Download Document

Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Bench to Clinic of Biotherapeutic
Molecules: Issues to Consider
By
Dr. Michael Meagher
Donald F. and Mildred T. Othmer Endowed Professor
Director, Biological Process Development Facility
Department of Chemical Engineering
University of Nebraska-Lincoln
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Objective of the Presentation
• Provide the discovery scientist with
information on what is required to move a
“discovery molecule” to a “clinical
candidate.”
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Starting Point
• Discovery has identified a protein with
therapeutic properties.
• Discovery wants to move the clinical
candidate into preclinical testing.
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Technology Transfer to Process Development
and cGMP Manufacturing
•
•
•
•
•
Product characterization
Identify production cell line-Pichia pastoris
Analytical methods
Raw materials
Bench-scale process




Fermentation (shake flask)
Recovery
Purification
Formulation and stability
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Product Characterization
• The more characterization the better.

N-terminal sequencing
 Tryptic digest and peptide map (LC-MS/MS)
 Mass spectrometry



Overall mass
Post translational modifications
Amino acid analysis
 Isoelectric focusing (pI)
 Bioassay(s)
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Cell Line
• Discovery is accomplished through highthroughput expression systems.
• Such expression systems are not intended or
suitable for high-level production and
cGMP manufacturing.
• Therapeutic gene may not be optimized for
scale-up and production.
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Cell Line
• Production expression system is determined
based on the post translations modifications
(PTM) that are required.
 Bacteria to transgenic animal
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Cell Line
• The cell line is the most critical component
of the production process.
• Thorough characterization of the cell line is
strongly recommended before moving a
process into scale-up.
• Prefer that a validated Master Cell Bank be
established prior to process development.
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Cell Line Evaluation
• Shake flask

SDS-PAGE and Western Blot (minimum)
• 5 L Bench-scale Fermentation

SDS-PAGE and Western blot (minimum)
 Stability of supernatant (extra) or homogenate
(intra)
• Small-scale purification
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Cell Line Evaluation-Case Study of
BoNTC Hc expressed in Pichia pastoris
• BoNTC Hc is expressed intracellular.
• By shake flask there was no “apparent” effect
of copy number on expression based on
Western blot.
• Evaluated 1, 2, 3 and 4 copy clones in a 5 L
fermentor.
 Standard basal salts media and trace minerals.
 Methanol set point during induction was 1. 5 g/L.
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
USAMRIID Botulinum Toxin Program
Directed by Dr. Leonard Smith
• Seven distinct serotypes (A-G)
• Current vaccine is a pentavalent toxoid of
serotypes A, B, C, E and F.
Botulinum Toxin
Zn Protease
Membrane binding and translocation domain
N
C
Light Chain (50 kd)
Heavy Chain (100 kd)
Vaccine
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Effect of Gene Copy Number on Cell Growth
During MeOH Induction
Cell Growth Vs Induction Time
5
One Copy
Two Copy
Three Copy
Four Copy
WB (old clone)
4.5
XV/XoVo (g/g)
4
3.5
3
2.5
2
1.5
1
0.5
0
0
10
20
30
Induction Time (hr)
40
50
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Effect of Gene Copy Number on Methanol
Consumption
Methanol Consumption Vs Induction Time
g Methanol consumed/XoVo
(g/g)
3.5
One Copy
Two Copy
Three Copy
Four Copy
WB (old clone)
3
2.5
2
1.5
1
0.5
0
0
10
20
30
Induction Time (hr)
40
50
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Effect of Gene Copy Number on
BoNTC(Hc) Production
unit/unit of BoNTC(Hc)
1.4
1.2
1
0.8
293-1
0.6
299-2
0.4
300-3
301-4
0.2
0
0
10
20
30
Induction time (hr)
40
50
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Effect of Gene Copy Number on MeOH
Growth Rate and Production of BoNTC Hc
Specific MeOH
Growth Rate (hr-1)
1
0.0544
2
0.0231
3
0.0054
4
Not Constant
WB (old)
0.0166
1.20
maximum unit/unit of
BoNTC(Hc)
Gene Copy
Number
1.00
0.80
0.60
0.40
0.20
0.00
0
1
2
3
Gene copy number
4
5
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
BoNTC Hc Cell Line Case Study
• The effect of BoNTC Hc copy number on
MeOH metabolism was unexpected.
• Essential to evaluate clones under
production fermentation (and purification)
conditions before deciding on a clone.
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Establishing a Seed Bank
• Purity
• Determine the cell line is mono-culture
• Identity
•
Phenotypic



•
Growth morphology
Carbon
Auxotrophic marker
Genotypic



Confirm and sequence gene insert
Restriction map
Ribosomal typing
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Establishing a Seed Bank
• Stability
•
Generational studies in shake flask and
fermentor




Analyze product
Copy number
mRNA
All aspects of establishing a seed bank must be
documented. Information is required for the
Master Cell Bank.
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Analytical Methods
• Product characterization assays
• Purpose is to sufficiently characterize the
product so as to create a reference standard.
• Bioanalytical reagents
• Antibodies, cell lines for bioassay, enzymes,
etc.
• Essential to insure sources of these reagents.
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Analytical Methods
• In-Process Test(s)
•
•
•
•
•
•
Fast
Reliable
Robust
Quantitative for product
Able to handle all types of samples
Provide an indication that process is operating
within specifications
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Analytical Methods
• Lot release assays for product
•
•
•
•
•
•
Amino acid analysis
Tryptic digest and peptide map
N-terminal sequencing
2 HPLC methods and size exclusion
Endotoxins
SDS-PAGE and Western Blot
 Reducing and non-reducing
• Host protein and nucleic acids
• Bioassay(s)
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Process Description
• Raw Material
• Anything used to produce or analyze the
product.
• Important to specify several vendors.
• Establish methods to I.D. raw materials.
• Understand “shelf life” of raw materials.
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Process Description-Fermentation
• Monitor Critical Parameters-Metabolic Activity
• pH
• Dissolved oxygen
• On-line sensors
• Off gas
• Calculate RQ and OUR
• Consumption of nutrients, acid or base
• Generate a fermentation history
• Move towards greater computer control
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Process Description-Purification
• Recovery
• The most difficult step in process development.
• Purification
• Identify critical parameters.
 pH, conductivity, temperature, protein
concentration, resin, membrane, etc…
• Determine scalability of each step
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
BoNTE Hc Purification
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Conclusions
• The most critical “raw material” is the cell
line.
• Essential to evaluate cell lines under
process development conditions.
• The greater the interaction of the discovery
scientists with the process development
scientists and engineers the faster and more
effective the transfer into the clinic.
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Credit Goes To
•
•
•
•
•
•
Mehmet Inan (Molecular Biology)
Vijay Jain (Molecular Biology/Fermentation)
Wenhui Zhang (Fermentation)
Mark Gouthro (Fermentation)
Rick Barent (Purification)
Joey Wu (Purification)
Biological Process
Development Facility
Department of Chemical Engineering
College of Engineering and Technology
Acknowledgements
• BoNT Hc work was funded by the United
States Army Medical Research and Materiel
Command.
Contract No.: DAMD17-02-C-0107