IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIUS007955605B2

(») United States PatentPrasad

(lo) Patent No.: US 719551605 $2

(45) Date of Patent: Jnn. 7, 2011

(54) MUI.TIVALI(fs T PNEUYIOCOCSOALPOLYSACCHARIDE-PRO FEIN COVJUOATECOMPO SITIOV

(75) Inventor: A. Krishna Prasad, ('hapel Hill. NC

(US)

(73) Assignee: Wyeth LL(.', Madison, NJ (IJS)

(1

) Notice Subject to any disclaimer. tbc tenn of thispatent is extended or adjusted under 35

U.S.(l 154(b) by 0 days.

(21) Appl No.. 11/644,095

(22) Filed: Dec. 22, 2006

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PUBLICATIONS

FORE)ON PATL'NT DOCUML'NTS

(65) Prior Publication Data

iJS 2007/0184071 A I Aug. 9, 2007

Related U.S. Application Data

(51) Int. CI.A 6JK 39/09 (200(&.01)

A 61K.39/305 (2006.01)A 61K 39/00 (200Cxol)A 6 IK 39/38 (7006.01)C0717 21/02 (2006.01)C07H 21/04 (2006.01)C07K Jd/0/J (ZOBC.OI)

C07K 1/00 (2006.01)C07K 17/00 (2006.01)

(52) U.S. ('I........... 424/244.1: 424/197.11: 424/184.1;536/23.1; 530/395

(58) Field uf ClassiTication Search .............. 424i244.1.424i184.1. 197.11: 536/23.1; 530/395

Sce application file for cmnplete smirch history

(56) References Cited

IJ S. PATENT DOCUMENTS

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(63) Contutuation-ut-part of application No. 11/395.593,filed on Mar. 31, 200(i. nosv abmidoned

(60) Provisimuil application No Cio/669.tios, lilcd sm Apr8. 2005.

Rexiosd et al 2002 C'ell Preservation imhnology vol I No 2 pp91-104 s

131ack, S, et al, "Laic acy, safety, and inununogensmty of heptava lentpacumococcal conjugate vaccine in childrcny Pediatr iaferr Dic A2000, pp 187-195, vol. Jry

Black S., et al., "Posf licensure evaluation of the eft'ectneness ofseven valent pneumosoccal coniugste vacancy Pediair hi/br( Du .1

2001, pp I lo(-1107, vol 20Block, 8 L, et al . "Pneumococcal scrotypes from acute otitis mediain rural fentuckyPPediaa iiifect Dii 3 2002, pp 85&3865, sot 21

Buckingham. 8 C, et al. "Incidence and etiologiec of complicatedpmapnmunonic effsssions in chilihen, 1996 to 20017 Pediair iiifecrDis A 2003, pp. 499-504. sot. 22.Butlm J, et al, "Scrotype distnbution of siicptoracrai paaaaamaemfestions mnong piesshool childien m the Lnited States, 1978-

1994p.i hifec(Di1 1995, pp 88&-889, sol 171. Byin ton, C., et aL,"An epnlemioloncal imestigation of a sustained high rate of pediat-nc pmapneumonx. empyema «sk factors and microbiological asso-

mationsy C'im iii/eri Dis, 200 c., pp 434440. vol 34("hoo, S, et al, "hnmunogenicity and Reacto enicity of aPneumosoccsl Coniu ate Vsccme Administered ('ombined with aHaemaphi ii&1 m//aeasae ispc b conjugate vaccine in t. ni ted KingdomInfants.*''ediair ia/eri flic / 2000, pp 854 SCi 2, 1 1st 19

Bltnngham, O, et al. '*Cssltsue-f(egattse (:hildhood Lmpyeme is

Usually Duc to Pcmci I I i a-Scnsi tive (irepiararnic paeataaatae Cap-sular Serotype 1 *L/ C'/m Jda 1, 2003, pp &2 I-(22, vol 41

(C'ontinucd)

Pnmary Ream/aer Robert A lemanvlssisiaa/L'raaiiiier Nitm A Archie

(74) .4/ioraci; Agent, or Firm Ropes R Ciray I.I.P

(57) ABSTRAC'I'n

inununo genic composition having 13 distinct

polysaccharid-

eidc-pnstcin conjugates and optionally, mt aluminum-basedadfuvant, is described. Each confugnte contmns a mipsularpolysaccharide prepared from a different serotype of 5/rep-iocoi cas pnemaaniae (1, 3. 4, 5, 6A, 6B, 7F, 9V, 14. )SC,19A. 19F and 23F) conjugated to a mirrier pmteui. The immu-nogenic composition, fonmilnted as a vaccine, increases cov-

erage against pncnmococcal disvasv. in inlants and youngchildren globally. and provides covemge fssr serotypes 6A and19A that is not dependent on the limitations of serogroupcross-pmtemion. Methods I'or making an inummogenic con-Jtiguts conlprislllg Strep/acarclis pl11'11 Iolllas.'efotype 19Apolysacchnridc me nlso prssvidcd in which thescrotype 19Apolysaccharide is co-lyopbtltzed with a camer proteus andconjugation is carried out in dimethyl sulfoxide (DMSO) viaa reshlcilvc ilniiililtlon incchanlslri

15 Claims, 3 1)rawing Sheets

US 7,955,605 $2Page 2

OTHER I&U BLICATIOFI S

Fskola J, ct at. "Fmcacy of a pncumococeal conjugate vam&ne

;i amsi acute otitis mediay HL'«gi 3 bird. 2001, pp 403 409, vol344Fagan. R I, "The epidemiology of incasivc pn

curn oc ocoaldisease in

children in I'u i(orth Oueenslandy 2 P«rdwrr Child Ihvirh. 2001,

pp 571-575,&ol 37G&eb&nk, (. S, et ai, "Irmnunogen&mty and eflicacy o5)irp&ocvrr«spar«i«v«i«r polysac&hm&de-pro(em conlu ste vacc&nes ago«us(homolo ousandhcterologousserotypes&nthechmchdlaot&t&smui&amodelF.I I«fr&i D&s, 1996, pp 119-127, vol 173llmisdorff W P. et al., *"A&tuch pneumococcal sero roups cause themost mvasive disease impbcations foi conlugate cacmne fomaila-t&on anti use, part IP Cii« Infer i Dis 2000, pp 100-121, vol 30

Hausdoiif WP, et al, "The contnbution of spcmfic pncumococcalserogroups to diffeient disease manifestattonc nnphcations fo& con-Jugate & acmne foimu let&on and use pmt IP I'Im Ivfixt Dis 2000, pp122-i40, vol 30Hausdorff, W,et al, "Cieog&aplucal d&tTerences &n invas&ve

pneumococcal disease rates and s«rot)pe fiequency &n young clul-drcny L««cet. 2001„pp 950-9 (2. vol 357Hausdoilf, WP, et al, "Mull&nauonal study of pneumococcalserot)pescaus&ngacuteot&t&smedkamchildrenPPed&«ti IiifeciDis J.2002, pp 1008-1016, vol 21

I 1 au ad oil', W P, et al . "In&as« e Pneumotoccat Disease &n (.'h& Idr en

()coy apluc mul 'Imnporal Vm&at&ons &n incidence and Serotype D&s-

tnbutiony F«hr .f Pad&air, 2002, pp 13(-139, vol i(it (Suppl 2)Hausdoilf, WP, et al. "Lp&dern&«log&cal DOTerences AmongPnmunococ&st Serotypesy L««& er hiiecr Drs . 2005, pp 83-93, vol.

5(2)ilofmann J, &4 al .

'"I he prevalence of dn&g-res&slant Sir«pi«roc& «s

pa& « «ion«it &n Atlanta." N F«gi I 1(eri 1995, pp 481 486, vol 333Jakobsen, H, et al "Pneumococcal serotype 19F conlugate vaccinemduces cross-protector &nunun&ty m seiotypc 19A m a mu&&nc

pneumococcal pneumonia modelybiiert I «mi&«,2003. pp 295Ci-

29(9,&ol 71

Joloba M L . et sl, "Pneumococcal conlu are vaccmc scrotypes ofSrrep(ococcus p&in«now«« icolatcs and the antimicrobial susccpti-brldy of such isohues in chrkh en &vith ot itis mech sy C Ini bifer & Drs2001, pp 1489-1494. vol 33Ke&teszD A, etal ."Invasivc Sireptocornih pmuwow«e infixtion in

Latin Amencan children, resulm of the Pan (&uencan llealth Orga-n&ant&on Sums&lienee StudyPCfi« lirf&ct D&s. 1998, pp 1355-1361.vol 26Flu rmm, I'. et al, '*A tnal of a 9-valent pneum«coeval &on) ugatevacmne in children with and those without HIV &nfectiony à F»gi,ibird, 2003. pp 12&4 i-I &48, vol 49blbefle, I(., et al., "Inununogemcity and Impact on VasophmyngealC'aniage ot'aK«navalent Pneumococcal Conjugate Vaccine.".Io«r-nvi offiifeciiv&is Dice«&ra, 1999, pp 1171-1176, &ol 180

Xahm. SI HH. ot al, "Identification ofcross rcac tive ant&bodies authlow opsonopha ocyuc act« ity for Sire«&oh vrriic p«e«mow«eF.IInfer i Dis, 199&7, pp 69tl-703, vol 17C&

( F Bn cn. K I., ct al, "Efljcacy and sati ty of cevcn valent co ntugatepneumococcal vaccine m 'unerican Indian children giouprandom&sed tnaly La&icet,200 k pp, 366-361, & ol. 362.Penn, RI., et al. "Ant&body rcsponsos &n adult volunteers topneumococcal polysacchande types 19F and i&)A admimstered sin-

lyand&ncombma«onyinficiimmwi.1982,pp. 1261-1262. vol,36Robbins, I B, ct al, "Cons&dmat&ons foi founulating the scvond-genenit&on pneumococcal capsular polysacchmule vacmne w&th

emphas&s on the moss-&sect&ve types w&tlun yoh&psy 3 i«fee& Drh.

1983, pp 1136-11(9, vol IAS

Rob&neon KA, et al FLp&dern&«logy of in&esne Sir«pi«roc&«spven«rv«i«« infect&ons m the I 'n&ted States, 1995-1998. Opportuni-ties foi prevention in the cong&gate vaccine crag 24JL( 2001, pp1729-1735.&ol 28(Rudolph. K M, et al. "Se& otype dh(t«but&on and antunicrobiel i esis-tance pattenrc of missive isolates of .(irrp(ororc«c pi&n&m«iv«e

Alaska, 1991-1998P,I I«fee& Dir 2000, pp 490-496, vol 182.Sniadack D H . et al "Potential intmventions for thc p&even&ion ofcluldhood pneumoma eograph&c and temporal diffe&ences m

sero&)pe and seiogroup &bat&&Eh&(&on of stenle s&te pnmimococcal

isolates from chd&hen &mph&at&one foi &ace&ne strateg&esy PrdruirI«far& L)i & K 1995, pp 503-510, col 14

Saeland. L', et al.. "Serum samples from infants &acc&nated w&th apneumococcal contugate vaccine, PncT, piotect m&ce aga&nst

in&esne &nfeclmn caus««i by S««7m«& oc&«s prie«wc«ivr acr«types6A aml 6BP,I Iiifrr i Dis .2001. pp 253.2&&0, vol 183Tan T, er al, '( hn&cal characteristics with comphcated pneumomacaused byS(rep&orvrrusp«ewvo«i««F Pedi« mr c, 2002, pp, 1-6, & ol

110Vakeva men, M, et al . "C rosc-react« ity of ant i ho&be( to type Cifl and6A polysacchm&des of Srrep(ococci&s pvemriom«e, evoked bypncumococcel c on) ugatc vacc mes, in m (ants.",I bifer( Dis,2001. pp78rJ-793. vol 1 84

Yu X,(3 B., et at, "hrunundy to moss-react« e serotypes &nduced bypneumococcal con) ugate &ace&nes &n &«fan&ay .If%& & Drs 1999, ppl(69-1576,&ol 180Whitney C.G, et al. '*Inmeas&ng prevalence of mutt&dnig-iesistantS(rep&or or nis pi i«««io«i«r m the I ln&ted St ates y W&Dipl I&H«d 2000,

pp 1917 1924,&ol 343,Whitney I'. et al, "Dec 0 ne in invasive pneumo cue cat disease afie&

the &nt&oduct&on of p&ote&n-polysacchande conlugate vacv&ne *'ei&&igi.&M«d 2003, pp 1737-1746, vol 34tl. bo 18

lanai(ca Tiomv, B et al, "Synthetic Polysaccharide type 3-RelatedD&-. Tn-, and letracacchande-C'RM,&- ('onlugates induce Protec-tion Agamst Sireprocorcuc piieivi&oirive Type 3 &n M&ccy I«dervI«n«iw. Jul 2001, vol 69(7). pp 4(i984701Hem&ksen, J.L.. et al, '*Vaccmat&on with Protein-C'onlu (ted andHat&ve lypc 3 Capsular Poly sacchm ate &n an Ethanol-Fed Rat ModelofPneumococcal Pneumoniayaffrohoi Cim Fhp Rrc. Dec 1997. vol

21(9), pp. 1630-1637.Mlxflc, H . ct al, "Immunogenicity and Impact on VasopharyngcalCau&age of a bona«alen( Pneumococcal Conlugate Vacc&ney .I

I«frrr Dm, Oct. Fy)&J. & ol, 180, pp. 1171-1176.Ada et al, "Cmbohyd&atc-piote&n Contugate Vacmnesy C7wic«i.(Cirro«i«I«RJ ««d I«frr«o«. 9(2) 79-85 (2003)sundekar et al, "Safety R luununogen&c&ty of a 13-valentPnmunococcal ("onjugate Vacc&ne in Healthy Infants G&ven WithRoutine Vaccmes &n Ind&a*k Abstract sub&outed Dec 2008 for the 7thIntl Symposnun on Anumxiobiai Agents and Rcs& stance, Bangkok,Thailand (Mar 18-20, 2009)Ands&son et al, "Immun&ration of 2-month-olrl infants w&th piolem-couplcd obgosaccbaridcs dm&vcd from thc capsule of H«ewopirdi«wiiue««e type b,".Ioihr««i of Pedi«(r«s&, 107(3) 346-3(l (198()Benmssa-I'rouw et al . "Synthet&c polysscchar&de type 3-related d&(

tn-. and tetrasacchandc-C'RM(197) conlugatcs induce protectionagainst .Sir«pter onuc p«eumo&n«e type 3 &n rn&cey Infer«v«v«dl&«mu«r&y. 69(7) 4698-4701 (2001)Buttciy ct al, "Immunogcnx&ty and safi:ty of a comb&nanonpneumococcal men&ngococval &a&c&ne &n &nfl nts. a iandonuzed con-tiofled tnaly JAMA, 293(14) I /(1-1758 (2005)Fauom et al, '*Ep&uhp&c ove&load at the site of mlecuon may result &n

suppiession of the umnune response to combined capsular polysac-chande conlu ate vacmnesy I'«crore, 17Q),126-133 (1999)Gatchahan et at. 17th Annual Meet&ng Of The F&u Soc Peed InfD&s I Lap&d), Poster N', Pla Poste& Sess&on I, L'stamsul, T&uqum,

Mar 27. 2001Hen«keen et al, "Vacmnat&on w&th protem-con)a ated and native

lype . capsulm polysacchmule in an ethmol-fed iai model ot'neumococcalpneum«may 4irohoium C7wir «I «vd Fi pen«ie«t«iRose«« it. 21(9) I Ci3(i-1637 (1997)K&enin cr ot al "Safety 9 Immunolog&c Sonmfcnonty of 13-valentPneumococ&al C.onjucate Vaccine I'ompared to 7-valentPneumococcal Conlugate Vaccine (liven as a 4-Dose Senes m

Healthy Infants and 'Ioddlcrs"& Pouxrpo&nt Prescntat&on gn:en at the2nd Annual Vaccine Cong&ass, Boston, MA (Dec 7 9, 2008)La os et al. '*Inununoio y of combin&ng CILM(I97) conlugates forSv'ephor«re&i& pi&eh«&ho«i«e, b'eisceri««iewi&gi(is and H«einopiviiihr«fivrrm«e &n C'h&lean &nfantsy Vacmne, 27(17) 2299)-305 (2009)Larsen et al.. '*Penodate-oui&oat&on Emit of al «nateF C«rboh) dr«relese«rrh. 10 186-187 (1969)L&nrlberg et al, "Stiuctural stud&es of the capsular poiysacchsndefrom Srreptorvrr«s pvc«moiu«r type IF C«rboi0 dr«re Ress«rrh,78(l) lll-ll7(1980)

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Mallet et al.. Immunogenimty and safety of a CRV(197 con)ugated9-& alen& pneumococcal and memngococcal C combinaimn vaccine

m healthy infant~, 25th Anmial Meet mg of the k'uropean Society forPaediauic Infectious Diseases (BSPID), Porto Portu al. May 2-4,

2007 (Poster)Hurkka ei al, "Inununogenicily an&i safety of the eleven valent

pneum»coeval polysacchmnle-protein D coniuyite &accine ininfants*'lic Pedio&i&i giger»o»i Disease .ioia»ui, 23(l I) 100y1014(2004)Pa)son et «I "Safety and Tolerabiliiy of 3 lots of ll-&alen(

Pneumococcal Coniuyiie Vaccine in Healthy Intbmts Given 90thRoutme Pediau ic Vacc matrons in ihe I ISAS Poster presentcst ai the

2nd Annual Vaccine Congress. Boston. MA (Dec 7-9, 2008)Peiiovsky et al, 'Vaccine adiuvanis current state and future tiend»P1»u»&»&o(oy u»d C'eii gi oiog) i 82(S) 488-496 (2004)Piymula et al, "Pneumococcal capsulai polysacchandes con)ugatedlo protem I& for prevention of acute otitis media caused by both.'(ovpiororr»s p»crim»in»r and non-typable Haemopl»i»siii/hie»r»e a iandomised double-blind egicacy study,** Lu»cii,367(9)512) 740-8(200Ci)Rennels M, Rmsm er R,. Pm(bore M, Corsaro. B., Ch'm *, I.Watson, W, for the C'ombination Conjuga te Vaccine Study Ciioup,Safety and Immunogenimiy of ('ombmed C'on)ugaie 9-valent S

pnemnomac-menmgococcal youp C (9»pn&-h(nCC) and H iiifii&-

e»ru b-9vPnC-MnC'C (HBO&xevpn&xMn&.C.) vaccine, 4(stInterscienceConferenceonAntimicrobialA entsandChemotherapy(ICAAC), Chicago. Illinois. Sep 22-25, 2001 (Abstract)Rexroad et ai, "I yophi ( i ration and the thermal stability ofvacanespC'eii Preiervu»oii 7»ch»oiogy. 1.91-104 (2002)

Sooth **Perodate oxidation, pKa and conf»unseen of hexuronic amdsin pol)suonides and mucopolysacchaudesy Bio& himica rif)&oph) iic» Br&a. 170(2) 471-473 (1968)Si urdardottir et al, "Safety and inununo enimty of CRM(97-con-g& ated pneumococcal-meningococcal C combination vacmne(9vPnCxMn&.C) whether yven in tuo oi three primary hoses.*'ac-mnc 26(33) 4178-4186(2008)Soyirdmdoitir et al, "1wo and 'I luce Doses of the 9vPncMnCC mInfancy, Pnme for C'omparable Booster Responses at 12 Months ofAgcy The 5th International Symposnun on Pneumococvi 8&

Pnmunococcal Diseases (ISPPD-5). Alice Spungs. Aust&aim. Apr2-6, 2006 (Abstract)Sigiu dardoitir et al, "Safely and inununogenw sty ol'. RM(9)7 con-

jugated 9-&alen( pneumococcal and menm ococcal ('ombumuonvaccine (9vPnCMn&Y) a&huts&stra(ed in bvo oi three pnmary dosesin infancyy 23rd Anmial Meetmg of the i,uropean Someiy foi

Peed iatrrc In fee tro us Diseases (BSPID), Valencia, Spain, May 18-2(i,

2005 (Abstract)Csonis et al, '*A elm&cat ural examining. the eifect of increased iota(

CRM(197) camm protem dose on tho antibody response to

Ha em opia(»&»ifhie rime type b C R M(197(conjugate vam inc P Vac-

cine 26(35) 4602-4Ci07(2008)Wcssclsetai,"StnictmaipiopmticsotgioupB streptococcal typeHIpolysacchmide conju ate vacmnes that inliuence irnmunogenicityan&I «tlicacyy pipe& »o» a»d (mmi &i»ri & 66t 5) 21862192 (1998).Ych ct al . "Hcptavalcnt pnoomococcal sacmnc conpigatcd to outamembrane prorein of Heissena memngitidis serogroup b andnasophmyngeal em»a e of Siryimrorcus p»eumoi»ue m infantsyP»cm»e. 21(19-21) 2627-2631 (2003)

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MUI,TIVAI,ENT PVEUYIOC.'OC.'C.'AI.

