Purification of cell-based influenza H5N1 viruses by ... · Purification of Cell-Based Influenza...

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Vaccine Technology IV Proceedings

Spring 5-24-2012

Purification of cell-based influenza H5N1 viruses by liquid chromatography technologies

Alan Yung-Chih HuNational Institute of Infectious Diseases and Vaccinology (NIIDV), NHRI

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Recommended CitationAlan Yung-Chih Hu, "Purification of cell-based influenza H5N1 viruses by liquid chromatography technologies" in "VaccineTechnology IV", Eds, ECI Symposium Series, Volume P17 (2013). http://dc.engconfintl.org/vaccine_iv/46

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Purification of Cell-Based Influenza H5N1 Viruses

by Liquid Chromatography Technologies

Vaccine Technology IV, May 24th 2012 , Albufeira, Portugal

National Health Research InstitutesNational Health Research Institutes

Alan Yung-Chih Hu, Ph.D.

Assistant Investigator

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

National Health Research Institutes (NHRI)

Since 1996

2National Institute of Infectious Diseases and National Institute of Infectious Diseases and VaccinologyVaccinology, NHRI, NHRI

2

Moved to this campus in 2004

�National Institute of Cancer Research

�Institute of Cellular and System Medicine

�Division of Aging-Related Musculoskeletal Diseases

�Division of Cardiovascular and Metabolism Medicine

�Division of Regenerative Medicine

�Institute of Population Sciences

�Center for Health Policy Research & Development

�Division of Biostatistics and Bioinformatics

Research Programs


3

�Division of Biostatistics and Bioinformatics

�Division of Gerontology Research

�Division of Mental Health & Substance Abuse Research

�Institute of Biotechnology & Pharmaceutical Research

�National Institute of Infectious Diseases and Vaccinology

�Division of Environmental Health & Occupational Medicine

�Division of Medical Engineering Research

�Division of Molecular & Genomic Medicine

�Center for Nanomedicine Research

Institution Objectives and manpower

�Improve the health and well-being

of Taiwanese

�Enhance the quality of biomedical

research and medical care

�Develop medical and


4

�Develop medical and

pharmaceutical technology

�Train and cultivate biomedical

researchers

�Promote health policy research and

implementation� Total: over 1600

� As of May 2012

Platform TechnologyDevelopment

cGMP Area

NIIDV Organization & StructureEffective, May, 2011

National Institute of Infectious Diseases National Institute of Infectious Diseases

and Vaccinology ( NIIDV )and Vaccinology ( NIIDV )

National Institute of Infectious Diseases National Institute of Infectious Diseases

and Vaccinology ( NIIDV )and Vaccinology ( NIIDV )

Vaccine R&D CenterDivision of Infectious

Diseases

PathogenicSurveillance Clinic Policy

13+58 12+125 ( 91 )


5

BC

G P

rod

uctio

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erin

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Fo

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r Pla

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An

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pro

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ss D

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rifica

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Qu

ality

Assu

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Re

gu

lato

ry A

ffairs

Ba

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rial V

accin

e P

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uctio

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Platform TechnologyDevelopment Regulatory

& Quality Management

Inn

ova

tion

M

an

ag

em

en

t

Strategic Planning

Management

•B

ud

ge

t Co

ntro

l •

Pro

cu

rem

en

ts

•P

roje

ct M

an

ag

em

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t

Qu

ality

Co

ntro

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Fa

cility

En

gin

ee

ring

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ou

se

an

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hip

pin

g

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e

Pro

ject

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cGMP Area

Production

Management

Facility

Management

•Te

ch

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r •

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an

ag

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rvic

e C

on

tract

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ics P

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Hig

h-th

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ue

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id

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tify n

ov

el a

nd

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kn

ow

n p

ath

og

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s a

nd

ne

w stra

teg

y

Mo

lecu

lar m

ech

an

isms o

f dru

g

resista

nce

, mo

lecu

lar p

ath

og

en

esis

Vira

l path

ogen

esis (E

V, In

fluen

za, H

IV)

Vira

l tum

orig

en

esis (H

BV

, HC

V, E

BV

)

TS

AR

/TS

AR

Y

Loca

l imp

orta

nt v

irus

(influ

en

za, E

V, H

IV)

Pathogenic

mechanisms

Surveillance

epidemiology

Clinic

trials

Policy

advocac

y

NIIDV programs

• Antibiotic resistance, microbial genomics and

infection control Advocacy

• Tuberculosis research and novel BCG/ TB

vaccine development

• Emerging virus infections and vaccine


• Emerging virus infections and vaccine

development

• Tumor viruses and therapeutic development

• Novel vaccine technology and bioproduct

development

6

Target Status 2012 Funding

Category 1:

