SabinSabin--IPV Development IPV Development in IMB, CAMS, Chinain IMB, CAMS, China

Institute of Medical Biology, Chinese Institute of Medical Biology, Chinese Academy of Medical SciencesAcademy of Medical Sciences

Reported poliomyelitis cases in China(1953~1999)Reported poliomyelitis cases in China(1953~1999)

Eradication

OPV useOPV use

VAPP in ChinaVAPP in China

250250～～500 cases of VAPP worldwide 500 cases of VAPP worldwide

per year by OPV use. per year by OPV use.

There is not statistical data of VAPP in There is not statistical data of VAPP in

China since 2000 to present.China since 2000 to present.

Cases by VDPV in Guangxi

Last case of poliomyelitis by wild virus

One case by wild virus in

Yunnan

1995年1995年

1994年1994年

3 imported cases by wild

virus in Yunnan

1996年1996年

1999年1999年

2002年2002年 2004年2004年

1 imported case by wild virus in

Qinghai

Cases by VDPV in Sichuang Cases by VDPV

in Guizhou

2005年2005年

Cases by VDPV in Anhui

2006年2006年

2007年2007年

Isolation of VDPV in health children

Identification of Identification of VDPVsVDPVs in Chinain China

Isolation of VDPV in health children

Trend of OPV replaced by IPVTrend of OPV replaced by IPV

The 61th WHA Resolution requests the The 61th WHA Resolution requests the DirectorDirector--General General ““to develop appropriate to develop appropriate strategies and productsstrategies and products…………including safer including safer processes for production of poliovirus processes for production of poliovirus vaccine and affordable strategies for its usevaccine and affordable strategies for its use””

Poliomyelitis: mechanism for management of Poliomyelitis: mechanism for management of potencialpotencial riscksriscksto eradication The 61th WHA Resolution WHA 61.1 2008to eradication The 61th WHA Resolution WHA 61.1 2008

WHO encouraged developing Sabin IPVWHO encouraged developing Sabin IPV

New manufactures are encouraged to explore the production of IPV from the live attenuated polioviruses developed by Sabin as alternative and safer seed viruses than the currently used wild type viruses.Requirements for Poliomyelitis Vaccine (Inactivated) WHO TRS 910 Annex 2 Revised 2000

"Attenuated poliovirus strains (such as Sabin strains) …used for manufacturing IPV, do not require containment in BSL-3/polio facilities provided they are produced under conditions that would make them suitable for oral vaccine use."

WHO Guidelines for the safe production and quality control of IPV manufactured from wild polioviruses (WHO TRS 926, 2004)

Developing Sabin IPV in IMB, CAMS

Started in 1983Started in 1983

Used bioreactor from 1994Used bioreactor from 1994

Got approval for clinical trial in 2007Got approval for clinical trial in 2007

Started phase 1 clinical trial at Aug.2008Started phase 1 clinical trial at Aug.2008

Started phase 2 clinical trial at July 2009Started phase 2 clinical trial at July 2009

Phase 2 was finished at June 2010Phase 2 was finished at June 2010

Phase 3 is predicted to start at Feb 2011Phase 3 is predicted to start at Feb 2011

Establishment of production

process and quality control

Flow chart of production of Sabin IPVFlow chart of production of Sabin IPV

Vero MWCB37℃ 7 days

1st passage Vero cell culture in 3 L scale

37℃ 7 days, Trypsinisation

2nd passage Vero cell culture in 50 L scale37℃ 7 days, Trypsinisation

3rd passage Vero cell culture in 350 L scale inoculating virus

Virus culture33℃ 2-4 days

DownDown--stream stream processingprocessing

purificationpurification

Virus Virus harvestharvest

Trivalent Trivalent bulkbulk

InactivationInactivation

ClarificationClarification

ConcentrationConcentration

GelfiltrationGelfiltration andand IonIon--exchangeexchange

FinalFinal LotLot

Bioreactor 550L

Quality controlQuality control

VirusVirus harvestharvest：：SterilitySterilityMycoplasmaMycoplasmaTTitration of infectious virus (CCID50)itration of infectious virus (CCID50)Virus Virus Identity testIdentity test

SabinSabin--IPV IPV monovalentmonovalent pools pools ：：SterilitySterilityInactivation tests (on cell cultures)Inactivation tests (on cell cultures)DD--antigen test with ELISAantigen test with ELISA

