ~ d l : c t i = n o f
i:,.;,: l::!cr:i.: ~ . r c : : : ~ i . : ; ~ i i c p i : ~ d s t:; sirme c:icen; ::pc?! tl-IF .joiu::;c c f
.
;-,.;L ,.. .. ,-_!-:c: C.:: :,c. ~"::duc::< -'.ad !-::a TIUBI!~(?U c?f h~tc!?t:s iieede:!.
BLG/UNDP/~ 7 . 3 Rev. l page 14
Maintenance of seed culture, preparation of inoculum, growth and recovery of organisms can be carried out by a graduate
m i c r o b i o l o g i s t with some years' experience and three t e c h n i c i a n s . Quality control of the b u l k vaccine and of the f i n a l
product will r e q u i r e a l s o a graduate scientist and one t e c h n i c i a n .
The animal facility should be supervised by a senior technician w i t h adequate experience and t r a i n i n g i n the main- tenance of small animals. Provision should be made for professional veterinary c o n s u l t a t i o n when required.
F i n a l l y , i t i s important t h a t i n every laboratory for vaccine production a t l e a s t one mechanical engineer should b e a v a i l a b l e , t o b e r e s p o n s i b l e for the equipment.
P4.2 Experience
I t is preferable t h a t the t e c h n i c a l s t a f f should have had an education beyond normal s c h o o l i n g where t h a t u s u a l l y f i n i s h e s a t 16 years of a g e . I n t h e i r f u r t h e r e d u c a t i o n they should have s t u d i e d microbiology and have a working knowledge of aseptic technique. The scientific s t a f f s h o u l d have spent a t least three t o s i x months in a produc- t i o n unFt actively engaged in t h e manufacture and c o n t r o l of bacterial vaccines. A l l the s t a f f should be trained for h e work t h a t they will be undertaking.
P4.3 Health
A l l s t a f f should be in good health, and s h o u l d h e immunized against or known to have n a t u r a l immunity against any i n f e c t i o u s organisms t h a t they way b e required t o handle. They s h o u l d b e i r m u n i z e d a g a i n s t tuberculosf S,
and not be s u f f e r i n g from any o t h e r respiratory or d i a r r h o e a 1 disease. Any s t a f f members having a s e p t i c wound should n o t enter the production area.
. .
I l ~ c t e c l ~ n i c a l s t a i - i l c i s k i I:!:; a f t e r - cne ::recdi.riy r e l u n y r:r
e s p e r i n x n t a l animals s h o u l d a l s o hr. ir: !:oocI ! ~ c a l . t h a c d i r e e fr;m any laten: i n t e c t i o n .
P 4 . 4 Organization oC a c t i v i t y
q u a l i t y contro: and produc t i 03 s h e i ~ 1.d be ad:nir.istrative Ly s e p a r a t e b u t it i s i.mpurran; t h a t q u a l i t y can:.roT s t a i r have first-hand knur.11edge and cxpcricnce of prc,duc t i o r . . Ik!"ncre i z no need f o r a n i s a l testing to i)c d u p l i c a t ~ d , b u t i t i s inpur!:ar::
c h a t t h e d e t a i i e d r e s u l t s of a l l "in ~ r o c e s s " a:id f i r i s i . t.ests s h o u l d b e available to b o t h p r o d u c t i o n aild q i l & i i . t y c v n t r c r l s t a f f .
I n any e v e n t , accurate and clear pe-rnanent rcccrds a i g i l e d l e g i b l y and d a t e d mist be kept, a z d must be nade avai1abj.e a t
a l l t i m e s to the production and quality c o n t . r o l staEF a s well as t o Lhe a p p r o p r i a t e i n s p e c t o r s from local h e a l t h a u t b o r i t i e s .
Examples u f f c m s s u i t a b l e for k e e p i n g s u c h records are shcr.il~
in Appcnuices P. 1.9 and P. 2 0 .
