Micro-scale vaccine development – A
tool set for success?
Dr Tarit K. Mukhopadhyay
Department of Biochemical Engineering
University College London
The World Vaccine Market
The Life Cycle
Disease Burden
Cause
World
Low-Income
Countries
Middle-Income
Countries
High-Income
Countries
Infectious and Parasitic diseases
23.1%
33.3%
13.9%
3.0%
Tuberculosis
2.4%
2.9%
2.2%
0.3%
HIV/AIDS
6.1%
9.7%
2.6%
0.7%
Malaria
2.7%
2.7%
4.5%
1.0%
46.1%
33.2%
55.5%
82.7%
Malignant neoplasms (cancer)
5.3%
2.9%
6.7%
14.4%
Cardiovascular disease
10.3%
7.7%
12.3%
16.4%
Non-communicable diseases
Sources: World Health Report, 2001 & World Bank, 2001
Vaccine Bioprocess Development
Product / Disease Based
Vaccines
Virus
Vectored
Vaccines
Inactivated
virus
vaccines
Polysaccharide
vaccines
Designed Vaccines
Novel
Analytical
Tools
Virus like
particles
Electrical
Impedance
Heat shock
proteins
Microwell “Bioprocess-on-a-Deck” Concept
www.tecan.com
96 DSW
24 SRW
96 SRW
[Lye et al (2003) Trends Biotechnol.; Micheletti & Lye (2006) Current Opin. Biotechnol.]
The scale down platform
Microwell (< 1 mL)
STR (5-100 L)
?
CFD Simulation of Fluid Shear and Energy
Dissipation in Shaken Microwells (24SRW)
120 rpm
150 rpm
200 rpm
250 rpm
300 rpm
400 rpm
[Zhang et al (2008) Biochem. Eng. J.; Barrett et al (2009) Biotechnol. Bioeng., in press]
Engineering Basis for Cell Culture Comparison?
Bioreactor Geometry
N
[rpm]
Ph
[-]
tm
(s)
kLa
[h-1]
P/V
[W m-3]
24 SRW
[800 µl]
160
4.33
420
6.3
~ 39
200
4.51
10
10.8
~ 41
250
4.69
5
18.5
~ 40
Shake Flask
[100 ml]
120
2.68
16
2.1
41
Stirred-Tank Reactor
[3 l]
200
na
6
41
21


 .(2n) df 2
ds 

Ph 
1

3
l
o
g

10 
df 
4
 





1

4  VL 3
1  1  
 df



2
2  
  
  
  


  

P
n3 .d4
1
= C.ρ. 2 / 3 . 0.2
VL
VL
Re
[Buchs et al (2002)
Biotechnol. Bioeng.]
Transient Transfection of CHO-S Cells with
PEI/DNA Complexes
• Compare Culture conditions
• cells (growth, viability)
• DNA (uptake, expression)
• Transfection conditions
• 24 well plates (800µl)
• shake flasks (60ml)
• STR (3 L)
Transient Transfection of CHO-S Cells:
Bioreactor Comparison at Matched tm
Cell Growth
Cell Viability
Transient Transfection of CHO-S Cells:
Bioreactor Comparison at Matched tm
DNA Uptake
SEAP Expression
The scale down platform for Wave
Bioreactors?
Microwell (< 1 mL)
WAVE (5L)
?
Lentivirus production in an inducible
producer cell line
DOX
+
HEK 293
Work conducted with Oxford Biomedica
Microwell plates (800µl)
Matching Cell Growth
and Viability between
Microwells and Wave
Bags
Wave bags (2L)
Scaling criteria
based on liquid
fill volume of the
Wave bag and
rocking rate
GUY et al - HUMAN GENE THERAPY METHODS 24:125–139
Microwell plates (800µl)
Predicting Optimum
harvest time between
Microwells and Wave
Bags
Wave bags (2L)
Maximum infective titre
correctly predicted, however
the Wave system produces
more particles overall –
stability issues?
GUY et al - HUMAN GENE THERAPY METHODS 24:125–139
ULTRA SCALE DOWN (USD)
CENTRIFUGATION
USD Principle for Centrifugation
Solids
Remaining
Q
= f


Discstack Centrifuge
(10 L at pilot scale)
Rotating Shear Device
(20 mL)
V
Q
=
t.
USD Principle for Centrifugation
Solids
Remaining
Q
= f

V
Q

=
t.
Discstack Centrifuge
(10 L at pilot scale)
Rotating Shear Device
(20 mL)
Σ lab 
Vlab  2 (3  2x  2y)
 2R 2 

