pet for pat? process evaluation tools for process analytical technologies in manufacture of...
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![Page 1: PET for PAT? Process Evaluation Tools for Process Analytical Technologies in Manufacture of Biological Products Charles L. Cooney Department of Chemical](https://reader036.vdocuments.mx/reader036/viewer/2022062308/56649db95503460f94aa9617/html5/thumbnails/1.jpg)
PET for PAT?Process Evaluation Tools for Process
Analytical Technologies in Manufacture of Biological Products
Charles L. CooneyDepartment of Chemical
EngineeringMIT, Cambridge, MA 02139
Advisory Committee for Pharmaceutical Science April 13, 2004
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SOME ISSUES
• What does the pipeline for new biological products look like?
• What will be the path for Follow-on Biologics?• How does the biological product respond to
physical process change?• Do we have adequate analytics to address the
uncertainties in the biological products industry?• How can we assure robustness to design and
operation of biological products manufacture?
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Where are we going?• Products
– Antibodies and Replacement proteins– Vaccines– Cellular therapies– Gene therapies
• Processes– Recombinant protein production– Tissue engineering - tissue repair– Transgenic plants & animals
• Challenges– Rapid and cost effective development &
scale-up– Continuous improvement & process
change– Follow-on biologics– Complex biologicals – cellular therapies
and tissue engineering
Multiple processes for the same or similar products
Complex processes for complex products
There is tension between the safety and economic agenda; where is the proper balance?
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Process for BiologicalProducts
Raw Materials
EnvironmentalConditions
Product
Parameter Control
Information Flow
How does the biological process respond to physical change?
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THE OXYGEN DILEMMA
•Required for efficient growth and recombinant protein expression•Potential in vivo or in vitro protein oxidation e.g. Met, Cys•Oxygen induced stress response
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O2 Gradients in Large-Scale Fermentors
10,000 L
DO
10%
40%
100 mL
Homogeneous
10 L
Homogeneous
• How do O2 gradients affect cell ?
• How does cell respond?• Effects on recombinant
protein production?
100l
Homogeneous
Heterogeneous
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Model System: 1-Antitrypsin• Elastase inhibitor (44 kDa) 10 met and 1 unpaired cys
• Activity lost with oxidation of active site MET358 Oxidation of met358 --> partial loss of neutrophil elastase activity & complete loss of porcine pancreatic elastase
• Use in protein replacement therapy• Cytoplasmic expression in E. coli BL21
methionine
methioninesulfoxide
H2N-C-H
COOH
CH2
CH2
CH3
S
H2N-C-H
COOH
CH2
CH2
CH3
S O
oxidation
M358
M351
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• Recombinant 1-antitrypsin (soluble at 30C) – Degraded in E. coli– Proteolysis is oxygen-dependent
• What is the connection between O2 and proteolysis?
Observed Problem in Synthesis
0
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N2AIRO2
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alize
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orm
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a1A
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Time (min)
WT ClpP-
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alize
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a1A
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Time (min)
WT ClpP-BL21
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N2AIRO2
Norm
alize
d a
1A
T
Time (min)
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N2AIRO2N
orm
alized
a1A
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Time (min)
WT ClpP-
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alize
d a
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Time (min)
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alized
a1A
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Time (min)
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Norm
alize
d a
1A
T
Time (min)
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orm
alized
a1A
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Time (min)
WT ClpP-BL21
Pulse-Chase Data (Laska, 2000)
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WHERE IS THE PROBLEM?
AND SOLUTION?
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• Proteolysis in E. coli– Majority requires ATP– ~70% by Lon and ClpP/AX– Current Strain BL21 is Lon-
• ClpP– Protease subunit of ClpP/AX
complex– Heat shock protein
• ClpP- strain (SG1146A)– E. coli BL21 ClpP-
Figure (Wickner & Maurizi, PNAS 1999)
Protease ClpP
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0
0.2
0.4
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1
1.2
0 10 20 30 40 50 60
N2AIRO2
No
rmal
ized
a1A
T
Time (min)
0
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1
1.2
0 10 20 30 40 50 60
N2AIRO2N
orm
aliz
ed
a1A
T
Time (min)
Wild Type SG1146A (ClpP-)
Some background degradation (~18%) remains• Other protease responsible
O2-Enhanced Degradation is Eliminated in ClpP- Strain
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Do we have the analytical techniques to probe a cell’s
global response to its physical environment?
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DNA Microarray Experiments
• 3,812 Genes representing 89% of E. coli genome
• Multi-Gene Groups– 167 protein complexes– 190 pathways– 333 transcription units
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Hyperoxic Stress Responses
• Increasing N2 → Air → O2
• Sustained Response
• Increasing Air → O2
• Short-Term Response
OxyR Regulon - ahpC /ahpF /dps /grxA /katG
-1.5
-1
-0.5
0
0.5
1
1.5
2
0 30 60 90Post-Induction Time (min)
RelativeExpression(Average
Log Ratio)
O2AIR
SoxRS Regulon - fur /nfo /sodA /soxS
-5-4-3-2-101234
0 30 60 90Post-Induction Time (min)
RelativeExpression(Average
Log Ratio)
O2AIRN2
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O2 Dependent Genes• SoxRS Response
– soxS, fur, sodA, nfo
• Iron Uptake– fur, sodA, fepB
• Fe-S Proteins– bioB, ilvD, leuB, mutY, fdx,
yfhI
• Fe-S Cluster Assembly– b2530 (iscS), b2531, hscA, fdx
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What is the right next step?
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When we introduce a process to make a biotherapeutic product do we know the “optimum” conditions
for quality and quantity?
During routine manufacture, do we improve the quality and
quantity of product?
SELF ASSESSMENT
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What is the way forward?•Is there a better way than incremental adjustment to optimize and scale a biological process?•We live with variance; have we taken adequate opportunity to observe it and learn?•Can we explore experimental space more effectively?•How do we embrace risk and manage it?•How do we assure ourselves that we have a robust process?
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PROCESS EVALUATION TOOLS
• Leverage analytical technology on process and product
• Look at the global system response• Explore how biological and parameter variance
propagate through the process?• Interrogate the cell at the molecular scale• Multi-scale analysis – scale down to scale up• Understand the interdependencies in experimental
space• Understand the connection between molecular
processes, process performance & product quality