7:he .sta!iI' sbouid be t r a i . n e d ir. t i l e p r o d u r : c i c r . . n f n r u n S c r of vaccines b e c a u s e t h e p r c d u c t i c c f a c i l i t i e s can b e cscd < c ; Che m n u f a c t u r c of a l l i:actcrial v a c c i : ~ e s except vacr:l.:ies p r s d n c ~ d ircx?. a n a e r o b i c , spore-bcarirg or:gani:;r:;s, c . g . !;c';an?!s.
Sinilariy, the quality c o n t r o l s t a f i : s b c u i d be c z p a b l e c1 c c n t r o l l i n g a 1 i b a c t e r i a i v a c c i n e s .
page 16
be p r e p a r e d as needed. The media used for he growth of B, p e r t u s s i s are not chemically defined and the i n g r e d i e n t s such as casein and casein hydrolysates are not reproducible from batch t o b a t c h . I t i s important, therefore, t o have a system f o r q u a l i t y control t h a t will ensure both the s t e r i l i t y of
the media and t h e i r ability t o support the growth of the organisms.
The composition of the media and the method of prepara- tion are d e s c r i b e d i n Appendix P . 1 .
P6. STRAINS OF BORDETELLA PEBTUSSIS
P 6 . 1 Characterization and maintenance (Part A , Section 3 . 1 . 1 , page 32) See Appendix P . 2 1
S trains o f B . pertussis used in the production of vaccine should be capable,oE yielding vaccine of acceptable quality. These may be f r e s h isolates or may be obtained from kn
P
wn r e p u t a b l e sources. Two or more d i f f e r e n t strains s h o u l d b e u s e d ; they should be maintained freeze- dried and before drying s h o u l d b e plated and examined under a plate microscope to verify their homogeneity. The f i n a l vaccine should include agglutinogens 1, 2 and 3. I n order t o ensure a long working l i f e of t h e s t r a i n s the initial drying of 50 t o LOO ampoules should be d i v i d e d into two parts in the r a t i o of 4 : 1 . The ampoules from the larger p o r t i o n a r e used a s seed stocks for production whereas those from the smaller portion are designated as the master seed s t o c k from which new d r y i n g s are made, This ensures that over a period o f several years the seed strains thernseLves are not subcultured.'~dvice on t h e source of s t r a i n s may be o b t a i n e d b y a p p l i c a t i o n t o t h e BHO Secretariat
( C h i e f , 3io2ogi.ca2s).
BLG/UNDP/~~. 3 Rev. l page 17
P6.2 E e p a r a t i o n of seed c u l t u r e s
C o n s i s t e n c y in manufacture i s a most important factor and in order to achieve this a s i n g l e medium should b e used
throughout the p r o d u c t i o n process. For the purposes of this manual medium o f t h e Cohen and Wheeler type i s r e f e r r e d t o ;
d e t a i l s of i t s formulation as well as of t h e f o r m u l a t i o n o f a l t e r n a t i v e media are given i n Appendix P . 1 .
To prepare a seed c u l t u r e , one ampoule o f a freeze-dried s e e d stock i s opened and the c o n t e n t s resuspended i n 5-7 m 1 o f l i q u i d Cohen and Wheeler medium. A l i q u o t s o f 1 m 1 of the suspension are then i n o c u l a t e d onto the surface of s o l i d Cohen and Wheclcr medium t o which charcoal has been added and c o n t a i n e d i n each of f i v e , screw-capped bottles leasuring 2 X 8 X 15 cm. These b o t t l e s have about 100 cm of medium s u r f a c e . The b o t t l e s are rocked gently by hand to e n s u r e that the a h o l e s u r f a c e of the medium i s i n o c u l a t e d . They are then placed i n an incubator w i t h the i n o c u l a t e d surface uppermust and left for three to four hours t o allow t h e medium to a b s o r b t h e e x c e s s f l u i d . When the surface o f the m e d i u m i s d r y the b o t t l e s are turned over so t h a t the inoculated s ~ r f a c e i s n o w downwards. T h i s must be done c a r e f u l l y e s p e c i a l l y w i t h large b o t t l e s t o avoid dislodging t h e agar. The procedure of drying and i n v e r t i n g b o t t l e s s h u u l d hp b l l o w c d wherever scZid medium i s b e i n
g
used.Zle bottles are i n c u b a t e d for f o u r days a t 35 - 3 6 C w l t h t h e screw-caps loose t o f a c i l i t a t e a c c e s s o f a i r . A r the end of t h e incubation p e r i o d the c o n t e n t s of the bottles are checked f o r p u r i t y by G r a m s t a i n (see Appendix P. 13).