SabinSabin--IPV trivalent bulk IPV trivalent bulk ：：SterilitySterilityDD--antigen test with ELISAantigen test with ELISAPotency by ratPotency by rat

Release tests for SabinRelease tests for Sabin--IPV final bulkIPV final bulk

Identity polio type 1, 2 and 3Identity polio type 1, 2 and 3AppearanceAppearancepH pH 22--PhenoxyethanolPhenoxyethanolFormaldehydeFormaldehydeTotal proteinTotal proteinResidual Vero DNAResidual Vero DNADD--antigenantigenBovine serum Bovine serum albuminealbumineSterilitySterilityEndotoxinEndotoxinAbnormal toxicityAbnormal toxicity

Pab

Rabbit anti Polio Rabbit anti Polio IgGIgG

anti-rabbit IgG-HRPO

Poliovirus

Detection of D antigen by using indirect ELISADetection of D antigen by using indirect ELISA

calf anti Polioviruses calf anti Polioviruses IgGIgG

D antigen Detection of WHO IPV Reference D antigen Detection of WHO IPV Reference 9191--574 (Type 1)574 (Type 1)

Comparison of DComparison of D--Ag contents of Sabin IPV Ag contents of Sabin IPV detected by our institute and NIBSCdetected by our institute and NIBSC

1703.11703.110001000SI071003SI071003

1053.31053.3825825SI071001SI071001

183.9183.9223223SI072001SI072001

558.9558.9700700SI073002SI073002

433.9433.9597597SI073001SI073001

213.0213.0200200SI072003SI072003

NIBSCNIBSCMy InstituteMy Institute

Rat potency test according to the Rat potency test according to the requirement of Salk IPVrequirement of Salk IPV44 standard dilutionsstandard dilutions : : undiluted, 1:3, 1:9, 1:27undiluted, 1:3, 1:9, 1:27To ImmunizeTo Immunize WistarWistar rats rats 10 10 rats/each dilutionrats/each dilutionBloodBlood sampling aftersampling after 21 21 daysdaysThe serumThe serum is foris for the virus the virus neutralizationneutralization testtest

NNeutralization eutralization test test with with Sabin Strains Type 1, 2 and 3Sabin Strains Type 1, 2 and 3

Immunogenicity, rat potency testImmunogenicity, rat potency test

Formulation Selection of Sabin IPVFormulation Selection of Sabin IPV

0

1

2

3

4

5

6

7

8

Ⅰ Ⅱ Ⅲ

WHO91/574

配比1

配比2

配比3

ED50

ED50

FormulationFormulation 1 : 40 32 60 DU; Formulation 2: 40 32 60 DU; Formulation 2 : 20 16 30 : 20 16 30

DU Formulation DU Formulation 3 :3 : 10 8 15 DU10 8 15 DU

Preparation of Sabin IPV product for Preparation of Sabin IPV product for clinical trialclinical trial

Virus suspensionVirus suspension

MonovalentMonovalent poolpool

Final lotFinal lot

Cultivation of poliovirus in 550 L bioreactorCultivation of poliovirus in 550 L bioreactor

TypeType Lot Volume MOI Temp. Time Titer Lot Volume MOI Temp. Time Titer (L) ((L) (℃℃) (H) ) (H) (lgCCID50/ml)(lgCCID50/ml)

SI00SI00--1001 370 0.025 33.5 66.5 8.401001 370 0.025 33.5 66.5 8.40ⅠⅠ SI01SI01--1001 353 0.010 32.5 68.5 8.001001 353 0.010 32.5 68.5 8.00

SI02SI02--1001 352 0.010 32.5 65.0 8.621001 352 0.010 32.5 65.0 8.62

SI00SI00--2001 362 0.028 33.5 75.5 7.852001 362 0.028 33.5 75.5 7.85ⅡⅡ SI02SI02--2001 358 0.011 32.5 67.3 7.632001 358 0.011 32.5 67.3 7.63

SI03SI03--2001 332 0.010 33.0 65.0 7.882001 332 0.010 33.0 65.0 7.88

SI00SI00--3001 380 0.019 33.5 59.5 8.383001 380 0.019 33.5 59.5 8.38ⅢⅢ SI01SI01--3001 344 0.019 32.5 64.2 7.753001 344 0.019 32.5 64.2 7.75

SI02SI02--3001 356 0.013 32.5 6.0 7.873001 356 0.013 32.5 6.0 7.87

Nine batches of Sabin IPV Nine batches of Sabin IPV monovalentmonovalent poolpool

