Parallel Integrated Bioreactor Arrays for Bioprocess Development

1
Parallel Integrated Bioreactor Arrays for
Bioprocess Development

• Harry Lee, Paolo Boccazzi, Rajeev Ram, Anthony
  Sinskey

2
Outline

• Bioprocesses and bioprocess development
• Alternative approaches and advantages of
  microfluidics
• Parallel Integrated Bioreactor Arrays (PIBA)
• Preliminary biological validation
• Applications
• Next steps

3
Bioprocesses
Human insulin
E. coli bacteria

• Microbial fermentation is used to produce
• Human insulin, human growth hormone
• Plasmid DNA vaccine, protein subunit vaccine

Monoclonal antibody
Mammalian cell lines
1000L Bioreactor

• Mammalian cell culture is used to produce
• Monoclonal antibodies, Protein therapeutics (ie.
  erythropoietin)
• Viruses for vaccines

4
Bioprocess development

• Optimal microbial strains or cell lines must be
  screened
• Growth conditions must be empirically optimized
• pH, temperature, nutrients, O2, induction, etc.

Conventional technology
Experimental Throughput
Process Knowledge
5
Properties of ideal system

• Controlled growth conditions (pH, DO)
• High oxygen transfer rate
• Online optical density and growth rate
• Parallelism of shake flasks
• Automation
• Improved data quality
• Ease of use
• ? Potential to predict performance
• in large scale bioreactor

6
Conventional approaches

• Miniature stirred tanks, enhanced well plates
• ? Online cell density measurements not reliable
  (bubble interference)
• ? Measurements require sampling
• Mechanical multiplexing
• ? minimal labor savings
• Robotic multiplexing
• ? Expensive

7
Microfluidic advantage

• Microfluidics enables high oxygen transfer rate
  without bubbles
• Online optical density measurements
• Online growth rate estimation
• Integrated sensors and fluidics
• Measurements do not perturb the fermentation
• Minimal mechanical parts
• Compact, bench scale instrument

8
PIBA device module (patent pending)
optical density
Integrated optical oxygen and pH sensors.
(Fluorescence lifetime)

9
E. Coli fermentation in PIBA
45.7
15
30.5
OD 650nm (1cm)
10
Cell density (g-dcw/L)
15.2
5
Similar to Flasks
0
0

• Highest oxygen transfer rate in mbioreactor array
• First pH and DO controlled mbioreactor array
• Growth to cell densities (13g-dcw/L) 4X higher
  than previous mbioreactors
• Online optical density enabled by bubble free
  oxygenation

7.5
7
pH
6.5
6
120
100
80
DO ( Air Sat)
60
40
20
0
0
1
2
3
4
5
6
7
8
9
Time (h)
10
Unique capability Real time OD monitoring
E. coli growth on LB medium
30
25
20
OD 650nm, 1cm
15
10
5
0
0
1
2
3
4
5
6
7
8
Time (h)

• Detailed growth kinetics are observable ?
  quantitative study of lag phase
• Identify nutrient limitations by change in growth
  rate
• Screening to high cell density is important to
  see nutrient limitations
• Important to isolate cell density dependent
  phenomena

11
Applications

• Standard platform for fermentation and cell
  culture
• Standardization allows sharing data, improved
  data interpretation
• Standardization was the driver for microfluidics
  in analytics
• Bioprocess development
• Improved process optimization
• Screening based on higher quality data
• Production scale conditions, growth rate changes
• Production bioreactor modeling
• Inhomogeneities, dynamically changing conditions

12
Value Proposition

• Improved process screening
• Screen under production scale conditions
• Early determination of production process yield
• Impacts investment decision on 500M - 1B
  production facility
• Production reactor modeling
• Time varying environment
• High cell density growth
• Faster manufacturing scale-up
• One year shorter time to market for a 500M
  product 30M

13
Next Steps

• Improved understanding of economic model
• Case-studies
• Beta prototype development
• Improved user friendliness ? fluidic interfaces
• Improved manufacturing process ? Injection molded
  layers
• Deploy Beta to collaborators/customers
• Rigorous biological validation
• Rank order of process screen the same in PIBA and
  bench scale reactor
• Production reactor modeling

14
Team

• Dr. Paolo Boccazzi
• Microbial Physiology, Molecular Biology,
  Bioprocess Development
• Dr. Harry Lee
• Electrical Engineering, Microfabrication, System
  Integration
• MIT 50K Entrepreneurship Competition Winning
  team member, 2005
• Prof. Rajeev J. Ram
• Electrical Engineering, Optoelectronic devices,
  Optical Spectroscopy
• Director, MIT Center for Integrated Photonic
  Systems
• Associate Director, Research Laboratory of
  Electronics
• Prof. Anthony J. Sinskey
• Biology, Health Sciences and Technology,
  Metabolic Engineering
• Co-Founder Genzyme, Merrimack Pharmaceuticals,
  Metabolix