If not needed i m m e d i a t e l y for further stages in t h e produc- t i o n the b o t t l e s are now stored i n rhc r e f r i g e r a t o r a t
0 -P 0
5 C
-
3 C with the screw-caps tightened.A l l t r a n s f e r s should be made from and to medium a t 35 0 C s o as t o ensure c o n t i n u o u s growth.
From t h i s point onwards i n production the amount of seed culture required i s determined by t h e method u f p r o d u c t i o n to b e f o l l o r ~ e d whether i t be on s o l i d surfaces, in liquid medium, in glass bottles or in fermenters.
BLG/IIKDP/~' 7.3 Rev. l page 18
I n order to a s s e s s the amvunt of seed required from one stage t o the n e x t , the c a l c u l a t i o n should be b a s e d on an inoculum
containing 2-5% u f the number o f organisms expected i n the harvest a t the n e x t stage. This, together w i t h t h e maintenance
0 D
of temperatures a t 35 - 3 6 C ensures t h e optimum ccnditions for growth.
P7. PRODGCTI ON OF CONCENTRATED SUS PENSIONS OF B. PERTUSSIS
P7.1 On s o l i d media
Two methods of production on s o l i d m e d i a are d e s c r i b e d .
I f mure seed is needed than is available from the f i r s t passage of the freeze-dried seed culture, an appropriate number of one litre Erlenmeyer flasks, each containing 150 m1 of medium to which 2 m1 of a 0.2% s o l u t i o n of g l u r a t h i v n e has been added, i s i n o c u l a t e d w i t h the calculated amount o f seed c u l t u r e . These are incubated f o r 24 hours, w i t h vigorous s h a k i n g , and t h e opacity ( s e e Appendix P.111 and purity ( s e e Appendix P.13) of the culture checked. I f pure, they may be used as the seed culture f o r a batch of b ~ t t l e s ~ c o n t a i n i n g solid medium and each g i v i n g a t least 300cm o f medium surface.
A s u i t a b l e bottle is about 20 t o 30 cm high, 12 t o 2 0 cm
Y
i d e and 3 t o 8 cm deep which provides a surface of 400 cm when 200 m1 of solid medium is introduced in alayer 0 . 5 cm deep. The optimal depth of medium can be found by experiment t o give the maximum yield o f organisms. The depth must be n o t less than 0.5 cm
otherwise the agar surface may be damaged during harvesting and i f i t i s too deep t h e medium may become detached when the b o t t l e s are inverted a f t e r being i n o c u l a t e d ,
A d h e s i o n of t h e medium can, to s o m e e x t e n t , b e i.mproved
BW/UNDP/~ 7 .3 Rev. l page 1 9
by increasing the agar c o n t e n t .
The bottles may be p r o t e c t e d from contamination by a cotton-wool plug or a screw-cap; if t h e latter i t be left loose during the growth to allow free access of a i r .
The contents o f one Erlenmey r flask, containing
I
150 m1 of medium, will seed about 12,0005
m of surface or 30 bottles each w i t h a medium surface o f 400 cm.