Production

contracts from

Taiwan CDC

BCG Vaccine Taking over Taiwan CDC orphan

vaccine production

DOH/CDC

Antivenin downstream

production

Category 2:

Emergency

manufacture

Flu Vaccine Enhance national Bio-security and

capability to produce vaccine under

emergency and /or pandemic disease

DOH Regular

Funding/ IndustryEnterovirus : EV71 (B4/C4)

Category 3:

Developing

Meningoccoccal Group B

Vaccine

�Complete product development up

to phase 2 clinical trial and then

MOEA/ Industry/

DOH

Pipelines of NHRI Vaccine Center


7

Developing

vaccine with

commercial value

Vaccine to phase 2 clinical trial and then

technology and product transfer

�Meningococcal group B using

lipoprotein technology is going to

enter into Phase I in Q4/2011

DOH

HPV Therapeutic Vaccine

Dengue Vaccine

Novel Adjuvant

Development

RSV/PIV3 Combined

Vaccine

Category 4:

Novel vaccine and

technology

development

Recombinant BCG/TB

Vaccine

Vaccine platform technology

development and novel vaccine R&D

to assist, reduce the cost and

timelines and enhance the product

pipelines in Taiwan vaccine industry

Basic R&D by

NSC/NHRI/PP project

Universal pneumococcal

Vaccine

Bioprocess Development

Vaccine R&D : NHRI cGMP Pilot Plant


8

viralBacteriaBCG

Central

service

Pilot Plant layout


9

Filling&

packingwarehouse

P3 animal

facility

Biologicals


10

Source from: N Engl J Med; pp. 2540~2543, 2008

Our choice

Pandemic manufacturing strategy for H5N1 preparedness

NHRI

cell-base technology

200,000 doses/ 3months

Frontline workers

Adimmune


11

Adimmune

egg-base technology

(30 million doses/year)

The nation

(23 million people)

Adimmune / Medigen

Cell-ase technology

(capacity ??)

Roller-bottle process flow

Ba

tch

0

10

14

21

Cell Production Bank

850 RB x 15

1700RB x 100 (20L)

Virus Infection

Harvest and Clarification

63

75 T-flask x 5

850 RB x 3

75 T-flask x 1

23

Bioreactor scale-up


12

Ba

tch

27

28

35

56

63

Harvest and Clarification

Concentration & Host DNA Removal

Purification

Diafiltration

Inactivation

Formulation

Immunization

QC assay

24

25

23

63 days

Process Development in VRDC

2.2L 7.5L 30 L 150 L


13

(a) Confluent microcarriers (b) Cytopathic effect (c) Purfied Virus particle .

(a) (b) (c)

Various scale of microcarrier cell culture bioreactors in Vaccine center.

2.2L 7.5L 30 L 150 L

Single-use bioreactors

Wave bioreactor

Cesco 200L

TideCell


14

Hyclone 50L/200L SUB Sartorius 70L Cultibag STR

Downstream Purification using Continuous ultracentrifuge


15

Bezonase

Concentration

Bezonase

HA yield

residual Protein

residual DNA

Depth filtration

Flowchart of downstream purification scheme

Filter selection

inactivationinactivation


ultracentrifuge

SEC

Sepharose 4FF

Diafiltration

Diafiltration

SEC

Sepharose 4FF

AEX

Diafiltration

16

AEX

inactivation

inactivationinactivation

Depth filter selection


17

*The use of DE: diatomaceous earth (硅藻土) had

significant effect on DNA and protein removal

(DE) (DE) (DE) (DE) (PP) (DE+PP)

Depth filter test on large scale


18

DNA removal by different anion resins

0

100

200

300

400

500

600

0

500

1000

1500

2000

2500

0 2 4 6 8 10 12 14 16 18 ng

/ml

HA

/50u

l

ViralQHA dsDNA

0

100

200

300

400

500

600

700

0500

10001500200025003000350040004500

0 2 4 6 8 10 12 14 16 18 ng

/ml

HA

/50u

l

CV

QXLHA dsDNA


19

CV CV

0100200300400500600700800

0

200

400

600

800

1000

1200

0 2 4 6 8 10 12 14 16 18 20 ng

/ml

HA

/50u

l

CV

POROS HQ50 HA dsDNA

050100150200250300350400

0

10

20

30

40

50

60

70

0 2 4 6 8 10 12 14 16 18 20 22 ng

/ml

HA

/50u

l

CV

POROS HQ20 HA dsDNA

Optimization of size exclusion Liquid chromatographyH

A u

nit

s (/

50 µ

l)

