BioProcess Engineering
“Role of a Chemical Engineer in the Biotech Industry”
Steven T Rose/Billy Allen ‐ Upstream
Harish Santhanam‐ Downstream
ISPE Biotech Presentation October 3 2010
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Manufacture of Drug Product
Drug Product
Excipients
ISPE Biotech Presentation October 3 2010
API
Fill/Finish
2
Biotech API Processes
Upstream
Downstream
API
ISPE Biotech Presentation October 3 2010
Produce aqueous process stream containing the bioproduct
Concentrate and purify the bioproduct
Active Pharmaceutical Ingredient for Formulation and Packaging
3
Upstream Process Objectives
• Provide a robust manufacturing cell culture / fermentation process that has the following performance capabilities
– Consistent product quality
– Consistent process yield
– Consistent level of process impurities
• Host cell protein
• DNA
• Product variants
– Consistent broth conditions for predictable downstream unit operations performance
• Clarification
• Primary capture column
ISPE Biotech Presentation October 3 2010
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Upstream Process Unit Operations
Master
Cell Bank
Thaw
Expansion
Working
Cell Bank
Antifoam
Feed
Glucose
er2
arg
Thaw
Expansion
Flask Expansion
ISPE Biotech Presentation October 3 2010
15L
NaOH
50L
250L
Seed Reactor Expansion
1000L
5000L
Production Reactor
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Key Steps in Upstream Process Development
•
Construct Cell Line – Genetically engineer host organism (mammalian cell line, E. coli, etc.) to produce desired bioproduct.
•
Clone Selection – Select from many hundreds of clones (genetically different cells) the most appropriate production cell line based on product quality and productivity.
•
Process Development – Optimize production process parameters to maximize productivity, obtain acceptable product quality and scale up process to production. •
Process Transfer – Enable information transfer (process and equipment) between Process Development and Production groups to minimize process issues and speed commercialization. ISPE Biotech Presentation October 3 2010
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Cell Culture Process General Requirements
• Phenotypic Stability
– The cells grow, metabolize, and produce with a predictable profile from batch to batch.
• Genetic Stability
– The cells retain the genetic code for the protein of interest over the population generations from the cell vial to the production vessel harvest.
• Axenic Processing
– The process is free of foreign growth.
ISPE Biotech Presentation October 3 2010
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Construct Cell Line
• Genetically engineer cells to produce monoclonal antibody of interest.
– Construct DNA for monoclonal antibody.
– Transfect Host Cell with DNA Construct.
– Select cells with desired performance capabilities.
– Create a bank of the cells with the desired performance.
ISPE Biotech Presentation October 3 2010
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Plasmid Construct For MAbs
•
Plasmid Contains
– DNA of interest
LC
• Antibody light chain
• Antibody heavy chain
Promoter
Promoter
Poly A
– Promoters
SV40
Intron
• Enhance DNA transcription
Selector
HC
– Selector DNA
• Glutamine Synthetase
• Without Glutamine Synthetase cells are not as likely to survive.
Promoter
ISPE Biotech Presentation October 3 2010
AmpR
ColE1 Ori
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DNA Incorporation into the Host Cells
(Transfection)
Plasmid
(Engineered DNA)
Host DNA
Insertion Sites
• Inserting Engineered DNA into the host DNA
•
Have the host cell make something it normally would not make. •
Same method that viruses use to infect us.
ISPE Biotech Presentation October 3 2010
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DNA Transfection
• Plasmid DNA inserts at specific sites in the host DNA
•
The insertion site is based on the codon sequence of the host DNA
ISPE Biotech Presentation October 3 2010
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DNA Transfection
ISPE Biotech Presentation October 3 2010
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DNA Transfection
• Plasmid DNA is now part of the Host DNA
•
The host system will read and process the engineered plasmid DNA as if it were its own.
ISPE Biotech Presentation October 3 2010
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DNA Transfection
• DNA insertion is in all practicality a random event
•
1000s of insertion sites.
•
Potential for multiple copy numbers.
• Outcome is not predictable.
ISPE Biotech Presentation October 3 2010
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Outcome of Transfection
•
Random Insertions can impact the cells.
– Inactivate Genes
• Affect the reaction network of the cells
– Negative or positive affects
– Location near Active Genes
• Production associated with cell growth
• Production associated with maintenance.
– Location in Inactive DNA
• Tends to be low productivity.
www.tulane.edu/~wiser/malaria/Summary.html
ISPE Biotech Presentation October 3 2010
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Protein Synthesis
stemcells.nih.gov/info/scireport/appendixA.asp
ISPE Biotech Presentation October 3 2010
http://scienceblogs.com/transcript/2008/03/whats_encoded_in_your_genome.php
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Protein Production
metabolism
• The amount of proteins produced is dependent on
– Availability and Use of Nutrients – Accumulated Number of Cells
– Specific Rate of Protein Production per Cell
ISPE Biotech Presentation October 3 2010
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Protein Production
metabolism
• Ideal Process
–
–
–
–
Product Quality Profile Consistent with Prior Proteins
High Specific Rate of Protein Production
High Cell Density
Low Metabolic Waste
ISPE Biotech Presentation October 3 2010
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Clone Selection
•
Clone Selection is the process of identifying the clones with the desired product quality, yield and phenotype.