POLYSAC'I'HARIDE-PROTEL'I CONJUGATECOMPOSITION

that of Prevnar, and provide coverage for 6A and 19A that isnot dependm&t on the limitations of serogroup cross-protec-n(iu.

C:ROSS-RFFFRFNC'F, TO RFI,ATFDAPPI,ICATIONS

This applimition is a continuation-in-p ut of IJ.S applica-tion Ser. No. 11('39(.593, tiled Mar. 31. 2006, wluch claimsthe benefit of lJ.S Provisional Application No 60/669,605,liled Apr 8, 2005. mich ol'«hich is hereby incorporated in itsentirety by reference herein.

FIL'Lli OV fHF INVENTION

The present invention relntcs generally lo thc iield ofmedi-

cinee.

Bnd specifically to microbiology, imnnuio logy, vaccinesand the prcivntion of infection by a bacterial pathogen byla«nun&rat&on.

BACICCJROUND OF THE INVENTION

S/rey/ucvccaiyiieoivvuiae &s o leadin cause ofmening&-tis. pneumonia, and severe ulvosive disease in infants andyonng children throughout tim world. Thc multivnlent pncu-mococcal potysacchande vaccules have been hcensed formony years mid lmve proved valuable &n preventing pneumo-mlccal disease &n elderly adults mid lngh-risk patients. How-ever. infants and young children respond poorly to most pneu-mococcal polysaccharides Tlm 7-valent pncumococcalcon) ugate vaccine (7vPnC, Prevna14& ) was the first of & ts kulddemonstrated to be lughly &mmunogen&c ond effective againstinvasive disctwsm and otitis media in infmits imd young chil-dren. This vaccuie &s no&9 approved &n many countnes amundthe world IYcvnor contains thc capsuLar polysoccharidesI'rom serotypes 4, 6B. 9V. 14, 18C'. 19F and 23F, each conju-gated to a carrier protein designated CRM& 97 Prevnar coversnpproximamls'0-90%, 60-80%9. Bnd 40-80% of invasivepneumococcal disease (IPD) in the IJS, Furope, aud olimrregions of the world. respectively [1.2]. Surveillance datagathered in the years following Prevnar's introduction hasclearly demonstrated a reduction of invasive pneumococcoldisease in US infants as expected (FICi. I) [3. 4J.

Surveillance of IPD conducted in US infants prior to the&ntroducnon of I'revnor denxmstrated tlmt o sigiuficant por-tion of disease due to serogroups 6 and 19 was due to the 6A(approximately one-tlurd) and 19A (approximately one-fourth) serotypes [5, 6]. Pneumococcal invasive disease sur-veillance conducted m tbe US aller licensure o I'Prevnar sug-ests that a large burden of disease &s still attnbutable to

serotypes C&A ond 19A (FtO. I) [3]. Moreover, these twospl'otvpcs account lor'lol'i'. cos(s ol ulvoslvc dlslilsc thonserotypes I. 3. 5. and 71'ombined (8.2 vs. 3.3 cases/100,000children 2 years and under) In addition. scrotypes 6A ond19A are ossomated lv&lh lugh rates of m&tlb&ot&c resist ulcc(11O 2) [7. 8, 9J. While it is possible that serogroup cross-protcction will result in a decline ol'vrotypc 6A mxl 19A

disease as more children are immumxed, there is evidence tosuggest tliot there will be a limit to the decline, and a sivniti-cailt burden of di seas( duc to thcsc serotypcs wit f rcmaul (scehalos& ).

Ciiventherelativeburdenondimportanceofinvnsivepneu-mococcal disease due to semtypes I, 3, 5, 6A, 7F, and 19A,adding these serotypes to the I'revnar formulation wouldincrcosi'. covcrogc fiif hlvoslve discase to &90% in tim IJS andLumpe, and os tugh as 70%9-80% &n Asia ond Latin Amenca.This vaccine would sigrnificantly expand coverage beyond

1 SUMt&(LORY OF TI ILI INVENTION

Accord&nay, the present invention provides generally amultivalent inununogenic composit«in compnsin 13 d&s-

unct polym&ccharide-protein conj«St&tea, wherein each ol'the«I coniugates contains a capsular polysacclrande from a ddTer-

ent serotype of Strpyivcuccai yueumaa/ae COnjugated to o

comer protein, together with a piiysiologimdly acccptnblevelucle. Optionally. an adluvant. such as an alununum-basedadjuvant. is includ(d in thc formulation More specifically,the present mvenl&on prov«les n 13-valent pneumococoalcojjugote (13vpnt'j composition comprising the sevmi sero-types in thc 7vPnC'accine (4, 6B, 9V. 14, 18CL 19F and 23F)plus six additional serotypes (l. 3, 5, 6A, 7F and 19A).

fhe present invention alan provides a multivalent immu-o nogmuc compositim&. &(herein the capsular polysaccluiridcs

are from serotypes I, 3, 4, 5, 6A, 6B. 7F, 9V, 14. 18C, 19A,19V ond 231'f Streptococcus yu pumvoiae ond the carrierplotcul Is CRM&97.

The present mvention further prov ides a nul it &valent immu-nogenic composition, wimrein the capsular polysacchmidcsare from serotypes l. 3, 4, 5, 6A. 6B, 7F; 9v, 14, 18C. 19 k,19F and 23V of5/reytvcvccaiyaeo moo/up, the camerproteinis CRM»7, mid the «ljuvant is an atua&foun&-bas(st adjuvant,such as aluminum phosphate. aluminum sulfate and ahum-

"o mun hydroxide In a particular embodiment of the invenliim,the ndjuvant is al&uuinum phosphate.

The present ulvent«ln also provides o nu&itivolent &nuuu-

nogcmc composition. comprising polysaccharide-proteincontugates together with a physiolog&cally acceptable

"» vcluclc. w lmrcin each of the cnnjugams comprises a capsularpolysaccharide I'rom a different serotipe of'/rcy/vcvrri(syac« mva/ ac cont ugated to a earner proieul. and the capsularpolysaccharidcs are prcpar(xf from serotype 3 ond at least oneadditional scrolvpc

4« In one embodiment of this nui itive lent imnumogenic com-position, the additional serotype is sclccted from the groupconsistin ofsemtypes 1,4. 5, 6A. 6B. 716 9V, 14. 18C, 19A,19V, and 23V. In another embodiment, the carrier protein is

CRM&97 In yet another en&bodi ment, the composition com-poses an adtuvont, such os an oluminmn-based adluvantselected from aluminum phosphate, aluminum sulfate ondalununum hydroxide. In a parncular embodiment. the adju-vBnt Is oduulluuul phosphate.

Thc pl(VPB& ulvi'.Buoil &&Iso plov«li'.s a uulltlvolcnt inuuu.o no eiuc composition, compnsulg polysacchande-protein

conjugates together with a physiologically acceptablevehicle. Ivherein each of the mmjugatcs mlmpnses o cnpsularpolysaccharide from a different semt)pe of 5'treyivcu(cusyapumvaiae cimjugated «I a carrier protein. and tlm capsular

s polysacchnrides ore prepar(xt from serolypes 4, 6B. 9V. 14,18th 19F, 23V ond at least nne additional semtype.

In onc embodiment of this n«iltivalcnt imn«ulogcnic com-position. the additi(ltud semtype &s selected froni the groupconsisting of serotypes 1, 3, 5, 6.4. 7F, and 19A. In another

(o cmb(idimcnL th( carrier pmtein is (..RM,77 In yct anotherembodiment, the composition comprises an adjuvant, such asan aluminum-bnsed adjuvant select&xi fnim alumimun phos-phate, aluminum sulfate and alununum hydroxide. In a par-ticular embodiment, the adjuvant is aluminum phosphate.

I'hc present invention also provides a method of inducingon unmune response to a Strep tv cv ca us yv e un(ua iu P capsularpolysaccharide conjugate, comprising a(bninistering to a

[js 7,95si,605 B2

lnulum an immunologically effective amount of any of theunmunogemc compositions lust descnbed.

Tfle present invention further provides that any of theimmunogcnic compositions administered is a single 0 5 ml,dose formulated to contain: 2 pg of each sacclmride, except s

for (iB at 4 pg; approxinmtely 29 pg CRM197 carrier protein;0.125 mg of elemental alumimun (0.5 mg alununum phos-phate) adjuvant; and sodium chloride and sodium succinatebliffer as cxclplcn(s.

Methods for making an immtulogenic conlugate compris- ui

ulg Sirciitococcas yneiononiuc serotype 19A (Pn 19A)polymiccharide covalcntlv linked to a carrier protein are alsoprovided. In one embodiment, the method comprises: (1)rencting purified serotypc 19A polysaccluiride with ml oxi-dizulg agent resulting ul m activated svrotype 19A polysnc-clmride: (ii) compounding the activated serotype 19Apolyssccharido isith a carrier protein, (iii) co-lyoplul izing thccompounded activated serolype 19A polysacchandc and car-rier protein: (iv') re-suspending the compounded activatedserotypc 19A polysaccluifidc and carrier protein in dimethyl so

sulfoxide (DMSO); (v) reacting the compounded, activatedserotype 19A polysaccharide and carrier protein with a reduc-ulg agent resulting ul a serotype 19A polysacchande:earnerprotein conjugate: and (vi) capping unreacted aldehydes inthe serotype 19A polysaccharide. carrier protcfin conjugateresultin in an inumulogeiuc contugate compnsing Srreyrv-i orcus pneumonia& serotype 19A polysacchande covalentlylinked to a carrier pmteul,

In a fiirther embodunent. the method for making an immu-nogcnic conjugate compiisiog,5'rreprococcvs pmmnomneserotype 19A polysacchnride covalently linked to a earnerprotein comprises: (1) reacting pun fied serotype I 9A polysac-charidc with sodium pcriodate resulting in an activatcdl scro-

type 19A polysacchande: (li) adlusting the pi I of the acu-vated serotype 19A polysacclbaride to 6.5+0 2; (iii)compounding tlm activated serotype li)A polysaccharidewith sucrose: (iv) compoundulg the activated serotype 19Apolysaccharide with a C'RMis carrier protein at n ratio of0 8.1, (v) co-lyophihzing the compoimdcd acuvatcd scmtypc19A polysaccharide and carrier protein; (vi) re-suspending cn

the compounded activntcd serotype 19A polysaccharide andcarrier protein ul DMSO; (vii) reacting the compounded,activated serotype 19A polysaccharide and carrier proteinwith sodiunl c)"itloboriiltl'dride resulting in a serotype 19Apolysaccharide:carrier pmtein conjugate: and (vili) capping mluueacted aldehydes in the serotype 19A polysaccharide:ear-ner protein. conjugate with sodium borohydride resultulg inan inununogenic conjugate comprising Srreyiococcai ynca-iru&mne serotype 19A polymiccharide covalently Iinkcxf to a

carrier protein. (1

Methods for making an immunogenlc conjugate compris-ulg a Srreytocoicos yocumiirnce polysacclraride covnlentlylinked to a corner protein in wluch the polysuccharide com-prises a phosplxidiestcr linkage between repeat units arc alsoprm ided In onc embodiment, the method comprises: (1) 1

reacting the polysaccharide with an oxidizing agent resultingin ml activatcxf polysaccbnride: (ii) compounding the acti-vated polysaccharide with a camel protein: (iu) co-lyophiliz-ing the compounded act ixmated polysaccharide and carrier pro-tcm: (iv) rc-suspmuling the compounded activated mi

polysaccharide and earner protem in DMSO; (v) reacting thecompounded, activated polysaccharide and carrier proteinwith a reducing agent resulting in a polysacchandeamrnerprotein conjugate: and (vi) capping unrimcted aldehydes inthe polysaccharide:carrier protein conjugate resuhing in an (ilnlnunogenlc coiitti ate conlprisln Streptococcus yncaoio-rri ae polysaccharide covalently linked to a carrier protein. In

further enlbodiments of such nwthods, the polysaccharidecomprising a phosphodiester linkage between repeat units is

Sirviitococcas yacoiuonicc polysaccharide serotype 19A,

19F, 6A, or 6B In a lhrtlmr mubodiment. the carrier protein is

C:RM,o,

BRIFF DFSC'RIPTION OF THF, DRAWIN(lS

FIO. I depicts the changes in IPD rates by semtype in USchildren&2 years of agc thorn baseline (1998)1999) to 2001

FICi. 2 depicts the distribution of pneumococcal isolateswith resistance to penicillin (PCN) in children&5 yenrs ofage(I 998)

FICi. 3 depicts the reverse clunulative distribution curves(R( D(:) of OPA post-third dose results from the D118-P16Prevnar trial.

DI',TAILIID DI',SCRIPTION OF TIIF, INVFNTION

Inclusion of'Prcvnar Smotypcs 4, 6B, 9V, I I, 18 . 19F, 23FData from IPD surveillance between 1995-1 998 estimated

that the seven serotypes in Prevnar were responsible tiiraniuud 82% of IPD ul children&2 years of age [5]. In North-ern California. the site of the efficacy tnal, the Prevnar sero-types accounted for 90'!o of all cases of IPD in infants and

young cluldren [10]. Sulce introducnon of the Prevnar vac-

cine in 2000. there has been a significant decrease in theoverall IPD rates due to a dcmrease in disease due to thevaccine serotypes [3. 4]. Therefore. there is no lustification atthis time to remove nny of the Yrcvnor acro types from the nextgeneration o I pneunmcoccal con) ugate i accuses but ralher to

add serotypes to obtain wider coverage.inclusion ol'i retypes I, 3, 5 and 7F

In the US, the rate of IPD caused by semtype I in childrenunder the age of 5 years is &2 /o. about tlm same as for each oftypca 3 mid 7F [l. 6[ Scrotypcs I and 5 accoiult liir higherrates of IPD in US populauons at high risk for invasive pneu-nlomiccal disease. Specifically, serotype I cmises 3.5% ofIPD in Alnskan lmtive cluldren&2 years ol'ge, and 18'/o ulchildren 2-4 ymsts of age [I I J. Both semtype I and serotype5 significantly cmlsc disease in otlmr parts of the world and in

uldlgenous populations in developed countnes [12, 13, 14].Serotype I may also be associated vvith more severe dis-

ease as compared with other pimumococcal scrotypes [15].Tlus observation ls based on the difference in rates of caseidentification bete can the US and I',urope, and the associateddifference in medical practice. Overall, the incidence of IPDis lower in L'urope than in the US. I low ever, the percent ofIPD causeil by serotype I ui Furope is dlspropouioiratelyhigher than in the US (6-7 /o. vs. 1-2%, respectively). Inburope, blood culnires are obtained predominantly from hos-pitnlizcd children. In tbe US. it is routine medimd pracuce to

obtain blood cultures ln an outpatient settulg from childrenpresenting with fi ver~39'. and elevamd white blood cellcounts. Ciiven the difference ul mmhcal practice. 0 is postu-lated tlmt the lower percent of disease caused by sero type I inthe US may bc diluted by higher rates of other svrotvpescausulg nulder disease. wlule the higher perceni in I',umpereflects more serious disnsse. In addition, seroepidemiologystudies of children with complicatnl pneumonia di monstratethat semtype I is disproportionately represented [1(i, 17, 18J.This sugogests that inclusion ol'erotype I mny reduce thcamount of severe pneumococcal disease. as v, ell as. contrib-ute to a total reduction in invasive pneumococcal disease.

I'hc nddition of scrotypes 3 and 71'ill increase coverageagainst IPD ln most areas of the world by approxuuately3 /o-7 /o, and in Asia by around 9%. Tlms. an 11-valent vac-

(JS 7,955,()05 B2

5cine would cover 50% in Asia and amund 80% of IPD in allother reyons [I, 2]. These serotypes are also important w&th

respect to otitis media coverage [19J. In a nuiltinational studyofpneunu&coccal scrotypcs causing otitis media, Hausdorffctal found semtype 3 to be the 8th u&ost conunon middle ear s

fluid isolate ovemll [20J. Serotvpe 3 accountcxf for up to 87%of pneumococcal serotypes assocuited w&th otit&s med&a.

I'hus, the importance of types 3 and 7F in ot&tis med&a, as w ellas in IPD. warrants &heir inclusion n a pneumococcal conju-gate vaccine. &o

1lowever, atten&pts to produce a multivalent pneumococcalcon)ugatc vaccine that exhibits significant inununogcnic&tyw&th respect to serotype 3 polysacchandes have been unsuc-cessful. For example, in n study of the imrnunogenicity andsafety ol'n 11-valent p&mun&ococcal protein D conjugutevaccine (11-Pn-I'D). no priming effect tv as observed for sefo-

type 3 in infants who had received three doses of the vaccinefollowed by a booster dose of either the same vaccuie or apneiunococcal polysaccharide vaccine (Nurkka et al. (2004)Ped. 1&ig D&s .A 23 1008-1014). In another study. opiwiopb- zo

agocytic assay (OPA) results from infants who had receiveddoses of 11-Pn-PD failed to show antibody responses forserotype 3 at levels comparable to other tested serotypes(C&atcha lian et al., 17'" Annual Meeting of the Eur. Soc. Paed.Inf. Dis. (ESPID), Poster No. 4, Pl A Poster Session I. Istan- "

bul Turkey. Mar. 27. 2001). In yet m&other study. wluchassessed the efTicacy of an 11-Pn-PD in the prevent&on ofacute on&is n&el&a, the vacwne d&d not provide protectionagauist episodes caused by serotype 3 (Prynu&la et al. (2006)I.ance&, 367 740-748) Accordingly, a pneun&ococcal cwiju- &o

gate vaccine c&xnpnsuig capsular polysaccharides from ser&w

type 3 nnd capable of eliciting m& immunogen&c response toserotype 3 polysaccharidcs provides n significant improve-ment over the ex&st&ng state of the artInclusion of Serotypes 6A and 19A

a Fpidwniology ol'Serotypes 6A and 19ASurve& fiance data u& the hterature suggest that sero types 6A

nnd 19A account ti&r more invasive pneumococcal disease inUS chifdrw&&2 years of'ge than serotypes I, '3, 5, aud 7Fcombined (I'IC&. I) [I, 5]. In addition, these serotypes are 4o

wmm&only associated with nntibiotic resistance (FICi 2) and

play an importm&t role &n otitis media [Cx 19, 20J. The abdityofthe currm&t Prevnar vaccine to protect against disease due toGA and 19A is not clear The rationale for inclusion of6A and19A components in a 13vPnC vaccit&e is discussed below.b. Responses to GA and 19A Induced by GB and 19F Polyrmc-charides

'll&e hcensed unconlugated pneumococcal polysacchaudevuccinis (li&r use in persons at lwist tv,o years ol'ge) haveconte&ned 6A or 6B capsular polysacchaude but not both u

[21J. Inununogenicity data generated at the time of fomuila-linn of the 23-vnlent pneiuuococcal polysacchnride vaccumdemonstrated that a 6B monovalent vaccu&e induced ant&bodyto both tfm 6A and 6B capsules The data I'rom several trialsassessui IgCI and opsonophagocyt&c assay (OPA) responses s

in a variety ofpopulations with free polysaccharide and withpnciunococcal conjugate vaccines suggested that IgCi

responses to 6A are u&duced by 6B uit& ens, but the responsesare gcnemlly lower, and the OPA activity with 6A organismsis diiTercnt thm& with 6B organisms [22, 23, 24, 25]. In add&- u&

tion, sublects respond&ng v;ith high 6B antibody may havehtth'. ol no &&ct&vfty'&gun&st 6A.

