sabin-ipv development in imb, cams, chinawho encouraged developing sabin ipv new manufactures are...
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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!