Type Sterile Filtration InaType Sterile Filtration Inactivation ctivation Volume (L) Volume (L) DU/ml DU/ml

Start End Start End Start EndStart End

I 0.80 0.90 1590I 0.80 0.90 1590 14001400I 0.80 0.90 I 0.80 0.90 1300 12001300 1200I 0.80 0.90 I 0.80 0.90 1310 10801310 1080II 0.80 0.90 II 0.80 0.90 176 160176 160II 0.80 0.90 II 0.80 0.90 395395 380380II 0.80 0.90 II 0.80 0.90 370 360370 360

III 0.80 0.90 III 0.80 0.90 706 600706 600III 0.80 0.90 III 0.80 0.90 780 700780 700III 0.80 0.90 III 0.80 0.90 780 660780 660

Production of Sabin IPV for Clinical Trial

Lot 20080301 45 64 67.5 DU/dose/0.5ml20080301 45 64 67.5 DU/dose/0.5ml

Lot 20080302 30 32 45 DU/dose/0.5ml Lot 20080302 30 32 45 DU/dose/0.5ml

Lot 20080303 15 16 22.5 DU/dose/0.5mlLot 20080303 15 16 22.5 DU/dose/0.5ml

Results of 3 Lots of Sabin IPV Used for Clinical Trial Results of 3 Lots of Sabin IPV Used for Clinical Trial Detected by Detected by NICPBPNICPBP

PassPassPassPassPassPassPassPassAbnAbn. . toxicitytoxicity≤≤1010≤≤1010≤≤1010≤≤10EU10EU∕∕mlmlEndotoxinEndotoxin

PassPassPassPassPassPassNegativeNegativeSterilitySterility

PassPassPassPassPassPassLess than Less than 5050BSA BSA （（ngng//dosedose））

PassPassPassPassPassPassLess than Less than 100100Residual DNAResidual DNA

（（pg/pg/dosedose））

7.497.496.386.389.989.98Less than Less than 1010Total proteinTotal protein（（μμgg//dosedose））

0.0110.0110.0150.0150.0240.024Less than Less than 0.10.1FormaldehydeFormaldehyde（（mg/mg/dosedose））

4.54.54.84.84.64.63.53.5～～6.06.022--PhenoxyethanolPhenoxyethanol（（mg/mlmg/ml））

6.76.76.76.76.76.76.5~7.56.5~7.5pHpHPassPassPassPassPassPassOrangeOrangeAppearanceAppearance

PassPassPassPassPass Pass With polio typeWith polio typeⅠⅠ、、ⅡⅡ、、ⅢⅢ

D AntigenD AntigenIdentityIdentity

200803032008030320080302200803022008030120080301StandardsStandardsItemItem

Results of Results of DAgDAg Contents (DU) Detected Contents (DU) Detected by NICPBPby NICPBP

272720202626484840403636737390907575Detection Detection resultsresults

2020～～4646

1313～～2929

1212～～2828

4141～～9191

2727～～5757

2424～～4646

6161～～136136

5454～～114114

4848～～9292

Release Release rangerange

22.522.51616151545453232303067.567.564644545Intended Intended DAgDAg

contentcontent

ⅢⅢⅡⅡⅠⅠⅢⅢⅡⅡⅠⅠⅢⅢⅡⅡⅠⅠTypeType

200803032008030320080302200803022008030120080301LotLot

Stability Test of SStability Test of S--IPVIPV

((High. middle, and low doses)High. middle, and low doses)

50

60

70

80

90

100

110

DAg %

Ⅰ 型 Ⅱ 型 Ⅲ　型

4℃,3years

20080301

20080302

20080303

Brief summary of phases ⅠⅠand and ⅡⅡ

clinical trialsclinical trials

Protocol of clinical trial phase 1

Infant group research should be carried out after no serious reactions in adult and children and approved by Ethic committee

Preparation and quality control of High middle and low doses of Preparation and quality control of High middle and low doses of trivalent trivalent Sabin IPVSabin IPV

After getting qualified report issued by NIDBC and clinical After getting qualified report issued by NIDBC and clinical tr ial protocol approved by Ethic committeetrial protocol approved by Ethic committee

phase 1 130 persons

Infants 90 test group 45 each 15 for low ,middle and high doses 3 times immunization at 2,3 and 4 months Control group 45