In contm&st to the chenucal composition of the 6A and 6Bcapsular polysaccharides where there exists a high degree ofsimilarity, tlm 19A and 19F capsules we quite ditT& rent due to &

the presence of two add&t&onal s&de chains &n the 19A polysac-charide. Not surprisingly, immiu&e responses measured in

lnmu&n volunteers immunized with 19)F polysaccharide vnc-

cine showed tlmt responses to 19F &vere induced in 80/v ofsubjects. but only 20'!Av of sub lee ts 1md a response to 19A [26].l,ow levels of'cross-reactive lgCi and OPA responses to scro-

type 19A sifter immuni/ation with 19F polysacclmridc lmvc

also been documented in trials with conjugate vaccines aswell [24, 26].

Internal data on cmss-react&ve OPA responses to 6A and19A hnve been gcncrated from thc 7vPnC bridging trinl

(D118-P16) conducted in US infants (11O. 3). 11&ese studiesare cons&stent iv&th the findings of others, and demonstrateinductiwi of cross-rwict&ve funct«mal antibody io 6Apolysaccharide after imnnuflzation with GB polysaccharide,although at a lower level. m&d very little functional antibody to

19A after inuuun&ration w&th 191'.

Impact of 6B and 19F Immuni&m&ion on 6A and 19A in

Animal ModelsAn&mal models have been used to evaluate the potential for

cross-pmtecuon w&th pulysaccharide immun&ration. In;mcutis &acedia& model developed by C&'iebink et al.. chinclullaswere immunized with a tetravalent polysaccharide outermembrane prote&n(OMP) conlu ate vaccine (conta&nin 6B,14. 19F. 23F sacchaudes) or placebo [27]. In tlus trial thereappearcxf to be sonw. cross-protection fiir 6A: however this didnot reach statistical significance and thc level of protectionwas lower than w&th 6B against ot&tis medm. In tlus smnemodel there was 100% protecuon aga&nst 191'nus media,bui only 17% protection agau&st 19A otit&s &uwf&a.

Saelm&d et al. used sera from infants immunizid with an

8-valent pneumococcal tetanus conlugate vaccine (contain-uig 6B tu&d 191') to passively immuiuze nuce pnor to m&

in&ranasal chaff&.ngc with 6A organisms. in a lung infiutionnu&del [28J. Of the 59 serum samples. 53% protected miceagainst bacteremia with 6B and 37% protected against 6A.

Mice pass&vely unman&z&d v, ith sera fram infants inumuuzedwith four doses ofan 11-valent pncumococcal conjugate vnc-

cine (containing 19F conjugated to tetam&s toxoid) were givenan &ntranasal challenge v;ith 19A organ&sms u& the samemodel [29]. Ol'100 mice passively uunmni/cd nnd then chal-lw&gcd, 60 mice had no 19A organisms deiccud in lungtissue. v,hereas organisms were idw&tified in all mice givensulu&e placebo. Ilowever. passive immun&zation d&d not pro-tect a ainst challenge &vith 19F orgamsms in th&s model„thorefi&ro, thc rclevm&ce of tlm n&odel fi&r scrogroup 19 isquestionable. In general these models provide evidence ofsome b&o logical impact of 6 B inunun&zation on 6A organisms&&i&hong& the effect on the heterologous serotypc was not as

great as tlmt observed with the homologous serotype. Theimpact of I'F immunization on 19&A organisms is not wellunderstood from these models.Impact of 6B and 19F Polysacchaude Conlugate Immun&za-

tion w& 6A nnd 19A Discase in EAicacy/Ffli:ctivcness Trialsfhe number of cases of d&sease due to the 613. 6A, 19T and

19A serotypes &n 7vPnC and 9vPnC (7vPnC plus serotypes I

and 5) eflicacy trials is noted in Table I [30. 10, 31[. Thenumbers of invasive disease cases are too snuff to allo&v anyconclusions to be dm&in for serotypes 6A and 19 k. However,

the 1&nn&sh ot&t&s media trial genem&ted a large munber ofpt&eiunococcal isolaies [32]. In thc per pron&col at&alysis7vPnC''as 84% (95% C f62%, 93 /6) eflicacious against oti &is

med&a due to serotype 6B and 57% (95% C124%. 76"!8) effi-

cacious against otitis media duc to serotype 6A (Table I ) In

contrast. semtype-spec&fic efflcacy with the 7vPnC &vns notdenumstmted for otitis media due to either 19F or 19A.

US 7,955,605 B2

TABLL'

('ases nf'Pneurtmcoccal D seve Due to Serorypes 6R, 6), 191',

,trit& 19 A ut Ffhc tcy Tr talc sr itlt the 7& PnC;tlirt &rr Pil('accmes

19F 193

PaC Contr PnC Cnarr PnC Conh PnC C ntt

Kaiser Eflicacy Tnal rvPnCIITTiXavalol&Tie&ca Tnal 7vPnC(ITT I

South Anrrc in Efncac) Tr tl-) PnCIR) (-)01'I)South Afncan 13ncacy I'»sf-9 I'nc HIV(+) (I'l l)I'trtttlslt Otttts Mnha lttal-7iPnC (PPI

I 7 0 I 2'3 (i I

0 i I i! I I I lt

I " I 0 (t I 3 I

I 7 3 1&) 2 3 2

9" 56 19' 43 8 17 26

Post-marketing IPD surveillm&ce data is also availablefrom 0 case-control tnal conducted by the Centers for DiseaseControl to evaluate the effecnveness of Prevnar (33j. Cases olpncumococcal invasivc discase ocmirring in children 3 to 23num(ha ofagc wmre identilicd in tlm surveillance laboratoriesand matched w&th tluee control cases by age and zip code.Aller obtainu&g consent, med&cal and unmun&ninon lustory

(sub)acts i&ere cons&dered unmun&zed if they laid race&ved alleast onc dose of I'revnar) was obmined from parmns andmedical providers for cases and controls. The prelim&naryresults were present»AI at the 2003 ICAAC meeung and a

summary ol'Ihe lindings for 6B, 19F 19A and 6A di)ernie is

presented in Table 2. These data indicate that Prevnar is ableto prevent disease due to 6A, although at u level tluit may besomewhat lower than serotype 6B d&sense. These data alsoindicate that thc cross-protection I'or invasivc discase duc to19A &s l united.

TABI F. 2

Prclrrnrnacy results of a Cas Centrol 'ical

Pc&fern&cd

6 the (.D(.'esentc&I al ICAAC 20&!3 I

)erob&re

Vaccute lypc. Au

Vaicrrtc Eelatcif All

go n-Vaio as 'li pe.: I I I

6R

191'A

19A

VP*

Inforrnatne Sets, ri i9i''094

(8 97)71

(3) 861

&-It&6. 48)94

f7, 99&i

7"

(16 9r)

(63 97)

(-87.80l

A published analysis (3) of the use of Prevnar also ind&-

cauxl that scrotypes (&B and 19F confi:rred a moderate reduc-Don in IPD caused by serotypes 6A and 19A mnong clnldrenunder two years of age (Table I in (3J). Disease rates I&along

unimmunizixf adults caused by semtypcs 6A, 9.4. 91., 9bk18A. 18B. 181'; 19rf&, 19B, 19C. 23A and 23B ("011 vacclne-related serotypes'*) were somewhat reduced (Table 2 in (3)).

These data establish that herd immunity from thc use ofPrevnar &n cluldren under Dvo years of age was modest forserotypes 6A and 1 9A, and provide a basta for the inclusion ofserotypcs 6A and 19A in thc 13vPn('accine of this inven-n&in,

Conclusion for Addition of 6A and 19AThe post-marketing surveill u&ce dani and tlm case-control

study results noted in FI(2 I and Table 2 v,ith the 7vPnC"0 vaccine suggest lhak consistent with tlm other information on

&nuuune responses aixl performance ln the ammals modelsdescribed above. there may be some cross-protect&on aga&nst

6A disease, but to a lesser extm&t thim to 6B &hscasc Further-more. 11 appears the protect&on ag oust 19A &s lun&ted There-

"» fore, a 13vPn('accine containing sscrotypes 6A and 19A

pnlvide) coverage that is not dcpendcnt on tlm limitationsol'erogmupcmss-protecuon by serotypes 6B and 191)

Accordingly, thc present invention provides a multivalentimn&uuo cnic compositnm con&pnsmg 13 distinct polysnc-

40 chande-protein conjugates, wherein each of the conjugatescontains a different capsular polysaccharide conjugated to 0

earner protein. and whereu& the capsular polysaccharides areprepared from serotypes l. 3. 4. 5. 6A. 6B, 7F, 9V, 14. 18(',

19.4, 19F and 23F of 5&rcp&vcvr mit pnevnrrmive. togetherwith a phys&ologically acceptable velucle. One such carrierprotein is the diphtheria toxoid designatixf ('RM»r. Theu&uuunogenic composlt&on may further compnse an adju-vm&t, such as an ahuninum-based adjuvant, such as aluminumphosphate. Slununum sulfate and aluminum hydroxide.

u Capsular polysacchandes me prepared by standard tech-niques known to those sin lied in the art. In the present inven-1&on. mipsular polysacchandes are prc7ared from serotypes I,3, 4, 5, 6A. 6B, 71', 9V, 14. 18C, 19A, 19II and 231'f5&rcplvrvccvcfmevrnvnive. These pncumococcal conjugates

3 are preparixf by separate processes and formulated into a

single dosage formulation. For example, in one embodiment,each pncumococcal polysaccharide )orotypc is grown in asoy-based med&um. The i&uiividual polysaccharides are ihenpurified through centrifugation, precipitation, ultm-filtrabon,

6tl and column chromau&graphy Thi purified polysaccharidh)are chenucally activated to make the saccharides capable ofrcaclfrlg wltll tlm catfyli:r pl'otchl

Once actn ated, each capsular polysacchande is separatelyconjugated to a carrier protein to form a glycoconjugate. Inonc em bod imm&t, each capsular polysaccharide is conjugatedto the same carrier protein. In thfs embo&hment. the conjuga-tion is effected by reductive amination.

[js 7,955,605 B2

10'lqm chemical activation of the polysaccharides and subse-

quent coniu ation to the carrier protein are aclueved by con-ventional means. See, for example, U.S. Pat. Nos. 4.673,574;md 4,902.50Ci [34, 35]

Carrier proteins are preferably proteuis that are non-toxic s

and non-reactogenic and obtainable in sufficient amount andpurity Camer proteuis should be unenable to standard con-)ugation procedures. In a particular embodunent of thepresent llivi:litton. C'RMioi is used as tfic carrier protein

CRM»,(Wyeth.Sanford,N C.)is anon toxicvaruutt(i e., ui

toxoidj of r/i phiheri u toxin isolated from cultures of C'ori ue-

buurerium diphtheria strain C7 ([1197) grimn in cmsamino

acids and yeast extract-based medium. CRM»i is puniiedthrough ultra-filtration, anmionium sulfate precipitation, and«m-exch m e chromatographv. Altcmatively, CRM„-, is pre-pared recombinantly in accordance with U.S. Pat. No. 5,(i14,382, which is hereby incorporated by refi.rencc. Other diplo-

/her!aa

toxoids arc also siutable for use as camer proteins.Other suitable carrier proteins include inactivated bacterial

toxins such as tctmuis toxoid, pcnussis toxoid, cholera toxoid iu

(e.g., as described in International Patent ApplicationW02004/083251 [38]). g co// Ll. Pb cali S'I: mid exotoxinAfrom Pseudatuuuas aerug/iiasa. Bactenal outer membraneproteins such as outer membrane complex c (OMPC), p orms,transferrin binding proteins, pneumolysin, pneumococcal "

surface protein A (PspA). pneumococcal adhesin pnitein(PsaA), ('5a peptide se from ()roup A or (iroup B strep/ororeris, or llaeuiulzhilus iiilluerzae pmtein D. can also be usixl.Other pmteins, such as ovalbumui. keyhole limpet hemocya-nin (KI H). bovine scnun ulbuniin (BSA) or puriliod protein su

derivative of tuberculin (PPD) can also be used as earnerproteins.

Al'tcr conjugation of tlm capsulur polysaccharide to (lie

carrier protein, the polysacchande-protein con)ugates are

puriiicd (miriched with respect to tlm amount ofpolysacclm- "»

ridc-protein conjugate) by u variety ol'echniques Thesetecluuques include concentration!diaiiltration operations,precipitation'elution, colunui chromatography, and depth fil-

tration See exmnples bdowAfler tlie individual glycoconjugates are purifisxl, they are su

contpoundcd to fomiulate the iminunogenic compositiim ofthe present invention. wluch can be used as a vaccine. Iior-

mulation of the inununogenic composition of the presentinvention can bc accomplished using art-recognized nicth-ods. Iior instance, the 13 individual pneumococcal cont ugates 4.

can be fomtulatixl with a physiologically acceptable velficleto prepare the composition, Examples of such vehiclesinclude, but are not limited to. water, buffered saline, polyols(e..g . glycerol, pmpylene glymil, liquid polyethylene glycol)and dextrose solutions. (i

In certain embodiments, the immunogenic compositionwifl compose mm or more adjuvants As defined herein. an"adtuvant" is a sub stmice tluit serves to enhance the inununo-gcnlclty (if ali illllliuliogi:i'Ilc Colllp(isltiillt of this ilivclitiiili

Thus.adjuvantsnreoilengiventoboost the unimuteresponse 5

and are well known to the skilled anismi Suitable adjnvantsto enhance efl'cctii encss of thc composition include, but arcnotluuited to.

(I) aluminum salts (alumk such as aluminum hydroxide,aluminum phosphate. aluminum sulfaie, ctc, ui

(2) oil-in-water enuilsion fomiulations (with or withoutother specific immunostimulnting ngents such as nnirmnylpeptides (deiined below) or bactenal cefl u.sll components),such as, for example,

(a) MF59 (PCT I'ubl No WO 90!14837), containing 5% (Squalene, 0 5/o Tween 80, and 0 5% Spun 85 (optionallycontaining various amounts of M'I'P-I'B (see below,

although not required)) fiirmulated into submicmn par-ticles using a microihndizer such as Model l)OYmicroflmdizer (Microfluidics. Newton, Massd

(b) SAF, containing 10'/o Squalenc, 0.4o/o Tween 80, 5%pluroiuc-blocked polymer L121, and tie-MDP (seebelow) either microfluidized into a submicron ennilsionor voitexed to generate a larger particle size emulsion,and

(c) Ribir" adjuvant ststmn (RAS). (Corixm Fiamilton,Mont ) cont using 2% Squalene. 0.2"/o Tween 80. andone or nxire bacterial cell v;all components fmm thegriuip consisting of3-0-deuylated monopluisphory lipidA (MPLr") descnbed in U.S. Pat. No. 4,912.094(('orixa), trchalose dimycolate ('TDM), and cell wallskeleton (CWS), prel'erably MPI,+CWS (Detox™),

(3) saponin adjuvants. sucli as Quil A or STIMULON™QS-21 (Antigenics. Framingham, Mass ) (U S Pat No.5,057,540) may be used or particles uncrated therefrom suchas IS('OMs (imnuuiostimulating complexes),

(4) bacterial lipopolysaccharidcs. synthetic lipid A analo s

such as mninoallql glucosamine phosphate compounds(A(ip), or derivatives or analogs thereof, which are avnifablefrom Conxa. and u luch are descnbed ui U.S. Pak No. 6.113,918; one such A()l's 2-[(R)-3-Tetradecanoyloxytetrade-canoylmnino]ethyl 2-Dcoxy-4-0-phosphono-3-0 [(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylaminoJ-b-D-

lucopyramisideo which is also know ns 529 (I'ormcrly kmiwnas RC52ifl), wluch is formulated as an aqueous form or as astable cnndsion, synthetic polynudeotides such as oligo-nucleotdes cootnuun CpO motif(s) (U.S Pat. No 6.207,646);

(5) cysokincs. such as interli:ukins (c.g, 11,-1, 11,-2, 11,-4,

11.-5. IL-6. IL-7. IL-12. IL-I b. IL-18, etc.), interferons (e.goganuna interferon), granulocyte macrophage colony stimu-latmg tractor (GM-C:SF), macrophage colony siimulating I;ic-

tor (M-CSI'), tniilor ilecrosts lhicmir (TNI ). costllliulatoryniolecules B7-1 and B7-2, etcz

(6) detoxified mutants of' buctcrial ADP-ribosylatingtoxin such as a cholera toxin (CT) either in a wild-type ornnitant forni, for exmnplq where the glutmnic acid at aminoacid position 29 is replaced by another amino acid, preferablya histidine. in accordance with published international patentapplication number WO 00/18434 (see also WO 02/098368and WO 02/098369), a pertussis toxui (PT), or an K i:ul/

heat-labile toxin (LT), particularly LlzK(i3, LltR72,CT-S)09. PT-K9/0129 (seeo e.g.. WO 93/13302 and WO92/19265): and

(7) iither substances that act as immunostimulating agentsto enhance the effectiveness of the composition.

Muramyl peptides include, bnt are not linuted to, N-acetyl-muramyl-i,-tlueonyl-D-isoglutamuie (tlu-MDP). N-acctyl-normunsmyl-L-ahmine-2-(1u2'ipalmitoyl-sn- lycero-3-hydroxyphosphoryloxy)-cthylamine (MTP-PF), etc.

The vaccine fomiulations of the present uivention cmt beused to protect or treat a human susceptible to pneumococcalinl'ection, by means of ixhninistcring tire vaccine via a sys-ienuc or mucosal route. These admmistrations can includeinjection via the intramuscular. intraperitoneal. intrademialor subcutaiwous routes, or via mucosal administration to theoml/alimentary. respiratory or genitourinary tmcts. In onemnbodiment, inrranasal adniinistrntion is used for tlm treat-ment of pneumonia or otitis media (as nasoplmryngeal car-

riage ofpneumococci can be more effectively prevented. tints

attcnunting infection at its earliest stage).The amount of c on) ug ate in each vaccine dose is selected as

an amount that induces an inununoprotsective response with-

(js 7,955,605 B2

12nur significmit, adverse efiiccts Such amount can varydependin upon the pneumococcal semtype. (Jenerally. euchdose will comprise O.l to 100 pg of polysacclmride, particu-larly 0 I to 10 pg, and more particularly I to 5 pg.

Optimal amounts of components for o particular vaccuie 6

can be ascertained by standard studies involving observationof appropnate imnume responses in subjects Following anInitial vaccination. subjects can receive one or several boosterimmuni&ations adeqirotely spaced

In a particular embodiment of the present uivention, the ui

13vPnC vaccuie is a sterile liquid formulation ofpneiunococ-cal capsular polysaccharidcs nf serotypcs l. 3, 4. 5, 6A. 6B,71'. 9V, 14. 18C, 19A. 191', and 23F Individually confugated to( RMI n I Icl'I 0 5 011 close is fonruil Itccf to conmufi' Pg ofeach saccharide, except for 6B at 4 ug. approxunately 29 pg i.

