Development of Cell-Based Influenza Vaccines for Pandemic ...nhidong.org.vn/Data/bvnhidong/bvnhidong/... · Mancera Gracia, J. (2017). Novel insights in the adaptation of avian H9N2

National Institute of Infectious Diseases and Vaccinology, NHRI 1

Alan Yung-Chih Hu（胡勇誌）, Ph.D.

Assistant Investigator

National Institute of Infectious Diseases and Vaccinology (NIIDV), NHRI

MOHW

NHRI

Industry

Development of Cell-Based Influenza Vaccines for Pandemic Preparedness


National Health Research Institutes (NHRI)

Established in 1996, and moved to Zhunan campus in 2004over 1600 people

Bioproduction plant


2009 pandemic H1N13

Mancera Gracia, J. (2017). Novel insights in the adaptation of avian H9N2

influenza viruses to swine.

European “avian-

like” H1N1

“Classical” swine

H1N1

Human seasonal

H3N2

North American

avian

North American

“Triple

reassortant”

H1N2

2009 pandemic H1N1


Timeline of influenza virus occurrences in human

4

Spanish Influenza Russian InfluenzaSwine

InfluenzaAsian

Influenza

1918 1957 1968 1977 1997 1998 2003 2009 2013

Hong Kong Influenza

2 lineages

H1

H2

H3

B

H5

H7

H9Avian Influenza

Se

aso

na

l

Influ

en

za

Pa

nd

em

ic

po

ten

tia

l

H7N9 human case

Survive Death

H5N1 human case

Survive Death

http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/

http://www.fao.org/ag/againfo/programmes/en/empres/h7n9/situation_update.html

http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/

http://www.fao.org/ag/againfo/programmes/en/empres/h7n9/situation_update.html


Current egg-based vaccine production

5

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV

Virus selection• Seasonal flu: announced by WHO in Feb

Production begins• Seed virus

• Large-scale production

https://medicalxpress.com/news/2009-10-production-swine-flu-vaccine.html

https://www.medindia.net/health-infographics/images/flu-vaccine-production.jpg

Partridhe J et al., 2010, Vaccine

FDA tests

yield, purity,

potency

Vaccine

licenced

Fill and

package

Distribution

Vaccination begins

2.46E+09

5.34E+08

0.00E+00

1.00E+09

2.00E+09

3.00E+09

6 month

estimated amount actual production

https://medicalxpress.com/news/2009-10-production-swine-flu-vaccine.html

https://www.medindia.net/health-infographics/images/flu-vaccine-production.jpg


Pandemic Influenza Vaccine Preparedness

Fast response:• Surveillance• Seed viruses• Sterility tests• Potency-test reagents• Fill-and-finish

• Before 2009: NHRI responsible for pandemic preparedness

• After 2009: NHRI acts as a backup for pandemic preparedness• Adimmune (egg_based)• Medigen (cell_based)


Process Development at NIIDV since 2005

7

2.2 L

7.5 L

50 L

150 L

microcarrier

BioNOC(Medigen)

200 L

R&D (7F) Bioproduction plant

Hyclone 50L

Sartorius 50L

Cultibag STRWave

bioreactor

aMDCK cells H7N9

50 L Bioreactor

Clarification

Virus inactivation

Concentration

Liquid Chromatography

Diafiltration/concentration

Formulation & Filling

Product

Bulk

Improvements updated:

1. Industrial scalability

2. Reduce bioriskand increase biosafety

3. Lower host cell protein/DNAresiduals

4. Higher recovery rate

1st generation: 60L roller-bottle technology

2nd generation: 50L bioreactor with microcarriers


Bulk specifications between H5N1 and H7N9

QC test items Spec. H5N1 H7N9

Sterility No growth Pass Pass

Endotoxin content < 100 EU/ml Pass Pass

Plague assay No growth Pass Pass

TCID50 No CPE Pass Pass

Egg embryo culture (in vivo) No growth Pass Pass

HA titer > 1024 4096 8192

SRD (HA protein content) μg/ml 36.41 61.73

Total protein content μg/ml 251.033 224.7

Total protein/HA content ratio < 6 6.89 3.64

Residual DNA content < 10 ng/dose 22.04 0.84

Residual benzonase content < 0.156 ng/dose Pass --

Formaldehyde content < 200 μg/ml 113.88 3.34

Sucrose content < 2% 1.3 --

http://www.google.com.tw/url?sa=i&source=images&cd=&cad=rja&uact=8&docid=id_4dIARS9m7YM&tbnid=kcOHzdeJ40kjzM:&ved=0CAgQjRw&url=http://www.epochtimes.com.hk/7/6/21/46748.htm&ei=vNUfU9_YHsWfkwWanYGoCg&psig=AFQjCNHbI3ii0R0T4hCinnOJz7q1zbYUjg&ust=1394681660567332




The development of sMDCK cells

aMDCK Bank (P62)DMEM+5%FBS

Direct adapted into SFM

Attached on Cytodex1 microcarriersSerial ad

apted

into

M1

Med

ium

+ <

0.5

% F

BS

A-M

DC

K ad

apted

to free su

spen

sion cu

lture

sMDCK Bank (P67)Freeze medium

Free suspension culture

Serial adapted into M1+M2+M3

sMDCK Bank (P70)M1+M2+M3

Virus Productivity of aMDCK

HA(units/ 100ul)

log10 TCID50/ml

H7N9(RG268)

574.0 7.6

H5N1(RG14)

612.7 8.6

Virus Productivity of sMDCK

HA(units/ 100ul)

log10 TCID50/ml

H7N9(RG268)

996.3 7.9

H5N1(RG14)

989.0 8.6

Attached MDCK Bank (P62)(DMEM+5%FBS)

1. Attached on Cytodex1 as

suspension culture

2. Culture Medium: SFM

3. Doubling time: 30~35 hours

50µm 50 µm

Suspension MDCK Bank (P70) (M1+M2+M3)

1. Free suspension culture2. Culture Medium: M1 + M2 +M33. Doubling time: 30~35 hours


Virus replication test (3rd-10th passages)

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

0 10 20 30 40 50

Ce

lls/m

l

Day

Cell growth

012345678910

,0,128,256,384,512,640,768,896

1,0241,1521,280

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

Log

10

TCID

50/m

l

HA

un

its/

10

0u

l

HA

DPI

H5N1

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

-5 5 15 25 35 45

Ce

lls/m

l

Day

Cell growth

012345678910

,0,128,256,384,512,640,768,896

1,0241,1521,280

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

Log

10

TCID

50/

ml

HA

un

its/

10

0u

l

HA

DPI

3rd 4th 5th 6th 7th 8t 9th 10th3rd 4th 5th 6th 7th 8th 9th 10th

H7N9


Tumorigenicity study in nude mice

• FDA guidance for industry:

– Test animals: BALB/c nude female mice

– Age: 8-9 weeks old

– Test cells:

✓ Positive control cells (Hela cells)

✓ ECP cells (No.1-4)

– Cell number: 1x107/1XPBS

– Injection site: between the scapulae

10 passages

aMDCK with OptiPro

Note: aMDCK was outsourced to the third party for characterization of a MDCK master and working cell bank. No tumorigenicity was found.


Improved downstream purification process

Clarification

Inactivation

Microfiltration

Concentration


Diafiltration

Sterile filtration

Harvest

Inactivation

Concentration


Diafiltration

Sterile filtration

Harvest 15K 30K 60K

HA (μg/ml) 122.7

Protein (μg/ml) 351.17

HA unit (HAU/50μl) 18428

Batch no: RG268-SM 20160524

The overall recovery yield: from ~ 30-35% up to 60-70%


Medium cost estimation in the USP

aMDCK(H7N9)

aMDCK(H7N9)

sMDCK2nd DSP

sMDCK3rd DSP

egg_based

MediumOpti-Pro

BalanCD MDCK

BalanCDsimple

BalanCDsimple

HA titer 438.1 512.0 797.0 1160

Medium required ratio(medium usage/harvest volume) 3.1 3.1 2.0 2.0one dose required HA (ug) 15 15 15 15downsteam recovery rate 0.3 0.3 0.3 0.7