For a batch of 100 suchbottles at least four seed flasks are necessary and, to allow for possible contamination, a t least six, preferably eight, flasks should be inoculated. The inoculum for each b o t t l e is about 5 ml and a r e must be taken to ensure that the total surface of t h e medium is inoculated. This is done by the method already described followed by absorption of excess fluid and i n v e r s i o n . Growth is allowed t o continue f o r two t o three days depending upon the t i m e a t which maximum yield of
organisms is achieved. Usually cultivation fcr more than 48 hours leads to a higher yield of organisms but a lower
p o t e n c y . The optimal p e r i o d s h o u l d be d e ~ e r r n i n e d experimentally.
At the a d of the growth p e r i o d , 2 0 ml of saline contain- i n g formalin a t a final concentration of 0.3-0.5% i s introduced into each bottle and t h e growth loosened from the agar surface by g e n t l e , manual rocking, c a r e being taken not t u damage the agar surface.
A smear is made from each bottle, Gram stained, and checked for purity. Bottles shown ts be contaminated are discarded. The conterlts of the non-csntarninated bottles are harvested by tilting them on t o one o f the luwer c o r n e r s and removing the s u s p e n s i o n of organisms by means o f a long, glass tube connected by t w o receiving vessels t o a vacuum l i n e . These vessels are connected in series and s t e r i l i z e d t o g e t h e r . The organisms as they enter the first bortle are f i l t e r e d through nylon gauze or orher suitable material in order to remove particles of agar,charcoal or other d e b r i s dislodged
HE,/I:KDP /.7 7 . 3 Rev. 1 page 20
from the m d i u m during harvesting. The uniform s u s p e n s i o n of organisms t h e n passes d i r e c t L y to t h e secocd receiver. When harvesting i s complete the two receivers a r e separated, a sample of the bulk suspension i s removed f o r tests f o r s t e r i l i t y and opacity and t h e b u l k p l a c e d in the refrigerator.
If the sterility t e s t s show nn evidence of contamination by the t h i r d day, p r o c e s s i n g may b e continued: it i s important, however, to allow the s t e r i L i t y t e s t t o proceed for the f u l l s t a t u t o r y period of 14 days. The c o n t e n t s o f the b u l k
container are centrifuged i n a refrigerated c e n t r i f u g e , the s u p e r n a t a n t f l u i d discarded and packed organisms resuspended in saline or phosphate b u f f e r e d s a l i n e , pH 7 . 2 , containing 0.01% thiomersal (ethyl-mercuri-thiosalicylate). The d e n s i t y t o which the s u s p e n s i o n i s adjusted must t a k e into account the f a c t t h a t it is t u be blended w i t h an equal part o f at l e a s t one other strain of 3. pertussis as well as w i t h d i p h t h e r i a and t e t a n u s t o x o i d s and a d j u v a n t . A reasonable figure for the a m i t y i s 500 to 1000 O . U . Method B
A s i m p l e method i n w h i c h the organisms are grown on
s o l i d surfaces and k i l l e d by heating alone may a l s o be used.
A t l e a s t t w o f r e e z e - d r i e d s t r a i n s w h i c h have p r e v i o u s l y been adapted t o grow on charcoal agar (Powell, C u l b e r t s o n &
Ensminger, 1951 : "Charcoal agar culture medium for p r e p a r i n g Haernophilus pertussis vaccine", P u b l . Hlth.Rep., 6 6 , 3 4 6 ) are reconstituted w i t h physiological s a l i n e
-
solution that has beenwarrned to 3 6 " ~ . The suspension is then i n o c u l a t e d on Powel.1 medium i n P e t r i - d i s h e s which
0 0
are then i n c u b a t e d a t 35 -36 C f a r 48 hours. The growth from these Petri-dishes is used as the inoculum for PoweZL medium contained i n t h r e e Roux flasks.
The Roux flasks are incubated for 48 h o u r s a t 35O- 3 6 O ~ a t which time t h e growth in each f l a s k i s c h e c k e d for p u r i t y and morphological characteristics ( s e e Appendix P . 1 3 ) . Into each of those c u l t u r e s shown t o be pure, a
B L G / U N D P / ~ ~ . 3 Rev. l page 21
volume o r 50 m 1 o f physiological saline is p i p e t t e d and the f l a s k s gently shaken t o e n s u r e t h a t t h e whole surface is covcred.