CRMI &&

carrier protein: 0. 1 25 mg of elemental aluminum(0.5 mng aluminum pluisphate) adjuvant. mid sodium chlorideand sodium succutate buffer as exmpicnts The hquid Is tilledinto single dose syringes svithout a preservative. After shak-nlg, tflL vaccuIc Is o honuigcncous, whlti: suspension ready zn

for intmnuiscular a&hninistration.lite choice ofdose level for the 13vpnC'accine is similar

to the marketed 7vPnC vaccine (Prevnar). 111e 2 pg saccha-ride dose level was selected for all serotypes, except for C&B,

which is at 4 pg per dose. The 7vPn(.'accine hos shown "

desirable safety. inununo en&city, mid efiicacy against IPD Inthe 2 pg sacchande dose level for serotypes 4, 9M 14. 18C,19F uid 23F. nnd at tlie 4 po dose for 6B.

The imnuuiization schedule cmi follow that designated forthc 7vPnC.'accine For example. Ihc routine schedule f(ir sn

uifants and toddlers against invasive disease caused by S.

/&nsinonn/oe due to the serotypes included ui the 13vPnCvaccine is 2, 4. Ci and 12-15 numths nf'age. Thc cmnpositionsof this invention are also suitable for use with older cluldren,adolescents and ndults

The compositions of this uivcntimi may further include oneor more additional ant&gens for use against otitis mediacaused by infection with other bacteria. Such bacteria includenontypable /!Cci»op/i!as /iif/neozc, Moroxc/!o larnrl!In!!&(formerly loiown as//faa/Ionic/a cu/alrha//s) and&I//n/0&ac Sn

cus Ofif/C&s.

L'xomples ofnontypable Huemoyh///I /o//oeozae antigenssuitable for inclusion include the P4 protein, also known asprotein "0*'IJ.S Pat. No 5,C&01.831, International PatentApplication %003/078453), the P6 protein, also knov,n as 4.

the PAI, or the PBOMP-I protein (U.S I'at. No. 5,110,9(fg;International Patent Application %00100790). the P5 p mt ein(U.S. Reissue I'at. No. 37,741), the Hue&Cop!I!!Os adhesionand penetcmion prntein (IJ S. Pot Nos. 6,245,337 ond 6.676.948), the LKP tip adhesui proteui (U.S. Pat. No. 5,643,725) u

and the NucA protein (U.S. Pat. No. 6.221,3(&S').

Examples of M&&luxe//c cufnrrhu!(s antigens suitnblc forInclusion Include the UspA2 protein (U.S. Pat. Nos. 5.552.146. 6,310.190). tlm Cf) protein (U S Pat No 5,725,862),the F, proteui(U.S. Pat. No 5,948,412) md the 74 lolodalton 6

outer membrane protein (U S. Pat No. 6,899.885).Fxamplcs nl'.4//0/ncncrns 0/I//Cis antigens suitable for

uiclusioo uiclude thnse idenulied ut international PatentApplication WO03&'048304.

TfIC Conlpositnuls Ol thiS invention nl'&1'lso include one or 6C

more pmteins from S/rcp/ococc us poco moo/ae. Examples ofSfrepfccncmis pncnmnmne pnIteins suitable for i&iclusio&1

Include those Identified In International Patent Application%002/083855, as well as that described in IntenmtioimlPatent Application WO02/053761. 6

The compos& tions of this uivention muy further include oneof moll'lot('.uls ffoul W/&'issci'lo alc17&iiglfldis type B.

I'.xmnples of Hei.i&emu meniag&f&dis type B proteins suitnblefor inclusion include those identified in International Potent

Apphcations WO03/063766, %02004!09459C» WOO I&85772, WO02&16612 and WOO I/87939C:o-lynphili&ation ond C'nnjugation Process I'or S pneumo-o!Oe Serotype 19A Polysaccharide

Serotype 19A Is much more prone to thermal degradationthan other S /meumon!Ce serotypes due to the presence ofphosphodicster linkages bctwccn its subunits In order to

improve the conjugation efliciency ond to control the stabilityof the inherently labile serotype 19A polysaccharide, o co-

lyophili&ation and conjugution pmcess in the presence ofdimethyl sulfoxide (DMSO) is used in the conjugation of theserotype 19A polysaccharide to the carrier pmtein ('RM, ».Tlus process provides Improved confugate charactenstics In

terms of molecular size and tlm pcrcmit age nl'ree sacclraridefor serotype 19A as compared to tlm usc ofconjugation pro-cesses involving discrete lyophihzation of polysaccharidesand earner proteins in DMSO or pnmesses Involving aqueousco-lyophilimition of polysa(mharides ond earner proteinswithout DMSO.

As described In more detail ut Exmuple 17 belo~, confu-gauon of the serotype 19A polysacchande to the earner pro-tc10, ( RMISI Ls o two reaction-step process Thc first stL7uivolves penodate oxidation (activation) to generate reactivealdehyde groups on the polysaccharide. The activatedpolysaccharide is then puriffexf by ultrafiltration &o removesaccharide frag&nents and smail molecule reaction by-prod-ucts. The acti vuted polysaccharide and (.RM&9& a& ( then cunl-bined and co-lyophdi&ed with sucrose as a cnsiprotectant.The conjugation step Is performed in DMSO via a reductiveaminaunn meclmnism in tfic prcsuncc of sodium cyonnbnro-hydride. Unreacted aldehyde groups are reduced (capped) bythe addition of sodium borohydride. Thc conjugate is thenpurified to rcmove unreactcd ('RMIS& and saccharide I'rag-

ments (e g., by diafiltration versus phosphate buffer followed

by buffi:red saline), giving a final botch concentrate of theglycnconl ugnte in buffered saline

Although a preferred carrier protein (vithin the processdescribed above is the nmtated diphtheria toxin ( RM&&»other earner proteuis nmy be used for conjugation with theserotype 19A polysaccharide within the present methods.(.amcl pn1&chw olc chosLn 10 &nclcasc thc ininlunogLnicit&'fthe bound serotype 19A polysncchande and/or to ehcit anti-bodies against the carrier protein which are diannostically,analytically and/or therapeutically beneticuil. Covalent link-

ing of an antigenic molecule (e.g., a polysacchande) to a

carrier em&I'ers enh incexf nnmmiogenicity and T-cell depen-dence (Pozsgay et al. (1999) PVIS. 96 5194-97, Lee et al.(1976) J./mmimo/.. 116:1711-18: Duitzisetal. (1976') PVBS,73.3671-75) As described hereui. useful carrier protemsuiclude Inactivated bactenal toxins such os tetanus toxoid,pertussis toxoid. cholera toxoid (e.o . as described in Interna-tional Patent Applicauon %02004!083251), /: cn/i I,T, Eco// Sl, and exotoxinA fmm pseud(miouos acrogioosu. Bac-terial outer membrane proteins such as outer nmmbrmie com-

plex c. purina. transferrin bmdiug proteuis. pueumolysm,pneumococca 1 snr face protein A, pnemn Ceo ccat adhesin pro-&cia. (.5a pcptul'Isc Inun C&OIup A of ((loup B sfi'I'pfocnr& us,or Hue&neph//os /4/loeaznc protem D, can also be used. Otherproteins, such os ovalbumin. keyhole fin&pet hemocyanin,bovine seruni albunun, or puntied pro&cut denvative of tuber-culin can also be used as carrier proteins.

Although this co-lyophilization mid conjugation process in

DMSO is descnbed for use with the serotype 19A pofysac-chande. tlfis process nmy also be used for serotypes that are

13

US 7,955,605 B2

14stnicturally similar to scrotypc 19A, such as 6&A, 6B, mid 19F

which also contain phosphodiestcr linkages benveen theirrepeat uruts.

The aboiv. disclosure gcnerafly describes the presentinvention. A morc complete understanding can be obtaincd byreference to the lbllmv&ng spec&lie examples. These examplesare descnbed solely for the purpose of & llustmtion and are notuitcnded to lima the scope of the intention

BXAMPI,FS

Fx&1&rlplc I

Preparation of X yne&oaomue CapsularPolysaccharide Serotype I

Preparation of Master and Working ('ell Basks

S yneamvmae serotype I was obtained from the AmericanI'vpe Culture Collection, AT('(', strain 6&301. Sceveral gaiem- zc

lions of seed stocks ivere created in order to expand the strainand remove components ofanimal origin(generations Fl, I 2,

and F3). Two additional generations of seed stocks wereproduced. The first addiuonal generation was nude from anF3 vial, and tlie subsequent generation vs as made from a vial -"'l'he

1irst addi«onal generation. Scvd vials were stored fro-zen (&-70'.) with synthetic glycerol as a cryopreservat&ve.In addition to frozen via la 1yophilized vials uvre prepared f«r

the I'4 generation. For cell bunkprep u ation, all cultures wererown ui a soy-based medium. Pnor to freezing. cells were ""

concentrated by caitrifugation. spent mcdiimi was rcntovcxi,

and cell pellets were re-suspended in fresh mednuu contain-ing a cryopreservatve. such as synthetic glycerol.

Fermentation and Harvesting

Cultures from the work&a cell bank were used to inoculateseed bottles containing a soy-based medium. Thc bottles wereuicubated at 36'. 2'. without a its&ion until rowthrequirements were met. A seed bottle vvas used to inoculate a ac

scend f&.rmen ter contai ning soy-bias ixl medium. A pH ofabout7.0 was maintauied i& ith sterile sodnun carbonate solution.After the target optical density was reached. the seed fennai-tor was used to inoculate tfm production fcnncntor containingsoy-based mednun. The pH was maintained with sterile ra

sodium carbonate solution The fermentation was terminatedafier cessation of grov th or when the uorkiiig volume of thefermenu&r was reached. An appropnate amount of s ten le 12%v

d&x&xycholaie sodium was ndded to the culinre &o lyssa thebactenal cells and release cell-associated polysacchande. u

After lysing, the fennentor contents vv ere cooled. 11&a pl 1 ofthe lysed culture bmth was ad)us md to appmxuuately pi16 6

with acetic acid. The lysate w as clanfied by continuous floivcentrifugation foflowixI by depth filtmation and 0.45 pmmicroliltrauon 5

In mi alternate pmcess. the fermentation pH of about 7.0was maintainal with 3N NaOH Afler the target optical den-sity was reached, the seed fennentor is as used to inoculate theproduction fennaitor containing soy-basal medium 1 he pHwasmaintainedwith3NNaOH The fcnucntation was tenm- ninatcd after cessation ofgrowth or v;hen the vv orking volumeof the fennentor was reach&&I An appropriate amount ofstenle 12% deoxycholate sodium was added to the culture toobta in a 0.12 iv concentration ui the broth, to lyse the bacterialcells mid release ccfl-associated polysaccharide. Afier lysing. rthe fermentor contents were held. with agitation. for a timeinterval betwcxn 6 and 24 hours at a temperamre benv ceo 7"

C. and ly', to assure tluit complete cellular lysis andpolysaccharide release had occurred. Agitation during thishold penod prevented lysate sedunent from settluig on thefennentor walls and pH probe. thereby allowing the pH pmbeintegrity to be nu&intaiued. Next, the pll of'he lysed culturebmth was adjusted to approxunately pl I 5.0 with 50% aceticacid Aflcr a hold time without agitation. for a time uitervnlbetu een 12 and 24 hours at a temperature between 15', and25'., a signiTicant portion of the previously soluble proteinsdropped on& ol'solution ns a sohd precipitate with little loss ordegradation of the polysaccharide, wluch remained in solu-tion The sohnion with the precipitate was then ciaritied byconunuous lkm centrifugation foliowcxi by depth filtrationand 0.45 pm micm(iltration.

Purification

Tire pun licntion of the pneumococcal polysacciiaride con-sisted of several concentration&diafiltration operations, pre-cipitationiclution, column cbmmatography.;md depth liltra-uon steps. All pmcedures were performed at roomtemperature unless otherwise specified.

Clanfied bmth from the fermentor cultures of S yneua&v-

n ine seroty pe I were concentrated and disfiltered using a 100kl )a MWF 0 (kilodalton molecular tv eight cutoff) filter 1)ia-

Iiltrat&on was accomplished us&n sodium phosphate buffer atneutral pl I. D&atiltranon removed the low molecular weightmed&u&u components fmm the higher molecular iveightbiopolymers such as nucleic acid. protein and polysaccha-

lid&.'liepolysacchandewasprec&p&tatedfromtheconcentrnted

and diafiltered solution by adding hexadecyltnmethyl anuuo-nium brmuidc (HB) from a stock solution to give a linn)concentration of 1% IIB (w!'v) The polysacchande/IIB pre-cipitate was captured on a depth filmr nnd the filtrate wasdiscarded Thc polysacclmride pnxipitate was resolubilizedand eluted by recirculating a sodium chlonde solutionthrouvh the precipitate-containing depth filter. The filterswere then rinsed with addiuonal sodiuni chloride solution

Sodiumiodide(Nal) vtasaddedtothepolysaccharidesolu-tion front a stock Nal solution to achieve a final concentmtionof 0.5'!v to precipitate IIB. I'he precipitate was removed bydepth filtration. The filtrate contains the target polysaccha-ride Thc prcccipitation vessel and thc tiber w crc rinsed &s ith a

NaCI/Nal solution and the rinse was combuied v;ith the par-tially purified polysaccharide solution Thc filter was dis-carded. The polys ecch vide was then filtered thniugh a 0.2 )unfilter.

Thc polysaccharide solutimt was concentrated on a 30 kDaMWCO ultra(ilter and d&aiiltered with a sodium chlondesulu&loll.

Tire partially purilied polysaccharide solution was I'urther

purdied by filtration &i&rough a depth tilter &mpreytated withactivated carbon. After filtration. tlm carbon filter was rinsedu«h a sodium chloride soluuon. The rinse is combuied withthe polysaccharide solution, which is then filtered through a

0 2 pm liltcrThe polysacchande solution was concentrnted on a 30 kDa

MWF 0 ultratilter and adjusted with a I M sodium phospliatebulf&.r to achie&u a liiud concentration ol'0025 M sodiuinphosphate. '11&e pH was checked and adjusted to 7.0+0.2.

The ceramic hydmxyapatite (HA) colunin wins equili-brated with sodium phosphate butfer cont aiiung sodium chlo-ride to obtain the appropriate conductivity (&15 pS). Thepolysaccharide solution &vas then loaded onto thc column.Under these conditions, unpunt&es bound to the resin and thepolysaccharide ives recovered in the flow-tlu ouPv from the

15

US 7,955,605 B2

16cohunn. The polysaccharide solution was filtered throngh 0.2

pnl infine filters located before and after thc colunm.11le polysacchande solution tv as concentmted using a 30

kDa MWCO tilter The concentrate was then diafiltercd withWater for Injection (WFI)

The dlahltered polysacchsmde solution was iilteredtlu ough a 0 2 iun membrane filter lnul polypropylene bottles.Smnples were removed for release testing and tlm punfied

polysacchande was stored frozen at -25''.Charactenzatlon

The '-NMR duta was consistent lv 1th the chemical struc-ture by the assiyunent of signals assigned to the protons oftlm polysaccharide nlolccule The 'H-NMR spectnnn showeda series of well-resolved signals (pmtons I'rom the methylgroup) for the quantitation of the 0-acetyl fimctiona I group inthc polysacchnride.

The identity of the monovalent polysaccharide tv as con-timlcd by countercurrent immunoelectrophoresis using spe-cific antlserm

High perfonnance gel filtration cluomatography coupledwidl refractive index and multimlgle laser liJott scattering(MALLS) detectors was used in con)unction with the smnpleconcentration to calculate the molecular weight.

Size exclusion chn(ntatoyaphy media (CI,-4B) was usedto profile the relative molecular size dlstnbutlon of thepolymccharide

Fxamplc 2

Following the cyanoborohydridc incubntions, tlm reactionmixture was diluted with cold saline follow cd by the additionof I M sodium carbonate to adlust the reaction mixture to pll9 0 Unrcacted aldehydes lvere quenched by addition ofsodium borohyd ride by incubation at 23''ior 3-6 hours

The reaction mixture was diluted 2-fold with saline and

trmisferred through a OA5-5 pm preiilter into a retentate ves-

sel. The reaction. mixture is dudiltered 30x with 0.15 M phos-phate buffi:r, pH 6, and 20x with saline Thc rctentate was

lafiltered through a 0.2 iun filter.

The conjugate solution was diluted to a target of 0. 5 mg 'mL

in 0.9% saline, and then sterile filtered into Iinal bull concen-trate (FBC) coiitainers ln a Class 100 hood The contugatewas stored at 2-8'.

Characterization

Size exclusion cluumatography media (CL-4B) was usedto profile the relative nullecular size distribution of the con-

"(1jugate

The identity of the conlugate was confirmed by the slot-blot assay using specific antisem.

Tile sacchande and protein concentrations were dcter-nuned by the unlnic acid and Low ry essays. respectively. The'tio of saccharide to protein in the covalently bonded conju-gate complex was obtained by the calculation:

gg/om eoe ebengeRoan=

gglmL gm(e(o

Preparation of Serotype I PneumococcalSaccharidci~CRM»7 Conjugate

Activation and Conjugation

Contauiers of puniied polysacchande were tlmwed andcombined in a reaction vessel To thc vessel, 0.2 M sodiumcarbmlate. pH 9 0 was addcxi lilr partial deacctylation (hy-drolysis) for3 hours at 50" (1Thereactionwas cooledto20'oC and neutrnlization w:as performed by 0 2 M acetic acidOxidation in the presence of sodium penodate was performedby incubation at 2-go I'.. nod the mixture was stirred for 15-21

hollrs11le activation reaction mixture was concentmted and dla- s.

liltercxI 1 0x with 0 9%9 NaCI using a 30K MWCO membrane.The retentate was 0.2 pm filtered. The activated sacchandewas filled into 100 nlL glass lyoplulization bottles and shell-I'rozen at -75''nd lyophiiized.

"Shell-freezing" ls a method for preparing samples for v

lyoplfilization (freeze-drying). Flasks are automaticallyrotatcxI by nultor dnven rol le» in a refrigerated bath contain-ing alcohol or any other appropnate fluid. A tlun coating ofproduct is evenly frozen around the inside "shell** of a tiask,permitunga greater volume ofmatennl to be safely processeii s

during each freeze-drying nm lhasa automatic, refrigeratedunits provide a simple and cflicicnt mcnns ol'prc-freezingmany flasks at a time, pmdumng the desired coatings ulslde,and providing sutficient surface area for eflicien freeze-dry-ing so

Bottles of lyophihzed material were brouJolt to room tem-perature and resuspended in I RM(97 solution at a snccluiride(protein mtlo of 2.1. To the sacchandeiprotein nuxture IMsodium phosphate buffer lvas added to a final 0.2M ionicstrength and a pH of7.5, then sodiiun cyanoborohydride was 9

added. 11le reaction was uicubated at 23'. for 18 hours.followed by a second incubation at 37" C. for 72 hours.

0-acetyl content was measured by the Hestnn method(Hestrin ct. al, I Biol 6'hcm. 19491, 180, p 249) The ratio of0-acetyl concentration to total saccharide concentration gavegmoles of 0-acetyl per mg of saccharide.

Example 3

Preparation of S Pneivnvnine CapsularPolysaccharide Serotype 3

Preparation of Master and Workuioo Cell Banks

S. 7(ncaa(ovine serotype 3 was obtained from Dr. RobertAustnan. University of Pennsylvania, Pluladelphia. Pa. Forpreparation of thc cell bails system. sec Fxamplc 1.