Transfection
Grow Out
Photomultiplier
Tube
Laser
•
Uses relatively rapid methods to quickly reduce the number of clones examined
–
–
–
–
–
•
------
FACS (Specific productivity)
Grow out (Growth Phenotype)
Titer (Yield)
1mL wells / 125mL flasks / 4L bioreactors
Full product quality package is reserved for final top four clone candidates.
Once clone is selected, the master cell bank is prepared.
ISPE Biotech Presentation October 3 2010
FACS
++
++ Electrical
++ Wire
++
++
Grow Out
High Throughput
Titer Screening
Titer and Growth
Screening
Limited PQ
Clone Selection
Titer, Growth, PQ
Process
Screening
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Clone Selection
• Challenges with Clone Selection
– Screening is performed in systems that are not entirely representative of the manufacturing process.
• Microwell Plates (1mL)
• Shake Flasks (20mL)
• Production Bioreactor (5,000L)
– Screening is performed on relatively young cells.
• Phenotype of cells may change with increased number of generations from the transfection.
• Nutrient and process requirements may change with cell age.
ISPE Biotech Presentation October 3 2010
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Process Development Roles for Engineers
• Process definition and optimization
– Final clone selection
– Empirical lab‐scale experiments to define process parameters using DOE approach
• Process scale up
– Translation of process from lab to production scale equipment for clinical trial production
– Equipment performance characterization
• Commercialization
– Development of increased process understanding through further DOE experimentation and process models
– Design of manufacturing facility and process equipment
– Translation of process to manufacturing facility
ISPE Biotech Presentation October 3 2010
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Process Development
•
Upstream process development is predominately empirical
– Complex reaction mechanisms not fully understood.
– The DNA integration modifies the reaction mechanisms.
•
Number of studies is manageable with platform processes
– Use a systematic DOE approach to gain understanding
– Parameters to explore process performance to be similar across projects
• Priority tends to be conserved.
• Similar studies to examine appropriate set points.
– Platform approach to cell culture process.
• Limits the need to revisit characterizing equipment, logistics, control or facility capabilities.
www.tulane.edu/~wiser/malaria/Summary.html
ISPE Biotech Presentation October 3 2010
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Process Development Approach
Y
Response Surface
Acceptable Limits
σx Manufacturing Capabilities
μx
Process Limits
X
Set point
ISPE Biotech Presentation October 3 2010
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Process Development Approach
Y
Response Surface
Acceptable Limits
σx Manufacturing Capabilities
Process will not meet required outcomes
μx
Process Limits
X
Set point
ISPE Biotech Presentation October 3 2010
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Process Development Approach
Y
Response Surface
Acceptable Limits
σx Manufacturing Capabilities
μx
Set point
ISPE Biotech Presentation October 3 2010
Process Limits
X
Process will have unnecessary deviations
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Process Development Approach
Y
μx(1)
μx(2)
X
For nonlinear responses, changing the process set point changes the process robustness.
ISPE Biotech Presentation October 3 2010
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Example of Process Development Data
Prediction Profiler
Titer
2.97035
1.698718
±0.2969
Trp(25) Oxi
0.51532
13
4.673895
±0.3968
3.7496
Ser(46)
Phosph
16.0132
13.28696
±0.6068
1.8
1.090141
±0.1659
0.57188
10
2.473784
±0.4437
DO (%)
ISPE Biotech Presentation October 3 2010
Initial
Temp (C)
T Shift (C)
N-1
Temp (C)
18
14
8
11
36
36
1
0
1
34
34
36
34
70
90
1.3738
50
% des H/HG
% Pyruvylated
6.24031
Day
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Stirred Tank Bioreactors
• Closed systems operations
• Better control than shake flasks
• Mass and energy transfers more complex
• Engineering role – Designing
– Controlling
– Scaling bioreactor
to conserve the operating space across scale, sites, and runs.
ISPE Biotech Presentation October 3 2010
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Scale Up / Scale Down
• Scale and Vessel Configuration Impacts
– Mixing
• Blend time increases with scale
• Heterogeneity of the culture increases with scale
– Gas Exchange
• Dissolved oxygen control and carbon dioxide accumulation scale by different parameters
– Time required to deliver or transfer materials
• Hold times will be longer with scale
– Controller response time
• Increased capacitance slows controllers at larger scale
– Interaction of process parameters
• Process parameters can fight each other. Need to balance control with scalability.