Children 20 each 10 for middleand high doses sequentially

Adult 20 each 10 for middle and high doses sequentially

Safety evaluation and antibody detection in infant groups

Antibody detection before and after immunization

Determination of serum normal values and function of liver and kidney

Baseline characteristics by study groupsBaseline characteristics by study groups

00909090901.09:11.09:1434347478.28.276.676.6Infant Infant **

00202020201.22:11.22:19911110.50.52.32.3ChildrenChildren##

00202020201.5:11.5:188121212.412.437.437.4Adult#Adult#

Lost RatesLost Rates(%)(%)

Complete Complete NoNo

Study NoStudy NoMale:FeMale:Femalemale

FemaleFemaleMaleMaleSDSDMeanMean

ConditionsConditionsSexSexAgeAgeGroupGroup

**: Days for infant: Days for infant; ; #: years#: years

Adverse Rates (Adverse Rates (%) in Infants%) in Infants

23.3323.331.111.11270270636333TotalTotal

14.0714.071.481.48135135191922ControlControl

31.1131.11004545141400LowLow

17.7817.782.222.2245458811MiddleMiddle

48.8948.89004545222200HighHigh

SystematicSystematicLocalLocalLocalLocalSystematicSystematic

Adverse ratesAdverse rates（（%%））Total dosesTotal doses

Adverse reaction NoAdverse reaction NoDosageDosage

Safety

Phase 1 clinical trial indicated no serious adverse Phase 1 clinical trial indicated no serious adverse reactions occurred with three doses of high, middle, reactions occurred with three doses of high, middle, and low and low DAgDAg contents of Sabin IPV groups or in contents of Sabin IPV groups or in each of relative placebo control groupseach of relative placebo control groups

Phase 2 clinical trial indicated no serious adverse Phase 2 clinical trial indicated no serious adverse reactions occurred either with three doses of high, reactions occurred either with three doses of high, middle, and low middle, and low DAgDAg contents of Sabin IPV test contents of Sabin IPV test groups or in OPV and wild strains IPV control groups or in OPV and wild strains IPV control groupsgroups

Anti poliovirus neutralization antibodies Anti poliovirus neutralization antibodies titertiterGMTsGMTs (Titer ≥≥1:81:8 %% ) in test infants group (phase I)

878（100）n=15

543（100）n=15

5771（100）n=15

High(45 64 67.5)

1357（100）n=15

301（100）n=15

7051（100）n=15

Middle(30 32 45)

343 （100）n=15

180 （100）n=15

2318 （100）n=15

Low(15 16 22.5)

Type ⅢTypeⅡTypeⅠDosage (DU)

Results of Immunogenicity of Sabin IPV in Phase 2 Clinical TrialResults of Immunogenicity of Sabin IPV in Phase 2 Clinical Trial

97.80 97.80 70670690.11 90.11 19219290.11 90.11 38638691 91 ww--IPVIPV

100.00 100.00 545545100.00 100.00 410410100.00 100.00 3315331592 92 OPVOPV

93.26 93.26 30730778.6578.6510110196.63 96.63 1789178989 89 Low doseLow dose

98.91 98.91 49249295.65 95.65 15515597.83 97.83 2981298192 92 Middle doseMiddle dose

98.82 98.82 88488497.65 97.65 339339100.00 100.00 6335633585 85 High doseHigh dose

SeroSero--conversion conversion （（%%））

GMTGMTSeroSero--

conversionconversion（（%%））

GMTGMTSeroSero--

conversionconversion（（%%））

GMTGMT

Type Type ⅢⅢTypeTypeⅡⅡTypeTypeⅠⅠ

No of No of infantsinfants

GroupGroup

Immune Protection and Cross Immune Protection and Cross Neutralization TestNeutralization Test

There is the a little bit of difference reportedly There is the a little bit of difference reportedly between the between the antigenicitiesantigenicities of Sabinof Sabin--IPV and wild IPV and wild virus;virus;

Cross neutralization is able to provide the Cross neutralization is able to provide the confirmation for the complete immune protection confirmation for the complete immune protection of Sabinof Sabin--IPV;IPV;

The neutralization test is processing.The neutralization test is processing.

Future work:

A: Phase III is preparing and is going to start at Feb. 2011.

B: To establish the recognized standard quality control

C: Capacity of production will be extended.

Thank you!

Thanks to WHO for her kind help to improve the work of Sabin-IPV development in China!