The bacterial suspension so produced i s then seeded fin
Powell agar f o r hatch production, u s i n g the suspension from one Roux f l a s k to i ~ o c u l n t e 20 Roux flasks each w i t h 2 - 3 m 1
6f the b a c t e r i a l suspension. After 48 hours of i n c u b a t i o n a t 3 5 - 3 5 0 C the b a c t e r i a l cultures are checked f o r purity and nic?rphological characteristics (see Appendix P. 13) a n d thcse found s u i t a b l e are harvested.
Harvesting i s performed by p i p e t t i n g 5 0 m 1 o f physio- l o g i c a l s a l i n e or phosphate b u f f e r e d s a l i n e , pH 7.2 into each Roux f l a s k , allowing them t o s t a n d for 30 t n i n u t e s and suspending t h e growth by gently rocking the flasks. The c o n t e n t s f r o m a11 flasks are collected i n t o a 10-litre 30trlc using a negative pressure i n a closed system.
T h e suspension syphoned frotn the culture flasks i s
f i l t e r e d through a gauze bag before ectering t!le 10-litre Crlliector bottle and the opacity of the harvested s u s p e n s i u n determined ( s e e Appendix P . 1L).
The suspension c o l l e c t e d from a single group of boctles and p o o l e d i n t h e 10-litre bottle i s inactivated by Seing p i a c e d i n a water bath at S 6 0 C f o r 3G minutes, w h i c h i s stirred continuously and t h e temperature
controlled. After inaccjva:ion a s o l u t i o n of thiomersal i s added co the b a c t e r i a l s u s p e n s i o n s o t h a t t h e final c o n c e n t r a t i n r . in the suspensJi>11 is 1:5000.
P 7 . 2 Tests on b a c t e r i a ? harvcsr
'?he ki. l l e d bacLeria l s u s p c n s i . o l ~ s harvested f rorn srjl.id meGia arc ~ t ~ e n t e s t e d f u r :
P i . 2 ( L 1 P c t e ~ l c y Appendix F . 4
E L G / E N U P / ~ ~ . ~ Kev.1 Page 2 2
Pi.Z(III) Abnormal toxicity Appendix P . 12
~ 7 . 2 ( 1 V ) I d e n t i t y A p p e n d i x P.5 P7.2 ( V ) S t e r i l i t y Appendix P. 15 P ~ . ~ ( v I ) Opacity Appendix P.11 p7.2(VII) Thiomersal content Appendix P . 7
The r e s u l t s of t h e s e t e s t s are recorded and the suspension h e l d a t 5O ~ O C and labelled, e.g. " S t r a i n X, f u l l y t e s t e d harvest ready for further blending". The label as well as the process production card should be signed and dared by an a p p r o p r i a t e s e n i o r member of s t a f f . S e e Appendices P.19 and P.20.
Where a number of t e s t s are a p p l i e d a t t h e same p o i n t Fn the production process, i t i s a d v i s a S l e t o carry o u t t h e t e s t s i n such an order t h a t those making t h e l e a s t demands on resources and giving the q u i c k e s t results should be performed f i r s t ( t h e s e are pH, opacity, s t e r i l i t y and mouse toxicity). These should b e shown to be s a t i s f a c t o r y before the more demanding and time-consuming tests are undertaken.
P7.3 In l i q u i d media Hethod A
-
in bottlesProduction in l i q u i d medium is, Tor p r a c t i c a l purposes, an extension of the development of seed cultures ( s e e
Appendix P . 2 ) and depends f o r its success on the u s e of a n inoculurn a t each stage of an adequate size such as 2-5%
of t h e y i e l d o f c e l l s expected in t h e following s t a g e of production. I t S a r e l a t i v e l y simple method, certain1.y advisable for a laboratory s t a r t i n g pertussis vaccine production.