ISPE Biotech Presentation October 3 2010
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Bioreactor Characterization
ISPE Biotech Presentation October 3 2010
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Oxygen Transfer Rate
Data
Process Model
120
100
Oxygen Transfer Rate = Kla x C*(1‐ DO)
)
(%
80
n
e
gy
x
O 60
d
e
lv
o
ss
i 40
D
20
0
0
20
40
60
80
100
Time (s)
ISPE Biotech Presentation October 3 2010
120
140
Downstream Operations Considerations
• The bioreactor is the source of many of the impurities that downstream removes.
– Lower culture viabilities release host cell proteins, DNA, and proteases that can impact regulatory requirements or product quality.
– Lower culture viabilities challenge the capability of clarification to remove the cells and cellular debris prior to the first chromatography column.
– The downstream platform may not always be capable to efficiently remove product variants.
• The development and operation of the cell culture process needs to take into account the impact to downstream processing.
ISPE Biotech Presentation October 3 2010
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Product Quality Attributes and Impact to Downstream Processing
• High Priority
– Product quality variants that impact potency and cannot be removed downstream
• Medium Priority
– Product quality variants that can be removed downstream and impact yield.
– Process yields.
– Harvest conditions; viability, cellular debris
• Low Priority
– Variability in cell densities or metabolites.
ISPE Biotech Presentation October 3 2010
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Downstream Operations‐ EColi Process
A
FERMENTATION
Chromatography
ISOLATION
Ultrafiltration
A
Crystallization
FILTRATION
Ultrafiltration
Chromatography
ISPE Biotech Presentation October 3 2010
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DRYER
Downstream Operations‐Cell Culture Process
ISPE Biotech Presentation October 3 2010
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Process Chromatography
Chromatographic Process‐ Block Diagram
Adsorption Chromatography (IEX, HIC, RØ, Affinity)
Equilibration
Load
Post wash
Elution
Regeneration
ISPE Biotech Presentation October 3 2010
Size Exclusion Chromatography
Column preparation
Equilibration
Sample application
Load
Residual sample removal
Sample separation
Elution
Column clean‐up
Regeneration
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Role of a Bioprocess Engineer Manufacturing Environment
Pressure profile of column
45
Pressure(psi)
40
35
30
25
20
15
10
ISPE Biotech Presentation October 3 2010
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Lot…
Lot…
Lot…
Lot…
Lot…
Lot…
Lot…
Lot…
Lot 9
Lot 8
Lot 7
Lot 6
Lot 5
Lot 4
Lot 3
Lot 2
Lot 1
0
Lot…
5
Column Hydraulics‐Continued
⎛ 150μu L ⎛ 1− ε ⎞2 106 1.75ρ (u )2 L 1− ε 1 ⎞ 14.7
0
b
0
b
⎟
⎟
⎜
(psi)
+
ΔP = ⎜
2
5
⎟
⎜
⎜ dp ⎝ ε b ⎠ 6
dp
ε b 3.6 ⎟ 1.013×10
⎠
⎝
Voidage trend at different steps
0.31
0.29
Voidage
0.27
0.25
0.23
0.21
0.19
Column
repacked
0.17
0.15
Lot number
ISPE Biotech Presentation October 3 2010
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Tangential Flow Filtration
RETENTATE
FEED
REVERSE
TFF OSMOSIS UNIT
Operation
PERMEATE
In general, Tangential Flow Filtration (TFF) operations are used for clarifying, concentrating, diafiltering, and purifying
proteins
ISPE Biotech Presentation October 3 2010
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Difference between TFF and NFF
Normal Flow Filtration
Tangential Flow Filtration
Reference: Millipore Technical Brief “Protein Concentration and Diafiltration by Tangential Flow Filtration”.
ISPE Biotech Presentation October 3 2010
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TFF Systems
ISPE Biotech Presentation October 3 2010
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Role of a BioProcess Engineer
Manufacturing Environment
Flux (GFD/psi
Flow(L/min),
Permeate Flow/Flux in during CIP
9
0.35
8
0.3
7
0.25
6
5
0.2
4
0.15
3
0.1
2
1
0.05
0
0
0
5
10
15
Data from CIP's
ISPE Biotech Presentation October 3 2010
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42
Permeate Flow (L/min)
NWP (GFD/psi)
Role of a BioProcess Engineer
Continued
Flux (J) = TMP /(Rm+Rf+Rg)
Rm=Membrane Resistance
Rf‐ Fouling Resistance
Rg‐ Gel layer Resistance
Monoclonal Antibody TFF Process
ISPE Biotech Presentation October 3 2010
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Disposable System ‐ Mixing
ISPE Biotech Presentation October 3 2010
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Conclusions
• The objective of the bioprocess process is to reproducibly produce bioproteins that are consistent in product quality, yield, purity and provides a predictable feed stream for downstream unit operations.
• This is accomplished with a team of scientists, engineers, statisticians, and other specialists.
ISPE Biotech Presentation October 3 2010
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