US $ /dose 3.5 1.51 0.63 0.22 0.5~0.8

1L= ~400 doses

1000L= ~400,000 doses

US Patent filed (#62248954 )

PCT Patent filed （IB16/56567）ROC Patent filed （#105135227）

16X reduction


The development of pandemic candidate vaccine virus

step1•Collection of specimens and disease/epidemiological data

step2•Diagnosis, virus isolation in MDCK, primary analysis

step3•Ferret antisera production

step4•Thorough antigenic and genetic analysis

step5•Review and selection of candidate viruses for vaccine use

step6•Reassortment of high-growth viruses using reverse genetics (full safety

testing)

step7•Evaluation of growth property

step8•Development of standardized reagents for inactivated vaccines

step9•Antigenic and genetic characterization of reassortants

Vaccine production

Vaccine strain fromCDC/NIBSC/NIID(H5N1/H1N1/H7N9)

Seed virus (wild type)Like H5N2 project

Synthetic HA/NA genesWith 6 internal genes

StrainPreparation(1.5 months)

Import permit(1-2 months)

PlasmidPreparation(2-3 weeks)

ReverseGenetics(0.5 month)

ViralAdaptation(1-3 months)

Eggderived

Eggderived

Safety inFerrets(1 month/3 weeks) Cell

adapted

Virus

receive

d

Cellderived

Cellderived

3.5~6.5months

>2months

aMDCK_Passage 1(6 well plate)

ElectroporationValidated Vero cells

aMDCK_Passage 2 (T25)

6 internal gens from MDCK-based donor virus

aMDCK_Passage 3 (T150 for bank)

HA assay TCID50 assayHI assay

Highly pathogenic Low pathogenic

Polybasiccleavage site;

RNSPLRERRRKRGLF

Monobasic cleavage site;

RNSPLGETRGLF

Synthetic HA/NA genes of H5N6/H7N9 viruses

~2months


Viral titers for pandemic CVVs

Viral strain

H5N1

1st

H7N9H5N6

NHRI-RG1

A/Guangdong/17SF003/2016

NHRI-RG3

A/Hong Kong/125/2017

NHRIR-G4

A/Guangdong/SP440/2016

NHRI-RG5

A/Taiwan/1/2017

NHRI-RG6

aMDCK 612 574 689 128 256 256 128

sMDCK 989 996 1409 1024 1024 1024 1024

Risk Group level reduction: P3 –> P2


Summary

• A combined technology of a new adapted sMDCK cell line with chemical-define (CD) medium was developed.

• No tumorigenicity was found in the nude mice study.

• The sMDCK cells are shown higher viral titers in both pandemic strains.

• This new combined technology of sMDCK with specifically-optimized CD medium could provide a low-cost vaccine production to the bottlenecks for establishing a large-scale cell culture using adherent MDCK cells

Tech transfer: Please contact Irvine Scientific, USA


Acknowledgements

• VC & NIIDV directors

▪ Dr. Pele Chong

▪ Dr. I-Jen Su

▪ Dr. I-Chun Chen

▪ Dr. Ching-lenLiao

• NIIDV collaborators

▪ Dr. Min-Shi Lee

▪ Dr. Min-Shi Huang

▪ Dr. Jen-Ren Wang

▪ Dr. Jerry Sung

• Bioproduction

• Worldwide collaborators▪ US CDC/BARDA▪ JP NIID▪ TW CDC▪ UK NIBSC▪ AU VIDRL▪ TW

MOST/MOHW▪ US Irvine

Scientific▪ TW Medigen

Dr. ChengPostdoc

MayRA

Chia-ChunRA

Dr. TsengPostdoc

PinWenRA

Japan NIID

UK NIBSC

Flu research group (3 PIs) at NIIDV

SinYiRA

SinYiRA

Chia-ChunPhD

ChunHsiangMSc

https://www.isirv.org/uploads/100015_boardPhotos/100058.jpg

Development of Cell-Based Influenza Vaccines for Pandemic ...nhidong.org.vn/Data/bvnhidong/bvnhidong/... · Mancera Gracia, J. (2017). Novel insights in the adaptation of avian H9N2

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