S i n c e B. pertussis growing in bottles must have an adeqilate a i r s n p p l y , any method used must s a t i s f y this demand. T h i s may be achfeved by s p a z g i n g , rolllng or
B L G / u ~ % P / ~ ~ . ~ Rev. l page 2 3
rotating at a s u i t a b l e angle, the method adopted b e i n g dcter- mined by the facilities available and the size of the u n i t a d o p t e d . The method described below for b o t t l e s is t h a t o f sparging.
The b o t t l e i s assembled, medium added, sterilized and inoculated a s described in Appendix P.2. It should contain e i g h t litres of m e d i u m to which 50 m1 u f a 0.22 sol.ucion of glutathione has Seen a d d e d prior t o i n o c u l a t i o n . An inoculum containing an a d e q u a t e number of organisms i s i n t r o d u c e d into the bottle, t h e contents of which should be at 35 0 C and
aeration through a f i n e grid (sparger) at the rate W E three l i t r e s p e r m i n u t e is started. Growth with aeration i s allowed t o continue for 30 hours or until maximum growth has been attained; d u r a t i o n o f growth will depend largely on t h e medium and should be determined by e x p e r i e n c e .
The bottles a r e then d i s c ~ n n e c t e d from the a i r s u p p l y and samples withdrawn for tests f u r opacity and purity (see Appendices P.11 and P.13). Ln t h i s method of produc- Lion, s e v e r a l b o t t l e s can b e cunneceed t o t h e a i r supply throrlgh a m a ~ ~ i f o l d system w i . t h a s i e r i l i . z i n g air f i l t e r j.n each line s o t h a t each b o t t l e i s t r e a t e d i n an i d e n t i c a l manner. If the contents of t h e b o t t l e or bottles are t o be used as seer! cultures f o r fermenters, t h o s e shoxn t o b e pure are transferred t u the r ~ f r i g e r a t o r u n t i l required or, if t h e y are to be used immediately are maintained at 35 0 C up CO the t i m e of i n o c u l a t F o n i n c o a ferncnter w h i c h i c h e l d a t t h e same temperature.
Kherc the c o n t e f i t s cf t h e bottles r e p r e s e n i the Sinal s t a g e i n produc:.ior:, the organisins arc killed l;y heacirle a t 56°C f o r ? @ n : i K u t e s t h e a d d i t i o n u f furmi..lFn t o g i v e a f i n a l ccncencratLcn c f 0.1;'.. A szrnple i s takeil f:!r a s t e r i l l . r y t e s t and t h e i ~ c t c l e s are h e i d iE t h e coid f o r t h r e e days.
I f at t h e end of this ti::ie the samp?e i s s t e r i l e t h e p e r t u s s i s '3fg211isin.r f r o m t ! ~ c - e : : e r i k h o t t J . c c a r c c e r l t r i f u g e d i n a r e f r i g e r a t e d centrifuge; it i s important, hovever,
!5K&/ihTf/77.3 Rev. l page 24
allow the s t e r i l i t y t e s t to run the f u l l s t a t u t o r y L4 days.
The supernatant fluid i s d i s c a r d e d and the packed cells resus- pended i n buffered saline containing 0.01% o f thiomersal t o a n o p a c i t y of 500 to 1000 O . U . A t this s t a g e , the f i n a l s u s p e n s i o n of organisas should be checked for :
P7.3(1) Potency Appendix P.4 P7.3(II) Mouse Eoxiry (mouse Appendix P . 6
weight g a i n )
P . 73(111) I d e n t i t y Appendix P.5 P.7.3(ZV) Sterility Appendix P. 15
P . 7 3 ( ~ ) Opacity Appendix P.ll
I t should then be stored at 5 O
5
3 ' ~ and labelled" 8 . pertussis suspension. Strain X . . Fully t e s t e d and available f o r further prcduction/processing~'. This l a b e l a s well as t h e process p r o d u c t i o n card should be s i g n e d and dated by an appropriate s e n i o r member o f s t a f f .