HA

unit

s (/

50 µ

l)


20

HA

unit

s (/

50

HA

unit

s (/

50 µ

l)

HA

unit

s (/

50

HA

unit

s (/

50 µ

l)

1 2 3 4 5 6 1 2 3 4 5 6

220

160

120

100

90

80

70

60

50

40

170

125

81

62

53

43


1: After Concentration

2: After Gel filtration

3: After Anion-exchange

40

30

25

20

15

10

32

25

17

14

Loading: 8µl /well0.65 µm

21

4.0 µm4: After Concentration

5: After Gel filtration

6: After Anion-exchange

Process scale-up in downstream purification

1000

2000

3000

4000

5000

6000

200

400

600

800

1000

1200

HA

un

it/5

0u

l

UV

280

HA unitXK 16/40 UV280 Hiscale 50/40 UV 280

XK 16/40 HA Hiscale 50/40 HA

5120

512

1024

XK 16/40

HiScale 50/40


2210x scale-up

-1000

0

-200

0

0 20 40 60 80 100

min

512

-2000

0

2000

4000

6000

8000

10000

12000

-200

0

200

400

600

800

1000

1200

0 20 40 60 80 100

ng

/ml

UV

280

min

ds DNA concentration

XK 16/40 UV280 Hiscale 50/40 UV 280

XK 16/40 dsDNA Hiscale 50/40 dsDNA

Comparison of purification using different schemes

TC001-1 ultracentrifuge

Bulk (0.65)

TC001-1 NB Bulk (0.65)

TC001-1 BBulk (0.65)

TC001-2 NBbulk (4um)

TC001-2 Bbulk (4um)

SRD (ug/ml) 76.1 32.32 33.18 24.67 29.81

BCA (µg/ml) NA 68.06 69.21 45.56 41.19

$ 282 $ 265


BCA (µg/ml) NA 68.06 69.21 45.56 41.19

µg/dose NA 30.9 31.5 28.5 20.6

residue DNA(ng/ml)

8.334 10.926 14.006 3.094 2.6

residue DNA (ng/dose)

1.6 5.0 6.4 1.9 1.3

Predicted Doses(based on 4L harvet)

1015 1465 1504 1118 1351

23

Before 0.2 µm

TideCell + Plus_MDCK

MC + Optipro

DNA variation from different culturing system


24

0 5000 10000 15000 20000 25000 30000 35000 40000

Roller bottle + Plus_MDCK

MC + Plus_MDCK

ng/ml

NBS

5-33

Gel filtration

NBS 33 run of 5L SEC 5040001:10_UV1_280nm NBS 33 run of 5L SEC 5040001:10_UV2_260nm NBS 33 run of 5L SEC 5040001:10_Cond NBS 33 run of 5L SEC 5040001:10_Fractions NBS 33 run of 5L SEC 5040001:10_Inject NBS 33 run of 5L SEC 5040001:10_Protein (ug per ml) NBS 33 run of 5L SEC 5040001:10_dsDNA by Qbit (ng per ml) NBS 33 run of 5L SEC 5040001:10_HA unit per 50ul

0

2000

4000

6000

Act

0 100 200 300 400 500 600 700 ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Waste

Opti-pro


Sub 3

column test 13 SEC 5040001:10_UV1_280nm column test 13 SEC 5040001:10_UV2_260nm column test 13 SEC 5040001:10_Cond column test 13 SEC 5040001:10_Fractions column test 13 SEC 5040001:10_HA unit per 50ul column test 13 SEC 5040001:10_dsDNA by Q bit column test 13 SEC 5040001:10_Protein by coomassie plus

0

2000

4000

6000

Act

0 100 200 300 400 500 600 700 ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Waste

0 100 200 300 400 500 600 700 ml

Plus_MDCK

Summary

• The use of diatomaceous earth dad significant effect on DNA

and protein removal, but mechanism is not clear how DE

absorbed the impurities

• Low DNA content can be achieved by SEC and AEX columns,

thus no addition of DNase is needed

• The use of LC chromatography showed a similar results that


• The use of LC chromatography showed a similar results that

compared to ultracentrifuge technology

• Variations from Upstream harvest could affect heavily on

downstream purification

• New method need to be implemented for improving viral

yield

26

Acknowledgements

• Department of Health

• National Science Council

• Taiwan CDC


27

Bioprocess team

• Center for Drug Evaluation

• Science Advisory Board

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