Fermentation and I larvesting

Cultures from the working cell bank were used to inoculateseed bottles containing soy-based medium. The bottles wereuicubated at 36" C. 20" C, without agitation unul rowthrcquirenlents werc nlet A seed bottle ives used to inoculate aseed fcrmentor containing soy-based medium A pi i ofabout7.0 was nmintained with sterile sodium carbonate solution.Aficr thc target optical density was reached, tfm sis'6 fi nncn-lor was used to inoculate an intenuediate seed fermentor.Afier tlm target optical density was reached, the intcnnedi ateaced li:nnenlor was used le in((cuiatc thc pr(xluction li:nnen-tor. 'lite pll was maintained with sterile sodium carbonatesolution 'I he fermentation was terminated at)ter the workingvolume of the fennentor w.ls reached. An appropnate amountof sterile 12% sodium deoxycholate lvas added to the culnireto lyse thc bacterial cells and reliase cell-associatixi polysac-chande. After lysuig. the fermentor contents were cooled.'lhe pH of the lysed culture brotll was adjusted to approxi-

17

(js 7,955,605 B2

18mately pH Ci.6 with ncctic acid. The lysate was clarified bycontimious flow centrifugation followed by depth filtmtionand 0.45 iun microfiltration.

Puritication

The purification of the pneumococcal polysacchande con-sisted of several concentration/dialiltration operatiims, pre-cipitation/elution. colunm cltiomatogmphy, mid depth filtm-tion steps. All procedures were perfonnixf at roomtemperature unless otherwise specified.

( 1arificd broth from the fernientor cultures of R pneaiiio-aiae scrotypc 3 were concentmtcd and diafiltered using n 100

kDa MEVCO filter. Diafiltration was accomplished usingsodium phosplmte bufTer ai neutral pH. Diatiltnition removedthe low molecular»ci lit medium cmuponents thorn the

higher molecular »eight biopolymers such as nucleic acid,protein and polysacclmride.

Poor to the addition of hexadecyltnmethyl ammoniumbromide (HB), a calculated volume of a Na(1 stock solutionwas added to the concentrated and diafiltered polysaccharidesolution ai give a fi mt concentration of 0.25 M Na('1. Thepolysaccliande u.as thmi precipitated by adding HB from a

stock solution to vive a tinal concentration of I'/o HB (w/v)I'he polysaccharide/HB precipitate »as captured on a depthfilter and the filtmte ii as discarded. The polysacchande pre-cipuatc v;as resolubiliied and cluted by recirculating, a

sodium chloride solution through thc prccipitati:-containingdeptli filter. The filters were then rinsed with additioimlsodium chlonde solution.

Sodium iodide (Nal) vs as addexf to the polysacchaude solu-tion front a stock Nnl solution to achieve a final mmcentrationof 0.5vz/) to precipitate HB. Hie precipitate was removed bydepth filtration. The filtrate contained the target polysacclm-ridc. The precipitation vessel mid the tilter ivere rinsixf with aNaC'I/Nal solution mxl thc iiliso uris coulbiucd with thc par-tially purified polysaccharide solution. The filter it as dis-

carded Tire polysacchande was then liltered tlu ough a 0 2 luufilter.

'Ilm polysaccharide solution was conccntratcd on a 30 kDaMWCO ultrafilter and diafiltered with a sodium chloridesolution.

The partially puri tied polysaccharide soluiimi u as li irtlmr

purified by tiltmtion through a depth filter impregnated withactivated carbon. After filtration, the carbon filter was nnsedwith a sodiiuu cltioride solution. The rinse was combinedwith the polysaccharide soluiimi. ivhich v as then litteredflu ough a 0.2 iuu tilter.

'Ilie polysacchande solution ii as concentrated on a 30 kDaMWC:0 ultratilter uid ndf osteal ii ith a I M sodiuni phospliatcbuffer to aclueve a final concentration ol 0.025M sodiumplxisphatc The pH was checkixf mid adjusted ui 7 0+0 2.

The cemmic hydroxyapatite (HA) column ives equih-brated tv ith sodium phosphate buffer containing sodium chlo-ride to obtain tlm appropriate conductivity (15 RS) Thcpolymicchande soluuon ives then loaded onto the columnUnder these conditions, impurities bound to the resin and thepolysaccharide v,as recovercxf in the flow-through from thecoliunn. The polysacchande was tin shed tlu ough the columnwith buffer and uas filtered tlwough a 0 2 pm filter.

The polysaccharide solution» as concentmted using a 30kDa MWCO filter. The concentrate was then diafiltered withWl'I.

The diafiltered polysacchande solution was filteredthrough a 0. 2 lun membrane filter into stainless stceel contain-

ers. Smnp Ics uvre removed fitr release testing and the puri tiedpolysaccharide ives stored I'rozcn at

-25''.'hamcterization

5

The I H-NMR data was consistent » ith the chemical stnic-nire by the assignment of signals assigned to the protons ofthe polysaccharide nxilecule.

I'he identity of the monovalent polysaccharide was con-fimied by countercurrent inununoelectrophoresis using spe-

i(1citic antisem.

High perfomiance vel tiltration cluomatography. coupledwith refractive index and nniltiangle laser light scattering(MAI.LS) detectors. » as used in conjunction with the sampleconcentmtion to calculate the molecular weight.

Size exclusion clwomatopaphy media (('1,-4B) was usedai pmtile the relative molecular size distribution of thepolysaccharide.

Fxample 4

Preparation of Serotype 3 PneumococcalSaccharide CRM„i Conlugaie

Acuvation and Conjugation

Cont uners of puntied serotype 3 micchande u ere thawedand combined ui a reaction vessel. To the vessel. WI'I and 2Macetic acid u ere added to a tinal concmitration ot'02M and 2

mg/mL saccharide. The temperature of the solution wasnusedtog5'C. tbronehourtohydrolyzethepolysaccharide.The reaction was cooled to =25" C. and IM magnesiumchlonde was added to a tinal concentration of 0.1 M. Oxida-tion ui the presence of sodium penodate was performed byincubation for 16-24 hours at 23' .

The activation reaction mixture was concentrated and dia-"" filtered lflx vnth WI'I using a IOOK MWCO membrane The

retentate was hltered tluough a 0.2-1un filter.For compounduig. 0.2M sodium phosphate. pH 7.0, was

added to the activated saccharide to a final concentration of 10mM and a pl I of 60-65. CRM,97 carrier protein »as nuxed

sa with the sacchande solution to a ratio of 2 g of sacclmride perI g of CRMivv The combined saccharide/protein solutionwas filled into 100 mL glass lyoplulization bottles with a SO

mL target fill, shell-frozen at -75''., and lyophilizixl.Bottles ofco-lyoplulized sacchande/protein material ivere

brought to room temperature and resuspended in O.IMsodium phosphate buffer, pii 7.0, to a final saccharide con-centration of20 mg/mL. Tlie pH was adjusted to CxS and thena 0.5 molar equivalent of sodium cyanoborohydnde wasadded. The reaction was incubated at 37'2 for 4g hours.I'ollouing the cyanoborohydride incubation. the reaction

-'" mixture ives diluted with cold 5 mM vaccinate/0.9"rt) salinebufl'er Unreacted aldehydes ivere quenched by the additionofsodium borohydride and incubation at 23" ('. for 3-ti bourn.The reaction mixture was transferred tluouvpt a 0.45-5 illllprehlter into a retentate vessel.

ss The reactiim mixture wns dialiltcred 30x v,ith 0 IM phos-phate bu ITer (pl I 9), 20x u i 1h 0.15 M phosphate buusr (pl I 6),and 20x ivith 5 mM succinate/0.9"A saline. 'I bc retentate waslittered through a 0 2-iun filter.

The con) u gite solution was diluted to a sac elm ride target ofso 0.5 mg/ml,. and then sterile filtere into I'B('ontainer: in a

Class 100 hood. The contugate is as stored at 2-g" C.

Chamctenzation

Size exclusion chromatography nttxtia (C:1,-4B) was usedto profile the relauve molecular size distribution of the con-pigate.

19

US 7,955,605 B2

20'ljm identity of tfm conjugate was confirmed by thc slot-

blot assay using spcmilic antiscraThe saccharide mid promin concentrations umre deter-

mined by the Anthronc and I oivry essays, respectively Thcratio of saccharide to protein in the covalently bondcxt conju-gate complex wmi obtaincdt by the calculation

pg/mt. 7 .hoverRoiio =

9 g/m( pro&em

L'xampfe s

Preparation of S. Poeomooi or CapsularPolysaccharide Serotype 5

S Poeoroooioe serotype 5 uas obtained from I&r (icraldSclutfman of the State University of Nev. York, Bmoklyn,N.Y. For preparation of the cell bank system, see Example 1.

For fenumitation, harvesting. purification and characteriza-tion of the polysacchande. see Example l.

Alternate Fermentation Process

Cultures from the working cell bank were used to inoculateseed bottles containulg a soy-based medium and a 10 mMsterile NaHCOs solution 'The bottles werc incubated at 36"

C. 2'. without agitation until gmwth requirements weremet. A seed bottle was used to uloculate a seed femlentorcontaining sov-bms&d mcxhum and a 10 mM stcrilc NaHCOssolutiou. A pii of about 7.0 uas maintained u 1th 3N NaOII.After the target optical density was reached, the seed fi:nnml-tor was used to inoculate the prndtuclion fi:numltorcontiuningsoy-based medium with a 10 ntM NaIICO& concentration.The pH was maintained with 3N NaOH. The I'ernmntation

wai terminated a fiercessation OI'growth or when the is orkingvolume of the fermentor was reachoxl. An appropriate a minuet

of sterile 12% sodium deoxycholate was added to tlm cultureto obtain a 0.12% concentration in the broth, to lyse thebacterial cells and release cell-associated polysaccharide.After lysing. the fernlentor contents were held, with agitation,for a time interval between 8 and 24 hours at a tempemturebetween 7" (3 and 13" (3 to assure that complete cellular lysisand polysaccharide release had occurred Agitation dunethis hold penod prevented lysate sediment from settling onthc li.'rmcntor &sails mid pH probe, tlmrcby allowing thc pHprobe integnty to be maintained Next. the pil of the lysedculture broth ivas adjusted to approximately pi I 4.5 with 50%o

acetic acid Aller n hold time without agitation. Iilr a timeinterval between 12 and 24 hours at a temperature between15" C'id 25'.. a signiticant portion of tho previouslysob&hie proteins dmpped out of so 1uli on &rs a solid prempi 1 atewith little loss or degradation of the polysaccharide, whichrcnlained in solution. Tlm solution with thc precipitate wasthen clamiied by continuous tlow centnfugauon follov ed bydepth filtration and 0.45 pm microfiltration.

l.ixampfe Ci

I'reparation of Serotype 5 PneunulcoccalSaccharide ('RIVI&97 ( oillllgatc

Activation and Conjugation

Containers of serotype 5 sacchande were thawed andcom-bined in a reaction vessel. To the vessel. 0.1M sodium acetate,

pH 4.7, was added followed by oxidation in tlm presence ofsodium pcriodatc by incubation for 16-22 hours at 23" ('.

The activation reaction mixture was concentrated and dia-

liltered 10x with WFI using a 100K MWCO membrane Theretcntate was liltcred through a 0 2 pm filter.

The serotype 5 activated sacchande was conlbined u.ith

CRM& 9- at a ratio of 0 8: I . The combined sac elm ride)proteinsoluti&ui wirs tilled uito 100 ml. glass lyophilwation bott)ca

(50 mL target till), shell-frozen at -75'.. md co-lyo-io philized

Bottles of co-lyophilwed material were brought to roomtemperature and resuspended in 0.1M sodium phosphate, pH7 5. and sodium cyanoborohydride was added Thc reactionuas incubated at 30': for 72 hour., followed by a sec&md

's addition of cyanoborohydride and incubated at 30" (2 for20-28 hours

Following the cyanoborohydride incubations, the reactionmixture ivns diluted 2-fold with saline and transferredtluough a 0 45-5 lun preiilter into a retentate vessel. The

"-" reaction mixture was diafiltered 30x with 0.01M phosphatebutTer. pH 8. 20x with 0.15M phosphate butTer, pH 6, and 20xwith sahne. The retentate was filtered tluough a 0.2 pm filter.

The contugate solution was diluted to a sac elm ride target of0 5 mgiml,. md then sterile lilternl into FBC contaiiwrs in a

-"'lass 100 hood. The contugate ives stored at 2-8'.For thL chm iiclol1zation of thc coillilgatL, sLL Fxanlple 2

Fxanlplc 7

Preparation of S Poco orv nine CapsularPolysaccharide Scrotypc 6A

S. Poeomooioe scrotype tiA ives obtained from Dr. CiemldSchiffmml of the State Hnivcrsity ol'eiv York. Brooklyn,

"" N.Y. I or preparation ol the cell bank system. see Example 1.

For fcrruentation, harvesting and purification of the polymic-clumdo.,ime I',xample I, except that during purification, the 30kDa MYYCO concentration step, prior to the cltiomatographystep, is omitted.

air

Fx'llnplc 8

Preparation of Serotype 6A PneumococcnlSaccharide C.'RM,„, Conjugate

Activation and ('oniugation

Serotype 6A polysacchande is a high molecular weightpolymer that had to be reduced in size prior to oxidation.

o Containers of serotype 6A saccharide were thau ed and com-bined in a reaction vessel. To the vessel, 2 M acetic acid wasadded to a final concentrauon of 0.1 M for hydrolysis for 1.5

hours at 60" C. The reaction was cooled to 23" C, and neu-tralization was perfimned by adjusting thc reaction mixture

1 uith I MNaOHtopH 6.0xidationinthepresenceofsodiumperiodate was perfonued by incubation at 23" ('or 14-22hours

The activauon reaction mixture was concentrated and dui-filtered 10x u:ith WFI using a 100K MYY('0 nmmbnne The

ol re)ca&ate was tiltercd fitrough a 0 2 pm filter.

Serotype CiA ives compounded with sucrose and filled into100 ml, glass lyophilization bottles (50 nil. taroet fill) andshell-frozen at -75', and lyoplulized.

Bottles of lyophilized matenal were brouodtt to room tem-pcmture and rcsuspcndcd in dimethyL&ulfilxide (DMSO) at a

sacchandeipmtein ratio of I;l. After addition of sodiumcyanoborohydride, the reaction mixturewas incubatied at 23"

21

(js 7,955,605 B2

22( . fiir I g hours Following thc cyanoborohydride incubation,the reaction mixture was diluted with cold saline. U'nreacted

aldehyde s were quenched by addition of sodium borohydndeby incubaumi at 23''iir 3-20 hours

Tlm dilutcsf maction mixture ivms trmisfi."rrcd through a 5

pm prefilter into a retentate vessel. The reacuon mixture uasdiafiltered 10x with 0.9% NaCI and 30x wttlt succinate-buff-ered NaC'I Thc rctmuate wms filtered through a 0 2 firn filter

The conjugate solutionisas diluted to asacchandetargetof0 5 mgvml, mid tlmn sterile filtered into FBC c(mtainers iri aC'lass 100 hood. The confugnlc uas stored at2-ft''or

tlie characterization of the conlugate. scm Example 2.

Example 9

Preparation ofS Pneumrttttae C'apsularI'olysaccharide Serotype 7F

The ceramic hydroxyapatite (HA) column was equili-bmted with sodium phosphate buffer contaiiung sodium chlo-ride to obtain the appropriate conductivity (15 ftS). 'Ifiepolysaccharide solution was tlmn londed onto thc colunm.

s Under these conditions. unpunties bound to the resin and thepolysacclmride was recovered in the flow-tlu ough from theroliunn. The polysaccharide uas

flushed

throng the columnwith buAer and was filtered tlu ough a 0.2 pm filter.

I'he polysaccharide solution was concentrated using a 30'" kDa MWsCO lilter. The concentrate isas then diafiltered uith

WFI.

The diafiltered polysaccharide solution was filteredtin ough a 0.2 iun membr me filter into st nnl ass steel contain-ers. Samples were removed for release testuig and the purifiedpolysaccharide was stored at 2m'" C.

For characterization of the polysacchande. see Example 3.

S, Pseronvni ue serotype 7F ives obtained fnmi Dr. Crerald

Schiffman of the State University of New York, Brooklyn.N Y. For preparation of the cell bank system. and for fermen-tation and harvestuig of the polysaccharide, see Example 3.

For an alternate fenimntation and harvesting process, see thealternate process described in Example l.

Punfication

kxample 10

Prcpmntion ol'Scrotypc 7F PncumococcalSacchande CRM»i Contugate

Activation and (.'onjugation

The purification of the pneumococcal polysaccharide con-sisted of several concentration/diahltration operations. pre-cipitabon/elution. column chromnlography. mid depth filtra-

tion steps. Afl pmcedures v ere perfonued at roointemperature unless otherwise specified.

C'larilicd broth I'rmn fcmnmitor cultures of 5'nemanmaeserotype 7Fwere concentrated and diafiltered usuig a 100kDa MWCO filter. Dinfiltration v as accomplished usingsodiuni phosphate bufli:r al neutral pl I. Dialiltration removedflie low molecular weight medium components from tirehigher molecular weight biopolynmrs such as nucleic acid,protein and polysacclmridi:

Serotype 7F does not form a prcmipttate with HB. Instead.impurities uvre precipitated from tlm cimcentrned imd dia-filtered solution by addui the IIB from a stock solution to a

final concentration of 1% HB. fhe precipitate was capturedon a depth filter and the filter wns discardisf The polysaccha-nde was contained in the filtrate.

Sodium iodide (Na1) was addixf to the polysaccharide

solut-

ion

from

aa

stock Nal soluuonto achieves final concentrationof 0.5% to precipitate HB. flic precipitate was removed bydepth liltration The librate contained lhe target polysaccha-nde. The precipitation vessel and the tilter v, ere nnsed with a

NaCI/Nal solunon and the muses were combined with thepartially purified polysaccharide soluuon. The filter was dis-carded. The polysacchande was then filtered tlu ough u 0.2 pmfilter.

The poly sac chan de solution was concentrated on a 30 kDaMW('0 ultmfilter and diafiltered u:ith a sodium chloridesolution

The partuilly punfied polysaccharidesolution was furtherpurified by tiltmtion through a depth filter impregnated withactivated carbon After filtration, thc carbon filter was rinsedwith a sodium cltioride solution. The rinse was combinedwith the polysacchnride solution, which was then filteredthrough a 0.2 iun filter.

'Ilie polysacchande solution si as concentrated on a 30 kDaMW(.0 ultrafiltcr and adjusted with a I M sodiiun phosphatebuffer to aclfieve a final concentration of 0.025M sodiumphosphate. The pH was checked and adjusted to 7.0+0.2.

Oxidation in the presence of sodiiun periodate was per-formixf by incubation I'or 1ti-24 hrs;u 23" (2

I'he activation reaction mixture was concentmitixl and dia-liltercd 10x with 10 mM NaOAc, pH 4 5, using a 1001(

MWCO inembr uie The retentate was filiered through a 0.2fun filter

Serotype 7F was filled uito 100 mL glass Iyoplultzattonbottles (50 mL target fill) and shell-frozen at -75" C. andlyophil ized.

Bottles of lyoplulized semtype 7F and CRM,» werebmughl lo room temperature and resuspmxlcd in DMSO at a

sacchande/protein mti o of I . 5 I . After the addition of sodiumcyanoborohydridc, the reaction was incubated at 23" ('org-10 hours Um cacted aldchydes were qucnchcd by the addi-uon of sodium borohydride by incubation at 23" C. for 1(i

hours

The reaction mmture was diluted 10-fold with cold salineand tmnsferred through a 5 iun prefil ter into a retentate vessel.The reaction nuxture was diafiltered 10x uith 0.9% salineand 30x v;ith succinate-buffercd saline. The retentate wasfiltered through a 0 2 pm filter.