6gln
 R1  R 2 
[Maybury et al (2000) Biotechnol Bioeng; Boychyn et al (2001) Chem Eng Sci]
USD (10 mL) Predictions and Pilot Scale
Verification (170 – 600 L/h)
10
high shear feed
Solids
carryover (%)
5
low shear feed
2
USD prediction
1
0.5
0.9
2
3
4
5
6
Flow rate/Equivalent settling area (m/s) x10 -8
[Hutchinson N, et al., (2006) Biotechnol Bioeng 95(3):483-491]
Purification: Human Papillomavirus VLPs
Virus Like Particle
(~20,000 kD)
Capsomere
(~280 kD)
L1 monomer
(~55 kD)
5 L1
~ 3 nm
Atomic Force
Microscopy
Image
~ 10 nm
(Crystal structure coordinates
courtesy of Prof. S. C. Harrison,
Harvard University)
Non-infectious (no nucleic acid)
Copyright © 2006 Merck & Co., Inc., Whitehouse Station, New Jersey, USA, All Rights Reserved
~ 60 nm
Micro-scale Chromatography
Mechanical Cell Disruption
Debris Removal
(Centrifugation or Microfiltration)
CEX Chromatography (80 μL)
Polishing Chromatography (40 μL)
1000-fold scale-down
[Wenger et al (2007) Biotechnol. Appl. Biochem.]
Minimal plate manipulations
(= reduced automation complexity)
Dynamic flow
(= Improved mass transfer)
[Wenger et al (2008) Biotechnol. Progr.]
Copyright © 2006 Merck & Co., Inc., Whitehouse Station, New Jersey, USA, All Rights Reserved
Engineering Considerations and Purification
Comparison
laboratory microscale
200 kDa
CEX Polishing
CEX Polishing
Flow Characteristics
Up, down
66.3 kDa
Screens attached to the
plastic tip body
i.d.
55.4 kDa
L1
h
36.5 kDa
i.d.
Separation media encased
between the two screens
5 - 20 L/sec = linear velocity of
270 – 1080 cm/hr (avg, 80 L)
14.4 kDa
Copyright © 2006 Merck & Co., Inc., Whitehouse Station, New Jersey, USA, All Rights Reserved
Gardasil®
Probing the Impact of Process Differences Using Atomic
Force Microscopy
Bioprocess #1
Bioprocess #2
Number of VLP Averaged
183
150
Diameter (nm)
58.6 + 6.2
43.9 + 8.3
ANALYTICS
Process Characterisation - Aggregation &
Particle sizing
• Dynamic Light Scattering vs. Nanoparticle Tracking Analysis.
– Hydrodynamic radius determined using Brownian motion
• DLVO theory
– Ionic strength of buffer dictates particle surface charge
DLVO Theory
Nanosight data for JEV inactivation study
500
450
- PEG
400
Size (nm)
350
300
250
200
150
100
50
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Sample
+PEG
Summary
• We want to develop efficient bioprocesses faster,
smarter and at lower cost, less time in the pilot scale
• A range of industry validated USD technologies are
available now
• The future will see microwell (and microfluidic?)
technologies currently used in discovery applications
being developed for early phase bioprocess studies
Bioprocess Enterprise @UCL:
Ultra scale-down (USD) Technologies
Supply and Support
• Training on USD technologies
• Supply of USD device for specified
period with offline technical support
Where can I get more information?
Dr Andrea CME Rayat
or
Dr Alex Chatel
Enterprise Fellows in Bioprocessing
USD-enabled Projects
• Support in research & process
development
• Help on data analysis and others
• Project-specific support
The Advanced Centre
for Biochemical Engineering
University College London
Bernard Katz Building, Gordon Street, London WC1
0AH
 +44 (0) 20 3108 4409
 usdtechnologies@ucl.ac.uk
Access these through:
Consultancy service | Industry-funded priority projects | TSB Collaboration | EngD Programme
Acknowledgments
The Students
Mike Hughson, Sara Nilsson, Kristina Schlegel, Aaron Noyes, Heather Guy,
Ann-Marie De Villiers
The Collaborators
Jeff Drew (Stabilitech), Mike Whelan (iQur), Rachel McKendry (LCN),
Khurrum Sunasara (Pfizer), Donald Low (Intercell), Mary Collins (UCL)
THANK YOU!
Label-free virus particle measurement using electrical
impedance
Is it possible?