Method II
-
i n fermentersP r o d u c t i o n in fermenters is a n e x t e n s i o n of production in bottles in t h a t c u l t u r e s grown in bottles can h e used e i t h e r as vaccine or as seed c u l t u r e s f u r fermenters. The contents of s u c h b o t t l e s i f held i n the cold r e t a i n both antigenicity and viability up to about four weeks a f t e r
the end of growth.
S t a i n l e s s - s t e e l fermenters can b e cf any size from 20 litres upwards. They need not b e h i g h l y instruinented since the growth of pertussis requires unly control o f i m p e l l e r s p e e d , a i r s u p p l y and temperature, and they may be purchased f r o m c o m e r c i a l m n u f a c t u r ~ r s or made t o o r d e r by a good f a b r i c a t o r o f s t a i n l e s s - s t e e l . Although t h e initial c a p i t a l investment may S e e m h i g h , they have an assured wurki.ng l i f e of a t l e a s t 10 years and are 1.ikely t o p r e s e n t
BLG/UNDP/~ 7.3 Rev. l page 25
the m i n i m u m i n demands on servicing i f c a r e f u l l y designed. To d a t e l i t t l e information i s available on the production of
p e r t u s s i s vaccine in fermenters larger than 150 litres.
The basic design of a fermenter for the p r o d u c t i o n of p e r t u s s i s v a c c i n e i s given in the l i n e drawing ( s e e Appendix P . 3 ) . There i s no need f o r c o n t r o l of pH, the temperature of i n c u b a t i o n i s 35 C , the s t i r r e r 0 speed 500 t o 600 rpm and
a e r a t i o n i s a t t h e rate o f 0.2 l i t r e s of a i r p e r l i t r e of medium p e r minute.
Ta seed 1 5 0 l i t r e s of medium i n a f e r m e n t e r , eight l i t r e s of s e e d r e p r e s e n t i n g a n inoculum volume of a b o u t 5% are used.
The growth period i s 30 to 40 hours and it i s advisable t o remove samples a t regular i n t e r v a l s ( e . g . 24, 2 7 , and 30 h o u r s ) t o a s c e r t a i n the s l u p e o f t h e growth curve. When the maximum growth has been a t t a i n e d a sample i s removed for t e s t s f o r p u r i t y ( s e e Appendix P.11) and f o n o a l i n is added t o t h e bulk t o givg a f i n a l concentration of 0.1%. The tank i s h e l d a t 35 C for 1
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3 days and checked f o r s t e r i l i t y ( s e e Appendix P.15). The organisms are t h e n s e p a r a t e d by c e n t r i f u g i n g i n a c o n t i n u o u s - f l o w c e n t r i f u g e (see Appendix P.18, i t e m 201, t h e contents of t h e fermenter being fed i n t o t h e c e n t r i f u g e a t a r a t e of 150 t o 300 m1 p e r m i n u t e , and t h e f l o w r a t e a d j u s t e d t o g i v e a c l e a r effluent. I f only one centrifuge is a v a i l a b l e , several s t e r i l e bowls should be o b t a i n e d s o t h a t a s one i s f i l l e d i t can be replaced. One bowl can hold the organisms from about 7 0 l i t r e s of c u l t u r e . The receiver i s removed from the c e n t r i f u g e u n d e r a s t e r i l e cover, f a b r i c or m e t a l , and transferred t o a s t e r i l e . c a b i n e t . The packed organisms a r e removed from t h e b o w l by means of a s t a i n 1 . e ~ ~ - s t e e l spatula. The p a s t e o f organisms is transferred to a Waring blender or s i m i l a r type homogenizer p r e v i o u s l y sterilised; the paste i s r e - saspended i n b u f f e r s a l i n e c o n t a i n i n g 0.01% t h i o m e r s a l t o a d e n s i t y o f 500 t o 1000 O.U. ( s e e Appendix P.11).Instead of s t a i n l e s s s t e e l also g l a s s can b e u s e d f o r fermenter vessels.