I'he conjuvesta solution was dilutcxf to a sacclmride target of0 5 mg/mf, 0.9%v saline. and then sterile ftltcrcxl into FBC'onuunersusa Class 100 hood The coniu ate uas stored nt

2 ft"('or

clmractenzation of the conlugite. see Example 4.5

I.xample 11

Preparation ol'S Paeaiaoniae C:apsularPolysacclrande Serotype 19A

S Paenmoaiae serotype 19A usa obtmned from Dr Crerald

Schiffinan of the State University of New York. Brooklyn,N Y For prepar uion of the cell bank system, sec fixample I.Iior fermentation, harvestuig and purihcation of the polysac-chande. see Example 7. For chamcterization, see Example 3.

23

US 7,955,605 B2

24Fxaniple 12

Prepamtion of Serotype 19A PneumococcalSaccharide CRM&97 CQIllugotc

Activation and Conjugation

Containers of serotype 19A sacclwride were thawed andcombined in a reaction vessel. Sodium acetate was added to10 mM (pl15 0) ond oxidou&m ii;Is carriini out in tlm presence Io

of sodnun penodate by incubation for 16-24 lu 1 at 23'.'llm activnti&111 reaction mixture was conccntmtcd and din-

hlterced 10x with 10 mM acetate, pH 5.0. usmg a 100KMWCO membrane. Tlie retentate was filtered throuoJI a 0.2ii&11 filter.

The activated sacchande was compounded iiith sucrosefollwved by the addition of ('RM»r. I'he serotype 19A acti-vat& 6 socclia110& olid (.RMISI mixture (0 8 1 mtio) ives tilled

into 100 mL glass lyophilization bottles (50 mL target fill)wid shell-frozen at -75" C. and lyophilized. "(I

Bottles of lyophilized matenal were bronah1 Io room tem-perature and resuspended in DMSO. To the saccharide'pro-tein mixnire, sodium cyanoborohydride (100 mg&'ml) wasadded. The reaction was uicubated at 23'. for 15 hours.Follow ing the cyanoborohydnde incubation, unreacted alde- "-'ydes

were quenched by the addition of sodium borohydndeby uicubation ot 23'. for 3-20 hours.

Tlm reaction mixture was diluted 10-fold with cold salineand trans ferrcxi throu h a 5 pm prelilter mto a re&entete vessel.I'he reaction mixture was diafiltered 10x with 0.9% NaCI, ""

0 45-pm liltered, wid 30x with dialiltration using 5 mM suc-cinate/0 9% Na( I buifer. pH 6. The retentate i&as filteredthrough a 0.2 pui biter.

The coifii ugate sulu ti&w vas dilutes) to a target O I 0 5 mg/mi.using 5 mM succuiate'0.9% sahne, and then stenle filtered ""

into FB('ontainers in a Class 100 hood The conjugate wasstored at 2-8'

I'or choractenzation of tlie conjugate, see Example 4.

Example 13

Preparatioo ofS Pncuwvniac CapsularPolysaccharide Serotypes 4, (iB, 9% 14, 18C, 19F

and 23F

Preparation of the R pncuui an/ac Seed ('ulturc

R pneumonia c serotypes 4. 6B. &916 18('. 19F and 23F v, ereobtantedl fi'onl Dr O'erald Sebi lTman, State l)niversity ol'N&sv

York. Bmoklyn, N.Y. S. pneumvniae serotype i4 ivas u

obtained from the ATCC, strain 6314.Sepnrately, one vial of e wh ol'lm desired serotvpes ol'/rep&vcure&» pl&en rrmni ncwas &ised tii start 8 fernlentait &oil

batch Two bottles conhsining a soy-based nwdium and phe-nol red were adjusted to a pH nwge of 7 4 0 2 usuig sodium Icarbonate. and the required volume of 50% dextmse/1%magncsi ion sulfate solutiwi wtws tfmn added to tlm bottles Thctwo bottles were inoculated i&1th dilTereot amowits ol'eedThe bottles were incubated at 3(i "+2" C iuitil tlm nuxfiumtumed yells Follov'in incubation, samples v cre removed cifrom each bottle and tested for optical density (OD) (0.3 to0 9)) and pH (4.(i to 5.5). One of the two bottles wos selectedfor inoculation ol the seed fermentor.

Soy-based medium was transferred to the seed fennentorwid stcrflind Thmi a volume of 50/v dextrose/1% magna- c

sium sulfate solution was added to the fermentor. I'he pl i andagitation of the seed fennentor were monitored and con-

trolled (pH 6 7 to 7.4) The temperature wos maintaincxi nt3(i" +2" (2 'Hie seed inoculum (bottle) was asepticnlfy con-

nected to the seed fermentor ond the inoculum was trans-ferred. The fennentor was maintained in pH control ondswuples were penodwafly renuived and tested for OD aod

pH. When the desired OD of 0.5 at 600 nm was reached. theintcrmcdiate femientor ives inoculwed with the li.rmwitatmnbmth from the seed femientor.

Soy-based mediiuu wos trwisferred to the intermediateli mien&or and sterilized Then a volume o(50'/v dextrose,'1%magnesium sulfate soluuon v'as added to the fermentor. ThepH and ngitation of thc internwdiate fimncntor were moni-tored and controlled (pH 6 7 to 7 4). The temperature wiismaintwned at 36 "+2" (2 'I'he contwits of the seed femientorwere transferred to the intermedi etc feniwntur I'he femwntori&as maintained in pl I wmtml a&xi swnples were periodicallyremoved and tested for OD and pH. When the desiried OD of0 5 at 600 nnl i&as reached. &he production fi.'rnientor ivasinoculated with the fermentation broth from the intermediatefemientOr

Soy-based medium was transferred to the productioti fer-

nientor and sterilized. Then o volume of 50/'v dextrose/I'/vmagnesium sulfate solution was added to the fcnnentor. 1 bc

pl I and agitauon of the production ferment or w ere mo tutoredand controlled (pH 6.7 to 7.4). The temperature i&os main-tained at 36''. The femientor wtws maiotained in pHcontrol and san&plea were p eood &cally removed and tested forOD and pH. until the fern&co&ation was complete

Deoxycll&ilatc sod&&lilt wils ill&led to the fermentor to a linulconcentration of approximately 0.12 "/v w /v. The culture wasmixed fiir a mininunn of thirty minute»i and thc tcmperaturcset pouit was reduced to 10'. The culture wns incubnted

overnight and follnwing confirmation of inactivation, the pH0 1'lc cillt ore lv'is adjusted to between 6 4 and 6 8. as neces-sary, with 50% acetic acid. The temperature of the fennentorwas increased to 20'+5" ('nd the contents were transfi:rredto the clarification hold tank.

The contents of the claniication hold tmd (including thecellular debris) w ere processed tlmsugh n centrifuge ot a tlowmte between 25 and 600 liters per hour (except Serotype 4,wlierein the cell debris was discarded ond the flow rate tight-ened to bctivecn 25 and 250 liters per hour) Samples of tlw

supernatant

ii ere removed and tested for OD. Ilia desired ODduring the centrifugation was ~0.)5.

Inn&ally. the supernatant was recirculated through a depthfilter assembly iuitil an OD off.05 0.03 was achieved. Theothe supcmatant v,tws passcdl through thc depth tilter assemblyaod tlu ough o 0 45 iun membrane filter to the Iihrate holdtank.

Subsequently. the product was transl'erred through closedpipes mi thc pllllilcatloll arc'I fot'rocessing.

All of thc above operations (ccntrifugntion, tiltmtion nnd

trwisfer) were perfomied bet~san 10' to 30'.For an alternate fermentation and harvesting process for

serotypes 4 and (iB. scc the alternate process described inExwnple I

Purilicatioo

Thc purification ol'ach pncumococcal polysacclwrideconsisted of several concentration/diafiltration operations,precipitation/elution, column chroniatogrnphy, and depth fil-

tration steps. All procedures were performed at mom tem-perature unless othe&wise specified.

('lariticd broth from the fi:rmentor cultures oftlm dcsirixl 5p&wnmvn)uc serotype was concentrated and dmhltered usinga 100 kDa MWCO tilter. Diaiiftration was accomplished

25

(js 7,955,605 B2

26using sodium phosphate bulfer at pH&90 l&iafiltnitionremovcxk the low molecularuvight medium components fromthe lunger molecular weight biopolymers such as micleicacid, protein aml polysacchmide

Tlmpolysaccharidewasprecipita&cd from thcconccntratedand diafiltered solution by adding HB from a stock solution togive a final concentnsnon of 1% IIB (w/v) (except Semtype23 Ii which had a final concentration. of 2.5%). Tile polysac-clmridc/HB precipitate was captured on a depth tilter and thc

&Ofiltrate was discarded. (Note: Serotype 14 does not precipi-tate; thereiilre the filtmte ivas retained.) Tbe polysacchandeprecipitate wiws resolubifizcd and eluted by recirculating a

sodium chloride solution tluough the precipitate-containingdc1&tb filter. 'Ihe filters were then rinsed witli additioimlsodium chlonde solution.

Sodiuni iodide (Nal) was addixk to the polysaccharide solu-tion front a stock Nal solution to achieve a iinnl mmcentrationof0.5% to precipitate FIB (except for Serotype 6B, which bada final concentration o1'0 25%) The prempitate is as removecl 9

by depth liltrauon Tile liltrate conuuned Ihe target polysac-charide. The filter uas discarded. The polysaccharide wasthen filtered tluou h a 0.2 iun tilter.

The polysacchande solution ives concentrated on a 30 kDaMIVCO ultrafilter and diafiltered with a sodium cliloride "-'olutum

The partially punfied polysaccharide solution was fiirtherpurilicd by tiltrati&m through a depth filter iniprcgnated withactivated carbon. Aller filtration, the carbon filter was nosedwith a sodium cldoride solution. The nnse was combuied ""

&lith the polysaccluiride solution. which was tlmn liltercdthrough a 0 2 gm filter.

11m polysaccharide solution was concmitrated on a 30 kl &a

MW('0 ultraiilter and the filter was rinsed ivith a sodiumchloride solution. The pH uas checked and adjusted to ""

7.0+0 3.

The ceramic bydroxyapntite (FIA) colmnn vila equih-brated is 1th sodium phosphate buffer containing sodium

chlor-

ide

untill

thc pH is 7 0+0 3 and thc conductivity u as 26+4 uSTbe polysaccharide solution iius then loaded onto the col- so

umn. Under these conditions. impurities boiuid to the resintmd the polysaccharide wtms rccovcred in tlm tlov; tfiroughfrom the cohmm. The polysacchande solution was filteredthrough a 0.2 tun filter.

The polysaccharide solution ives concentrated using a 30 ss

kDa MWCO filter. The concentrate was then diafiltered withWFI until thc conductivity was &15 FS

The diafiltered polysacchtmde soluuon u.as filteredthrough a 0.2 iun membrane filter into bulk containers anilstored at 2-8'. o

Fxmnple 14

Preparation of Pimuniococcal Saccharide ('Rlul&77

Conjugates For Serotypes 4. 6B. 9V. 14, 18C', 191'

and 23F

Activation Process

Thc diff&.'r&ait svroiypc saccharidcs folloivdil'fcrent path- mi

ways filr activation fftydrolysis or no hydrolysis prior to acti-vation) and conjugation (aqueous or l&MSO reactiims) asdescnbed in tlus example.

Polysaccharide was transferred from the bulk containers tothe reactor vessel Tlm polysaccharide uas then diluted in (WI1 and sodium phosphate to a final concentration range of1.6-24 mg/mL.

Step l.For serotypcs 6B, 9V, 14. 19F and 23F, pH wns adjusted to

pH 6.0+03For scrotypc 4. hydrochloric acid (0.01 M final acid con-

centration) was added nnd the solution was incubated lilr25-35 minutes at 45''('iydmlysis vas snipped by cool-ing to 21-25" (2 and adding I M sodium phosphnte to a targetof pH 6 7 0.2 An ui-process test was done to confirm mi

appropriam level of dcpynivylation.For smrotype 18(., glue&&i acc&lc acui (0 2 M flllal acid

concentration) was added and tlm solution was incubated for205-215 minutes at 94'''. Temperature was ihmidccrcased to 21-25''. and 1-2 M sodium phosphate v asadded loa target ol'pl168 02Step 2: Pcriodnte Reaction

The required sixlium peri odate molar cquwa lent s for pneu-mococcal saccharide activation was determined using totalsaccharide cmiten! (except lilr svrotype 4). For serotype 4. amtio of 0 8-1 2 molcs of sodium pcriodate per mole of sac-chande was used. With thomugh mixing, the oxidation reac-tion was allowed to proceed benvcen 16 to 20 hours at 21-25"

C. for all serotypes except 19F for wluch the temperature u.as'=.15" C.

Step 3: l)ltrabltrationThe oxidized saccharide was concentrated and diafiltered

with WFI (001 M sodium phosphate buffer pl I 60 for sero-type 19F) on a 100 kDa MWCO ultralilter (5 kDa ultrafilterfor 18C). The permeute was discarded and tbe retentate wusfiltered through a 0 22 gm tilter.

Step 4: I,i opl»izationFor semtypes 4. 9V, and 14 the concentrated sacchande

uas mixedl with ('RM197 mirrier protein, lillcd into glassbottles, shell-lm!Cn md stored at =-65'. The I'nizen con-centratixk saccharide-('RM,97 uas lyophilizixl and thenstored at -25''(.'or

sero&)&les 6B. 19F, and 23Fa specified mnount ofsucrose was added which was calculated to achieve a 5%+3%sucrose c&mcetl&ratiotl in illa conjugation reacii&m mixture.Serotype 18C did not require sucrose addition. The concmi-trated sacchnride was then tilled into glass bottles, shcll-frozmi and stored at =--65'. Ilie frozen concentrated sac-charide was lyoplulized and then stored at -25 "+5" C.

(.'Onjugation process

Two conjugation processes were used: aqueous conjuga-tion for serotypes 4, 9V. 14 and 18(1 and DMSO conjugationfor serotypes 6B. 19F and 23F.Aqueous ConlugationStep 1: Dissoluiion

For serotypes 4. 9V and 14, the lyoplulized activated sac-chande-('RM&7 lnixture was tlmived and equilibrated atnmm temperature. The lyophilizcxi activated sacclraride-

CRM&9 'u Is tllcll I'ecolls'll'tilted ill 0.1M sodblnl pllospll'I'tebutfer at a typical nitio of

11. of buffer per 16 24 g ol'saccharide for serotype 4 a&xi

9V

11, ol'buffer pcr 6-10 g ol'saccharide lilr svrotype 14

The reaction nuxture was incubated at 37''. unul totnldissolution fiir the scrotype 9V and at 23"+2'. for serotypes4 aud 14

For serotype 18C, tbe lyophil ized saccharide ivas reconsti-nited in a solution of ('RM„, in IM dibasic sodium phos-phate at a typical ratio of 0.11 L of sodium phosphate per I Lof CRM197 solution. The reaction mixture (8-1 2 g/L saccha-ride concmitration) was incubated nt 23"+2" C. until totaldissolution.

The pH v;as tested as an in-process control at this stage.

27

(js 7,955,605 B2

28Step 2: ('onjugation Reaction

For serotypes 4 and 9V, the coniugation reaction was iiu-tiated by adding the sodium cyanobomhydride solution (100mg/niL) to achicim 1.0-1.4 molcs sodium cyanoborohydridcper mole of saccharide. The reaction nuxmre w as uicubated 1

for 44-5Z hours at 37"+Z" (2 The temperature was thenreduced to 23'" C'nd sodium chloride 0.9% v.as added tothe reactor. Sodium borohydride solution (100 mgmL) wasadded to achieves I 8-2 2 molar equivalents of sodium boro-hydride per mole saccharide. The mixture w.as uicubated for ni3-6 hours at 23''. The mixnire was diluted with sodiumchloride 0.9/'9 and the reactor was rinsed. The diluted conju-gation nuxture was iiltered usin a 1.2 pm pre-filter uito aholding vessel.

For serotypev 14 mid 18C', the conjugauon reaction wasinitiated by adding the cyanoborohydride solution (100mg/mL) to nclucve I 0-1 4 moles of sodiuni cynnoborohy-dride per mole of sacchande. The reaction mixture was incu-bated for 12-24 hours at 23"+2" C'. I'he tempemture wasincrcnvud to 37'" (. mid lhe reaction was incubated for 9

72-96 hours. The temperanire was then reduced to 23'+2'.and 0.9/9 sodium chloride vvas added to the reactor. Sodiumborohydnde solution (100 mg/mL) was added to aclueve1. 8-2. 2 molar eqmva1ents of sodium borohydride per mole ofsacchnride. The mixture was incubated for 3-6 hours at "23''. Tile mixture was diluted with 0.9% sodium chk&ride and the reactor v,as rinsed. The diluted conjugation mix-ture was then Iiltered usmg a 1.2 luu pre-(ilier uuo a holdingvessel.Step 3 Ultraiiltration 100 kDa "(1

Tllchliluthxli olltu lulhulllllxturcwas collcclltl iledmiddla-filtrated on a 100 kDS MWCO ultrafilter with either a miiu-mum of 15 1'olumes (smrotypc 4) or 40 volumes (scrotypcs 9V,

14. and 18C) of 0.9% sodium chloride.llm pernmate was discardcxI

For svrotype 4. the retentatc wm lilteredi through a 0 45 pmfilter.

An in-process control (ziccharide content) was performedat this stepStep 4: HA Column puritication Sc

This step was only performed for thc scrotype 4 conjugateThe IIA column v;is first neutralized usuig 0.5M sodium

plxlsphate buffer (pH 7.0+0.3) nnd then ixpiilibrated with0 1/% sodium chloride Th'iltered retcntatc (scrotype 4) wasloaded onto the colunui at a flow rate of 1.0 L/min. The 4.

column was washed w ith 0 9'/9 sodium chloride at a flow rateof ==2.0 L/min The product ives then eluted with 0 5Msodiiun phosphate buffer at a flow rate of rg2,0 L/nun.

The HA I)action was limn concentrated and diaiiltered on n

100 kDa MWCO membrane with a nunimumof 20 volumes o

of 0. 9% sodium chloride. The permeate was discarded.Step 5 Sterile Filtrnuon

The retentate after the 1001 Da MWCO diaiiltration wasfiltered tfirough a 0 22 pni filter. In-process controls (sacclm-ride content. frhee protein. free saccharide and cymiide) were 1

performed on the filtered product. In-process controls onIiitcrhvi rctcntate werc pcrliinnCd ui dctcnninc whetlmr addi-tional concentmtion, duiiiltration, and'or ddution werenecdhxI to meet FB('argets. Tliese mid additional tests wererepcatchl 111 FB(. salllplcs shl

As necessary. the filtered conjugate was diluted with 00)'!9

sodium chloride in order to achieve a tinnl concentration ofless than 0.55 g/L. Release tests for sacchande contenh pro-tein content and saccharide:protein ratio were performed atthis stage 6

I'inally. the conlugate was filtered (0.22 pm) and filled into10 L stainless steel canisters at a typical quantity of 2.(i4

g/canister At this stage. yield, saccharide content, proteincontent, pH, saccharide protein ratio and lysiim content 11 ereperformed as in-process contnlls. Release tesnng (appear-ance, frhw proteui, I'rhx. saccharulc. midotoxin, molecular siredetermination, residual cyanide, saccharide identity, C'RM

197

identity) ivas performhxi at tlus stage.DMSO Coniu etonStep I; Dissolution

Thc lyophilizhxI activated saccharide serotyphs 6B, 19F,

23F and the lyophihzed CRM,7, earner protein v ere equili-bmted at mom temperature and reconstmited in DMSO. Thedissolution concentration typicafly ranghxl I'rom 2-3 grams ofsaccharide (2-2.5 g protein) per liter of DMSO.Step lh ('onjugation Reaction

Tile '11 "tlvltcd sacchallhlc still CRM1 9 7 earner protein weremixed I'or 60-75 mimnes at 23'+2" ('. at a ratio range of 0.6g-I 0 g saccltaridc/8 C'RM, » filr serotypes 6B and 19F or 1.2

to 1.8 g saccharide/8 CRM197 for serotype 23F.

The conjugation reaction was inittathx) by adduig thesodium cy moborohydride solution (100 iug/mi.) at a ratio of0.8-1 2 molar hxluivalmits of sodium cyanoborohydridc to onemole activated sacchande. WI'I ives added to the reactionmixture to a target of I /9 (v/v) and the mixture was incubatedfor over 40 hours at 23'+2'.

Sodium borohydride solution. 100 mg'mL (typical 1.8-2.2nailer equivalents sodium borohydnde per mole activatedsaccharide) and WFI (target 5%i v/v) were added to tlm reac-non and the mixture was uicubated for 3-6 hours at 23'" C.This procedure reduced tmy unreactedi aldchydes prcscnt onthe sacchandes Then the reaction mixture ives transferred to

a dilution tank containing 0.9'/9 sodium chlonde at &15'.Step IH 100 kDa Ultrafiltcation

The diluted conlugate nuxture was filtered tluough a 1.2

pm filter mid concentrated and diatiltered on n 100 kDaMWCO membrane with a minimum ol'15 volumes ol'0 9'/9

sodium chlonde (001 M sodium phosphate/0.05M NaCIbut)'cr was used for serotype 23F) The pcrmcatc was dis-curded. Thc retcntate was filtered thmugh a 0 45 pm tilter Anin-process saccharide content sample was taken at this stage.Swp IV DFAF, C'olumn Puriiication

I'his step was only performed for serotype 231(

The DLAH colunm v,as equilibrated witli 0.01M sodiumphosphate/0 05M sodiiun chlorido bulfer Thc fihered retcn-tate (serotype 2317 was loaded onto the column and washedwith 0.01M sodium phosplmte/005M sodium chloridebuffer. The colunui was then washoI v ith 0 01M sodiumphosphate/0.9% NaCI buffer. The product was then elutedhvith 0 01 M sodium phosphate/0 5M sodium chloride bu lfer.

Step V: 100 kDa UltmiiltrationThe retentate from 6B and 1917 was concentrated and dia-

iiltered with at least 30 volumes ol 0 9% soihum chlonde. Thcpermeate was discarded.

Tlm eluatc from serotype 23F was cohtcclttratcxI and diafil-tered with a mim noun ol'0 vohunes of 0 9% sodium chlo-ride. The pennhite vvas discarded.Step VI Sterile Filtration

The retentate after the 100 kDa MWCO dialliltration w.is

filtered through 0.22 lun filter. In-process controls (saccha-ride cmitcnb free protcm, free sacchnridc, residual DMSOand res idim 1 cyanide) ivere performed on the filtered product.ln-process controls on filtered retentatc were performhxI todeternune whether additional concentration. diaiiltmuon,anChor dilution were needed to meet FB('argets. These andadditionnl rests werc rcpcatcd in I'BC'amples.

As necessary, the filtered conlugate 11 as diluted v ith 0 9%sodium chloride to achieve a final concentration of less than

29

(js 7,955,605 B2

300.55 g/I. Release tests for saccharide content, protein contentand saccharideprotein ratio were performed at this stage.

Finally, the confugate was filtered (0.22 gm) and filled &nto

10 L stainless steel canisters at a quantity of 2.(i4 g/canister.At this stage, yield, sacchande content, pmteui content. pll,saccharide:pmtein nttio and lysine content were performed asui-processcontroln Rclodsetcituig(appearmiCc, frecprOtein,free saccharide, endotoxin, molecular size determination.residual cyanide, residual DMSO. saccharide identity andC'RMO„ idenuty) wni perliinned at this stage

Ex unple 15

Fornnrlation of a Multivalent PneumococcalCoafugate Vaccine

The final bulk concentrates ol'he 13 coniugatcs contain0.85"/o sodnun chlonde. Type 3, 6A. 71'nd 19A bulk con-centrates also contmn 5 mM sodium succinate buffer at pll5 8. The requirixf volumes ol'bulk concentratls &vere calo&&-

lated based on the batch vohune and the bulk sacclmndeconcentrations After 80'/o of thc 0 85'/o sodium chloride(physiological snluie) and the required uuount of succuiatebuffer were added to the pre-labeled formulation vessel, bulkcollci:&31&d1cs v clc added. The preparation was then stcrilciil tered through a 0 22 pm membrane utto a second containerby using a Mil lip ore Du rap nrc membrane filter unit. 'I lm tirstcontmncr wins wasfmd with thc renmining 20'/o oi' 85'/o

sodiiun el&for&de and the solution was passed tluough thesanm filter and coffcctixf into the second container. Tlm liir-nnilated bulk iias mixed gently dunn mid follow&a theaddition of bulk aluminum phosphate. Tire pH was checkedmid adjusted if necessary The formulated bulk product wasstored at 2 8" C.

'Ilia fornnrlated bulk product was filled into Type I boro-silicate glass syringes obtauied from Bee&on Dicknismi. Thevaccuie was monitored at regular intervals for turbidity tocnsurc the unifiirmity of thc filling operation Thc filled vac-

cuie (I'uml Product) ives stored at 2-8'.

L'xample 16

Inununogmiicity of thc 13-'1'slant ( onjugatc Vaccine

Tb date, the preclinical studies performed on the 13vf'n('accuie

have been in nnbbits. Studies RHTOI-0021 and//Hl'01-0036 were designed to independently examine theeffect of cbcnucal con)ugation of cupsular polystccharides(PSs) fmm S. Paeamonine to CRM, 9, and the effect of alu-minum phosplmte (Al I'04) adjuvant on the immune response

to the 13vPnC'accine in rabbits Tlmsc cffcccts were charnc-tcrized by nntigcn-specific FI,ISA for senun IgCi cmiccntm-tions and for antibody function by opsonophngocytic assay(OPA).Study "HTOI-0021

Study 4HTOI-0021 exmnined the ability of the 13vPnCvaccine iv&th AIPO, adjuvant to elicit vaccine serotype-spe-cific immune responses. The pneumococcal serotypes repre-sented in the 13vPDC vaccine uiclude types l. 3. 4. 5, 6A, 6B„

io 7F, 9V. 14. 18C. 19A. 1911 and 231. Secondary obfecuvesincluded m eval&mtron oi'he kinetics and dumtion ol'heantibody response New Zealand White rabbits were inunu-nized intramu scufarly at week 0 and week 2 w ith the plannedlnmian clinical dose ufo&&eh polysacclmndc (2 pg nf'each PS.

i- except 4 pg of 6B) formulated vuth or without AIPO4 (100gg/dose) Sera werc collected at various time points Scrotypespecific IgCi wns mmisured by ELISA and functional activitywas assesscxf by OPA

Table 3 shows tlm gcomctric mann titer (CiMT) achieved in

so pooled senun samples. I'ollowing two doses ol'he 13vPDCvaccine. A mtio of the IgCr'iMfs was uscxf to co&lip(&le

res ponsui from week 4 to iv eek 0. These (hiui demonstrate thatthe inclusion of AIPO4 in the 13vpn('ornn&lation elicitedhigher levels of Ig(i antibody in comparisnn to the same

; vaccine v, ithout adjuvmtt. Afthougt the anubody responseswere greater when AIPO, mas included in the fornuilation,these increases were not statistically significant.

F'unctional antibody responses were also assessed in rab-bits following inununization with the two 13vPnC formula-

Tn tions (Table 4). )&r'hen compuring vaccine fornnilations withor without ad 1u vent, lugher OPA GMTs mere observed in the13vPnC'+AIPO vacciim trcatmmit gmup. OPA titcrs wercdetected ut iveek 4 serum pools to all vaccine serotypes inboth groups. For the mafonty of the serotypes. OPA t&ters

",- niemsured at vvok 4 &&ere at leasi 4-fold higlmr than those at

week 0 (baseline)The kinetic responses to each of the 13vPn( vacrine sero-

typcs werc cvaluatcd fmm serum pools of both trestmcnt

gin&1 ps. IgC& titers to each sero type (vere measured from blooddn dniwsatwmekfiandweeks1,2,3.4,8.12,26,nnd39midtlmn

coulpalccl With tile except&&ill of si'.Ouype I, &lntibody

responses in animals receiving adjuvanted vaccine weresuperior to those that received non-adjuvnnted vacciim andpeaked at week 2 of the inununization schedule (data not

4- shown).Overall, tlm data indicato tfiat tlm 13vPnC'accine liirnm-

lated with aluminum phosplmte &s inununogen&c in rabbits,eliciting substantial mitibody responses to the pneumococcalcapsular polysacclmndes contained in the vaccuie and these

u responses are associated ivith functional acuvity. Theresponses observed to thc sevmi core serotypes follmiingutununization ivith 13vPnC+AIPO, are consistent with lus-uirical responses of rabbits to the heptavafent formulation.

TABLF. 3

Ross&& I C'rnnrunc Responses tomls) 1 unuinp,!&nnrumzsiion

&iitk Too Doses ol'.'-r,iten& Pneurnoorccst O&kcocon/us&re

Diluent ri ili ALPO„" 1 PnC 1 PnC'+ AI.PO&"

R&1&le Week4 Rsno W e&4 Parle

semi p» Week tr &Veek 4 Wkd Wt(l Week &1 &9" Ctr V;kd &Vkll Week 9 mrs'I ('1) Wk4 iVka

&&le: &rill 1 (l o 9,926 1 &9 Srr

&2, &&-12.211) (sn'2 2" 993l

31

(js 7,955,605 B2

TABLL 3-continued

32

R&bbn Igo lmmut&e Respnnses (CAITs) Feline tig Immunm&l onuithTno Oocesof tl valent Pt&eurnococcal ol&cncon u re

D luenr n tb )I PO "'1&i nC" 11&Pnt + 41 PO

R.'tt&o Wack 4 Rat o W ek4 R.&t 0

8 r type )Vccl u '4'eek4 O'Inwktl )Veal tl (9&'I) 9'14u'IU V'eekt& (9 'l) V'14'6'Ift

71

14

lsC

"31'.1&tft

elo&t

"I&tu alta&

e inn

141 elnfi

elf&tl c)DU

3,85&J 79

89 995

4'7 177

~ l&JD 'eo

:l&tf )UD

. I tnt

10&l lett

f& 4

f& 4

I 0

02

ID

I 0

10

«0

171

50

121

6,647i .77'l-l «&2)

tx 54fg.&&31-2Z87&l

&2A. fi.14 tt(91

22AI(11.9t!7-41.914)

8, ul811.&64 18A44)

4&, 91

&26,911 7&t, 57)

is 7N&

f, 193.34 616&

6,9f&6

isAIG-IR9(2&ging)

(1,7 7 (1- 2 8,7 2 5 i

113 599

(54 18.236,7rl14 16&

17.146 "8.&&9&tt

S121(1.894-14.962 &

271

117

3&J3

12&

lls

939

287

106

ss 16,443t7,&!96-18,1061

29.1113

(l&d42-&I0 16.714

(6 9 9-4(t.!4(&l

tu 6R734

( I 141-1& .1461

54 3, 5 tl S

ill,286 48,955)14'I 84,&i88

(46A45 15 .15 0&)8 4&. 31*

mb 6.71 ltli7ft 16 f&7G

I'I 649- 6,78)1.D 35.DGD

(24 708-49 6921

144 280.976(1 19 587 66&t,)6&)

Sit 243&12

(9R41 67,141)50 I &.041

(4, 11-4).ftltll

17

701

I WJ5 I

TABIig 4

,I pi&* rr fclfrrfe OPA 0 vl Is t'oi SZ)v R&bb&f Sent&&1

Pnols I olio u&g Ir&unuruzat&or& ith 1 o no&ca

o(1 -». lent I'ucuu&peacoat Ol cocun unate

a dose of 2.2 gg of each PS (except 4.4 go& of 6Bh Animalsreceived one of three vaccuie prep u &tions (a) 13vpnC (PS

directly conjugated to( RM&& 3 (b) 13VPIIPS, (f lee PSlor(c)13vPRI S+( RM»7 (free PS mixed with ( RM&9&j All vflc-

cine preparations contmned AIPO4 as the adluvantat 126

gg(dose

11& Pnr" 13& P&iC + AI PO'471.

9')

'4

1st:19419F

. 11

e8 64 16

e8 16 4es If(

8 128 16

8 2&G 64

16 3

8 256 32

8 n 64'8 Izfl 0

8 64 8

8 64 16

il 16

&8 12 128

8 I 648 l,&124 I 8

8 128 16

8 128 16

16 3':8 25G 64

8 I,n 4 2(68 &12 I?8

&8 56 64

Study )(IIT01-0036

Refit) Pnf&o

ser type )veel tl weel 4 wk4wkf& )veel f& v eek4 u'14 wk(tScrotype specific immune responses for all vaccine prepa-

rations were evaluated in an Ig(i L'LISA and complement-medtated OPA measuring functional antibody. 11)e mumuieresponses ivere conipared between the treauneut groups.

I'able 6 prcscnts CiMT data obtained from uvck 4 bloodsa&uily&id &n uuigen specilic IgCi EI,ISAs Addtttotud analy-ses show the ratio of OMI values at wueek 4 to week 0. Thedata indicate tlmt thc conjugate vaccine preparation elicitedgreater serum I Ci titers than I'ree PS or fr&m PSGC RMmnvaccine With the exception of 6 pueamoman type 14, the13vPnC'accilm was able to induce fimctional antibodies to

the representative strains of S (&ircamartian in an OPA (labiaCih After two imnnunzations with cddtcr the 13vPnPS or13vPnPS+CRM&97 vaccine, neither could ulduce OPA ttlersmg-fold at v;eek 4 relative to week 0 for 10 out of the 13

scrotypcs mcasurucd (Table l&)

60

Study aHT01-003(i compared rabbit inumme responses tothe polysuccharides (PSsj contauted in the vaccine, afterunmtunzatton with the I 3vPnO vaccine u ith or v ithout con-

lugation to thc C'RM„„protein N&:u 7(eland White rabbitswere immunized intramuscularly at week 0 and v;eek 2 with

In conclusion, these results indicate that con) ugation of the13-valent pneumococcal vaccine polysaccharides produceshiglmr serum Igfi titers mtd overall greater fimctional anti-body activity than seen with free polysacchande alone orfnlxefl lvlth unciullug(fied (- RM& 97.

33

US 7,955,605 B2

34

TABUI 5

Rabbit Ippi Responses(OC(Ts) to PnPS b ) I!)A I r I lou ing Immun rationu th T» n Doses nf I'f valent Pnenmncnccat Olvcncon n ate

I I'nPS fires P',(

I lvPnPS = CRNI»1PS mined ith C RA I PnC

C(eek4Serohp 'ipeek 0 (95', Cll

Ralio C(eek4 Ratio'IV1,4 Wkb W'eek il (9 ', Cl) Wkd 9,'l,il '8'cek f)

(9 "k4 Ratio19' ('1) WRA lpkl

6A

GB

14

18('9I

2)F

(0

154

5(

Cil

66

(WV

„9fi(843- .7'if))

(64-9081

)79(15(l-9'(9)

(11)-4C(i)

466()16-688)

727

()84 1,)7()61

I 9-9 )104

(48 19(i

298(117-7771

I '17

(t) ( 3,688)tl9

(44-179)1.)62

9-),3171

1.0tl

(4t(7- .4Bi)

(8 39

42 89

1 6 'itl

4 0

RU 9)l

11 6 6"

il

I

4 50

17( 66

18 0

61

149 121

1.9(9(Bti9-4.7 9)

16

174-3(8)

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167

174 39(i)

195,(71-C't()

i 61

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(31(l-2 6

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161

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)2 I (0

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I!A,414-16.67G)

I .89(l

(9.1!i-! b.724)

)5 SG4

124.467 (0.824)734 '14

(167.)(2-3 f!. 83)'3 99

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(12..'(5.'-33.)D)

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TdtBI.F, 6

5 pire'iiiiiotil i OPA Titcrs for Ra(bit Seruni Pnol» Pellouutp, Innnunizatron

uirh Tiio Doses et'13 valent Pneumoco cal Va" ne(

OPA Titerc

No

1)vl'nPS 4 C1MistIyi" P(rru«ediiith

I glvPiiC

1 7 icilrti eiil Ritii'ai iit Ral.io

Ccr type Weef: lr' ek4 (Vk4 W)0 Week 4 Wk4 (Vkb Week 4 (V84 Wl il

6A

7k

9V

14

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19A

191

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(js 7,955,605 B2

35Fxan&pie 17

Alternate Procedure for Serotype 19A PnemnococcalSaccharide/ CRM„, (.onjugation

Overview

The fit liow ing example describes a process for making annunnU&ogcnic col&JUga&L'compl1s&n Sfrepfocot'I'Ils /nit'If&lio-

n/Ue serotype 19A polysaccharide coi alently linkLUI to a car-rier protein. In vencral, fi&llowing periodate oxidation (acti-vation) to generate reactive aldehyde groups on thepolysaccharide, the serotype 19A polysacclmride was c&1-

lyophi lived &Lith the

earner

protein m&d con jugntion was cer-ned out in d&methyl sulfoxide (DMSO) via a reductive anu-nation mechanism in the presence of sodiumcym&oborohydnde. As opposed to coigtugat&on processesinvolving discrete lyophilizat&on of polysacchandes and car-rier proteins in DMSO, or processes involving aquenus co-lyoplultmt&on ofpoly sacchandes and earner proteu&s w&tlxiut

DlvlSO. the follow&ng process improvement providedunproved conjugation efiiciency nnd comrol oivr thc stabil&ty

of serotype 19A polysacclmrides as measured by molecularsize and the percentage of free saccharide. Although thisprocess is describLUI ft&r the sero type 19 A polysacchande. tlusprocess may also be used for serotypes that are structurallysimilar to scrotypc 19A, such as 6A, 6B. Bnd 19F which alsocontau& phosphod&ester linkages bet&veen fi&eir repeat units

Ac&tv&It&(ul

36'thc ctl lv(&phv'1&zctl ac&'&va'tLd polvsacchaf&ttc/( RM&vi &nate-

rial in I)MSO was perfornwd at 2 mg/mL in the 1yophili mtionbottles. The activated polysaccharide/CRM197 DMSO solu-

uon was then trm&sl'erred to the coifitugation vessel and stirredfor 60-75 min at 23B2''t 70-120 rpm With stirring. onc

molar Lxiuivalent of sodium cyanoborohydride was thenadded, followed by 1% WI'I. Thc IL"&et&on SURIt&tu& was thenstirred at 23 2', Ibr 8-16 bours One molar equ&valent ofsodium borohydride was addcxl to thc conjugation vessel for

I (Ithe capping reaction and the solut&on was stirred at 23 2'.for 3-16 hours. The reaction solution was then d&luted 10-foldin cold (2-8" C.j 0.&)vgs sodium chloride.

The 10-fiild d&luted react&on solution was passed tlu ough a1.2'5.0 iun filter and then concentrated to 1-2 g/L on anultraiiltratim& system cquippcd w&th a 300K or 1000KMWCO re enerated cellulose I ft membrane. A filter chal-lm&ge of 1-2 gmms of polysaccharide per ft ol'men&branc

area was used Iiir the purification A 10x d&aiiltratmn of thecoIljugate solution was then performed against 0. 9 "/o sodinnlcldoridc IJpon nILnibrane cleaning. a 30x diatihration of theconiugate solution tvas then parfum&ed ag unst 5 mM sodiumsuccinatef0.9/6 sudium chloride buffer (pH 6) The purifiedconjugate was then pnsscd through a 022 pm filter and

„- samplesforpre-ifBCtest&ngremoved/ihecontugatesolutionwas then stored at 5+3'2 for up to 30 days until prepamtionfor the I'u&al Batch Concentmte (I'BC).

Prcvaration of Final Batch Concentmte

Containers oi'vrotype 19A polysaccharide werc Ihawcdand comb&ned in a reaction vessel. Oxidation react&ons werepcrfiinned in 10 mM sodium acetate (pH 5) by the addition of100 mM sodium acctatc bulfcr (pH 5) at a polysaccharide ""

concentration of 2 mg/mL. Ox&dat&on was carried out &n thepresence of sodium periodate by incubation at 23+2" C fi&r

16-24 hours w&th 150 rpm nuxing.

Punficat&on ofActivated Polysacchande St t

The pre-FB('onjugate solution was diluted to a targetconcentration of 0 5 Jvl, using 5 mM sodium succinatc/0 9%sodium cldonde buffer. The solution &i as nnxed with mag-netic stirring for approximately 15 minutes and then passedthrough a 0 22 pm Iilter intti sterile polyp&opylene FBC'ottles.The FBC contugate solution was then stored at 53''.

Characterizat&on of the conjugate was performed asdescribed in Example 4.

( oncm&&ration and diafiltration of the activated serotypc19A polysacchande was performed w&th 30K or 100KMIV('0 I'all (.'entremets polysulfone I ft ultrafiltrabon cas-settes. A target membrane challenge of 2 gams of polysar.- SS

clmride per ft of membrm&e area was used for punfication ofthc active&cd polysaccharide Thc ultrafiltration system wasequ&l&brated in 10 mM sodium acetate buifer (pll 5) poor tothe addition of the oxidauon reaction solut&on. The oxidationreaction solut&on was then concentrated and diaiiltered SU

agmnst 10 mM sod&um acetate (pH 5). The activated polysac-cbaridc was tlmn stored at 5+3' fiir up to 14 days iu&til thcmaterial w ms compounded li&r lyoplulization.

Co-lyoplnbzat&on md ('onjugauon Process

Thc pH of thc activated scrotvpc 19A polysaccharide wasadi usted to pH 6.5 0 2 by the addit&on of d&basic IM sodiumphosphate. I'he activated polysaccharide was compoundedw&&h BUcn&sc Il& ii Iatlo of 25 gran1s of mlcrt&sc pLI'ian& oi st1

polysaccharide, followed by compounding with CRM&vf at I

0 8 saccharide/protein ratio. Ihficr shell freezing. the 100 mi.lass bottles were placed in -25" C. storage.

'Il&e bottles of co-lyophilized polysacclmride/CRM»1were rcnx&vcd from thc -25'. freezer m&d allowed to equili- Ibrate to room te&upemsture &n a lanunar fiow lx&od. The bottleswere randomly sampled for moisture m&alysis. Dissolution of

Co-Lyophyl&zat&on vs. Discrete Lyophyl&zat&on &n

DMSO

Twelve 1-6 gmm batches of serotype 19A conjugate w ereproduced. with six pmduccd using tlm co-lyophyilizationwith DMSO method described above and six pmduced usinvg

a discrete lyophyl ization with DMSO method as described &n

Fxnmple 8 above (.Imractt:rizatitm of tho conjugate was per-formed as descnbed u& Example 4, including use of sizeexclusion chromatography media (("L-4B) to

profile

th rela-t&ve molecular size distribution of the contugate and a uron&c

acid assay to measure saccharide concentration. With respectto thc king-tenn stab&lity oi'he serotype 19A co&fitugate, n

decrease in molecular size of about 10% w&th a concom&tantincrease in free sacclmride levels of about 10'/s is expectedover a period of 18 months Acrordin ly, in the product&on ofserotvpe 19A contugates, a preferred value for contugateuiolem&lar size is about 70% 0 3 Kti, with a prcii:rrcd frccsacchande level ofbelow about 20-25%

As shov;n in 'I able 7. characterization of the smrotype 19A

coniu ates pmduced by both methods showed that the co-

lyophylization process provided significantly improved con-

jugate chamsctcri sties in tern&s of both molecular size as wellas percentage of free sacclmnde us compared to the d&acrete

lyophylization process.

tjs 7,955,605 B2

37TABLL& 7

& ompaf)sonc,)t Kev emmmafe eh m&etensl os for Sefo&ype

is&co f o)h & iaf onmD»&SOV) D)safe&efvo hvf )moon

ro &.yof hyfoannn D corefelyoph to&innfn = 6&

Oha&.)a&ensue

Sfanda&d a&and»&

V&Mil Devlauon Moan De)la)flan

'4&" Kd fr&.-48) 6, 7 ss &3 &l

saoohandeFree Ss "&unde o&S ".1 3& O2f" I

REFI')REN( ES

1. Hausdorff W P, Bryant J, Paradiso P R. Siber Ci R. Which

pnmnno cue & el sero@cups m&use &1&e most iuvas&vc d&sc»sc

&mphcat&ons for conlugate vacc&ne fomn&lation and use,part I (7m /&ifrri D&s 2000; 30:100-21

2, I lausdorffW P, Bryant.1. Kiosk C. Pan&d&so P R. Si bar Ci R.

71&e contmbut&on of spec&lic pneumococcal serogroups toditT&:rent disvdsv. manifestatim&s implications Ior co»ju-gate vaccine formulanon and use, part I. C//» I»/eri Dis2000: 30 122-40.

3 Whitney (. G. Farlcy M M, Hadlcr J. e& al Decline inutvas&ve pneumoco)ccal &hsease after the uttroducuon ofpromin-polysaccharide conjugate vaccine New F»g/ ./Med 2003: 348(18) 1737-46

4. Black S. Slfinefield H. Hen san 3. et a i. Postl i censure evalu-ation of thc elTcLctivcness of seven vnlent pneumococcalcor&JB'te vaccine. Pedioir hi/pr/ Di& J 2001; 20: 110&-7.

5. Robinson KA, Bauglunan W, Rotluock (i, et al. L'pidemi-

olf)gy of invasive Sirepiororcns p»eumo»inc inf&.ctions inthe United States. 1995-1998: Opportu&utes for preventionin the conjugate vaccine em J IM4 2001: 285:1729-35.

6 Bu&ler 3, Breiman R. I.ipman I L et al Serotype d&stribut&on

of Sil'e/yiororriis pari&ufo»foe u&feet&ons a»t&ong p&'cscl'&001

children in tlm I lnited Stn&es, 1978-1994. I/&ifrri /)is 1995.171:88&-9.

7. Whitney C Ci, Parley M M, Hadler J. et al. Increasingprcvnlence of multidru -msistm&t Sirepiororrus p»em»o-uiue &n the United States. NLr&&g/ JMed 2000: 343: 1917-24.

8 Hol'mann.l, C'etron M S. Farley M IVI. et al. The prcvalenccol'rug resistant Sirepior orr»s p»emu»»inc in Atl uun. N/'»g/ J Med 1995'33'481-(i.

9 Joloba M I„Windau A, Bajaksoudim& S, Appclbaum P C'lausdorlTWP,.lacobs M R. Pneumoc&mcal conlugate vnc-

cine serotypes of Sirrpiororcus p»sumo»&oe isolates and&hc nntimicrobial suscept&bility of such isolates in childrenwith otitis med&a. C//» fii/«7 Dis 2001: 33:1489-94.

10 Black S, Shincf&cid H, I-'ireman B, et al Efiicacy, safety,and unmunogenic&ty of heptavalent pneumococcal conlu-gatevaccineincluidren.Ped/oirInfectDis J2000;19:187-95

11. Rudolph K M, Parlonson A J, Reasonover A L. Bulkow L

R. Parks D J, Butler 3 C Serotype distribution and antim&-

It should be understood that the forego&ng d&scuss&on andexmuples nmrcly present a detailed descnp»on of certninembodinwnts. It therefore should be apparent to those ofordinary skill in the art that various mod itications and equiva-lents can be made w&thout depart&ng from the sp&rit and scopeol'hc invention

All joun&al article~. other references, patents and patentapphcat&ons that are identifie in this patent applicat&on areincorpnrated by reference in their entirety.

38crobial resistance patterns of invasivc isolntes of,&irrpio-rvrcusp»emnvuiov.Alaska, 1991-1998. Ji»/er&Dis2000;182:490-6.

12 Sniadack D H, Schwartr. B, Lipman H, et al. Potcntinl6 &ntervent&ons for the prevention of ch&ldhood pneunum&a:

geographic and temporal differences in serotype and sero-

group distmbution of stemle s&te pneumococcal isolatesfrom children: unplicntions for vaccine strategies. Pedi oirIofe& 7 Dfs.1 1995. 14 503-10.

I 3. Fags n R L. Hanna 3 N. Me sacr R D, Brooke s D L„MurphyD M. The epidemioloooy of invas&ve pneumococcal diseasein childrm& in Far Nor&h Queensland ./ Pocdioir (.6//dHe»if/i 2001; 37:571-5.

14 Kntesx DA. Di Fabio.l IsdcC'unto Brm&dileone M(., er

Bl InvBB&vc Sirepio&'ocru& p&7&'lu»o»lu&'&feet&on h& I,atu&

American children: results of the Pan Junerican HealthOrgm&ization Su&vcillancc Study C//» fiiferr Dfs 1998,26.1355-61

15 Hausdorff W, Siber Ci. Paradiso P Cicm)graphical differ-sn m&ces in invasi«'.'pncumococcni d&sL»sc &ates &u&d acro&ypc

frequency in young cluldren. Lancet 2001; 35 / 950-52.16. Buckingham S ('. King M D, Miller M I,. Incidence and

enolo ies of compl&cated parapnemnonic efihs&ons &n chil-dren. I')96 to 2001. Pedi»fr/»fer&D/s J2003: 22:499-504.

17. Byington ('. Spm&ccr L, Jolmson T, et al. An epidemio-lo ical invest&gation of a sustau&ed lugh rate of ped&a&Dc

pampneumonic empyema: risk factors and microbiologi-m&l associations. ( I/o Infer& Dis 2002, 34 434-40.

18. Tmt T, Mason i'ald L', et al. Cl&nical chantctenstics with"6 con&plicnmd pneumonia caused by Sirrpiororrusimrumri-

»ioe Pediuirirs 2002: H0,1-619. Block S L, I ledrick I, liarnson C J, et al. Pneumococcal

serotypes from acute otitis media m rural Kent&mky Pedi-

»fr 1»feei Di 3 J 2002; 21 859-6&."» 20. Hm&sdnrff W P, Yothers Ci, Dagan R, et al. Multinational

st&dy ol pne&unococcal scrotypcs cm&sing acute otitismedia in cluldren Ped/ofr I»/eri Dis f 2002, 21 1008-16.

2 i. Robbins I B. Austrian R, Lac(..ls et al Considerations forI'ormulatin tlmcmcond-genernfionpncu&nococculcapsular

nn polysaccharide vaccine vvith mnphasis on the cross-reac-tive types within groups ./ /&ifi. ri D&s 1983, 148 1136-59

22. Nahm M II, Olander J V Magyarlaki Ivb Ident&lirttion ofcross-reactive antibodies with low opsonophagocyticactivity for Sirrpioro& roe p»eru»omoe,/ i»f'err Dis 1997;176:698-703.

23. Yu X. Cirey B, Chang S, Ward J l. I idwards K M. N ahm MII Immunity to cross-reactive serotypes induced by pneu-mococcal conjugate vaccu&es in infants. J Io/krf Dis 1999);

180 1569-76

o 24 Vakevauten M, Eklund C, Eskola J, Kayhty H, Cross-react&vity ofantibodies to type 6B and GA polysaccharidesol'irepiurorrus posu»u&mue. evoked by pneumococcalconfi&gate vacc&nes, u& u&fants. I I»/eri Di& 2001; 184:789-93

6 25 Lkstrom N, Kilp& T, I.dhdenknri M. I,ehtonen H. AhmanH. Kayhty, H. Immune response to cross-reacting pneumo-coccal scrotypcs 6A/6B and 19A/19F in tlm FinOM »ac-e&ne tnal. Third World of C&x&grass of Ped&at&SC Infect&ousDiseases, Santiago, ('bile, Nov. 19-23, 2003.

6&& 2(& PennR I„I cw&BF B.Douglas R(i,3r.. SchifliuanCi, I LL

C J. Robbins J B. Antibody responses in adult volunteers topnc&unococcal poly sacclmride types 19F and 19A admin-istered singly and &n combu&anon. I»/cc/ Ioo»mi 1982;36:1261-2.

27 (iicbi&d& (i S, Mcier I D, Quartey M K, l,iebclcr C I„ i,c CT. Inununogen&dty and efiicacy ofSiren/oroci us p»em&m-

moe polysacchande-pmtein conjugate vaccines against

39

(js 7,955,605 B2

40homologous and heterologous scrotypes in the chinchillaot&t&s med&a model. I Infect Di s 1996; 173; 119-27.

28. Saeland k.', Jakobsen H, lngolfsdottir (1. Sigurdardottir S

T, .Ionsdottir I Serum samples from infants vaccinatediv&th a pneumococcal coniugnte vaccu&e, Pncl; protectmice against invasive infection em&sad by Srreprorocc»ip»e»n(»nine serotypes 6A and 6B. JI»/kcr Dis 2001; 183.253-60.

29 Jakobsen fi, S&UUI'ilsso&I V D, S&gilrdardottir S, Schulr D,

Jonsdou&r I Pneumococcal serotype 191'ontugate var.-

cine induces cmss-protective inumu&ity u& serotype 19A ina murinc pneumococcul pneumonia model. Infect Ammm

2003: 71:2956-9.30 Klugmmi K P. Madhi S A, Huebner R P„Kohbergcr R,

Mbelle N. Pwrcc N A trial of a 9-valent pneumococcalconjugate vaccine in children with and those without HIVinfi:ction VF»gl.I(ffed 2003, 349 1341-8

31. O'Bnen K I,. Moulton I, IL Re&d R, et al I:ilicacy andsafety of seven-valm&t conjugate pneumococcal vaccine inAmcricmi Indimi children: group rmidoniiscd trial. I r»Ice&

2003: 362:355-61.32. kskola J. Kilpi 1, Palmu A, et al. kiff&cacy of a pneunxi-

coccel conj uolte vacmne aga&nst acute ot&tis med&a. IygnglJMed 2001: 344:403-9.

33. Pili shvili T. Pmley M. Varrf &&or M, Rcingold A, Ny(luis&A,et al. Bffect&veness ofheptavalent pneumococcal conlu atevaccine in children. Abst O-1079. I('M(:, ('hicago, Ill..2003.

34. U.S. Pat. No. 4.673,574.35 U S Pat No 4.902,506.

%1&at is claimed is:I A nwthod fiir making an imnnmogcnic conjugate com-

prising Srreptococc»s pueunnr»iue scrotype 19A polysaccha-ride covalmitly linked to a carrier protein, thc method com-

prising:(a) reacting punfied serotipe 19A polysacchande u &th an

oxidizing agent resulting in an activatixl scmtypc 19A

polysaccfiande.(b) compounding the act&vatied semtype 19A polysacclm-

ride with a carrier protein,(c) co-lyoplul&z&ng the compounded activated serotype

19A polysaccharide and carrier protein;(d) rc-suspending the compounded activated scrotypc 19A

pofysacebande and carrier pmte&n u& dimethyl sulfox ide(I&MSO)

(e) reacung the compounded. Oct&vs&ed serotype 19Apolysaccharide and carrier pmte&n with a reducing a»Cent

resulting u& a serotype 19A polysaccluiride earner pro-te&n coniugi&te; and

(f) capping imreacted aldehydes in the serotype 19Apolysacchande:carrier protein conjugate resulting in anUIUUUnogcn&c co&11&lg'itc coulp&ls&ng SII'17&roc»i'I" uspnemnoniuc serotype 19A pi&lysacclmride covalentlyliukcxf to a camer prote&n: whereu& siep (c) &s performerlnher step (b) and before steps (d) and (e).

2 The method ol'laim 1, wlmrein the carrier protein isCRMI()7.

3.

firn

method of claim 2, ivhcrein the activated serotype19A polysacchinide and ('RM197 ari con&pounded at a mtioof 0.8: l.

4. The method of clnim I, wherein thc pH of the activatedserotype 19A polysacchande &s adjusted to 6.5 0.2 prior tocompounding v, ith the carrier protein.

5. Thc nwthod of claim I, wlmrein thc activated serotype19A poiysacchande &s compounded w&th sucmse prior tocompounding with the carrier protein.

6. The merhod of c lain& I, fur tlmr comprising purifying thei&un&unogcnic coniugiltc.

7. The method of claun I, wherein the ox&d&zu&g agent is

so(frit&rl pinodaIc8 The nmtlmd ol'claim I, wherein the reducing agent is

soditm& cyanoborohydnde.9 The method ol cia&m 1. v'hereu& mppu&g unreacted alde-

hydes composes reactu&g the semtype 19A polysacchaude.carrier protein conjugate u:ith sodiiun bomhydride.

in10. A method for making an unmunogeruc coniugate com-

prising S&rcyrucoccui y»enmo»inc serotype 19A polvsaccha-ride covalently lu&ked to a carrier protein, the method com-

prising,

(a) reacting purified serotype 19A polysaccharide withsodium penodate result&ng &n an act&vated serotype 19 A

polysaccharide,(6) adjusring the pH of the activated sero type I 9A polysac-

charide to 6.5+0.2:

&o (c) compo&uidu&g the activated serotype 19A polysaccha-ride w&th sucrose;

(d) compounding the activated serotype 19A polysaccha-ride w&th a earner protein at a mtio of 0.8: I;

(e) co-lyopluhzu&8 the compounded act&vated serotype19A polysaccharide nnd carrier protein.

(f) rc-suspending thc compounded activated scrotypc 19A

polysaccharide and carrier protein in dimethyl sultiixide

(DMSO):

(g) rencung the compounded, acuvated serotype 19A"o

polysaccharide and carrier protein v, ith sodiiuncyanoborohydride resulting &n a serotype 19A polysac-charide:earner protein confugate; and

(h) cnpping unrcactcd aldchydes in tlm scrotypc 19A

polysaccharide:carrier protein conjugate with sodiumhorohydride resulting &n an immunogenic conlugatecou&pr&su&g,u&vp&ococcui puca»&omne scmty'pc 19A

polysaccharide covalently linked to a carrier protein,wherein step (e) is perfom&ed after step (d) and before

sn steps (f) and ( ).

11 The mithod ol'clmn& 10. ulmrein the carrier protein isCRM197

12. fhe method of claim 10, further comprising purifyingthc &iumiinogcn&c conJUgatc

ss 13. A method fiir mak&ng an uumunogeruc coniu ate com-

prising a Streptococcus pueimio&i&ue polysaccharidecovalently linked to a carrier prote&n wherein smd pofysac-chande comprises a phosphodiester linkage between repeatunits, ihe metlmd composing.

(a) reacting said polysaccharide with an oxidizing agentresulting in an activated polysaccharide:

(b) compounding the actuated polysacchande v, ith a car-

rier proieu&,

(c) co-lyophiliring the compounded activated polysaccha-:(

ride and camer protein:

(d) re-suspending the compounded activated polysaccha-ride and currier prote&n in d&methyl sulli&xide (DMSO),

(e) reacting the compounded. nctivated poly sac chnride and

carrier protein with a reducing agent resulting in a

polysacchande.carrier prote&n coniugate, and(I') capping unrcactid aldel)ydes in thc polysnccl)aude

carrier protein mmjngate resulting in mi immnnogeniccontugate comprising Strep&ococcus pneum»ninepolysaccharide covalently fi&dCcd to a cmricr protein;where&n step (c) is performed after step (b) and beforesIi'.ps (d) and (c).

41

US 7,955,605 B2

4214. The method of claim 13, wherein snid polysaccharide

compristn a phosphodtester leakage between repeat units isStrefnocorcus pneumoniae polysaccharide serotype 19A,191'1 6A, or 6B.

15. The method el'claim 13. wherein the carrier protein isCRM197.