Bench to Clinic of Biotherapeutic Molecules: Issues to Consider

1
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

2
Objective of the Presentation

• Provide the discovery scientist with information
  on what is required to move a discovery
  molecule to a clinical candidate.

3
Starting Point

• Discovery has identified a protein with
  therapeutic properties.
• Discovery wants to move the clinical candidate
  into preclinical testing.

4
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

5
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)

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Cell Line

• Discovery is accomplished through high-throughput
  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.

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Cell Line

• Production expression system is determined based
  on the post translations modifications (PTM) that
  are required.
• Bacteria to transgenic animal

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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.

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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

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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.
  

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USAMRIID Botulinum Toxin ProgramDirected 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
C
N
Light Chain (50 kd)
Heavy Chain (100 kd)
Vaccine
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Effect of Gene Copy Number on Cell Growth During
MeOH Induction
13
Effect of Gene Copy Number on Methanol Consumption
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Effect of Gene Copy Number on BoNTC(Hc) Production
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Effect of Gene Copy Number on MeOH Growth Rate
and Production of BoNTC Hc
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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.

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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

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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.

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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.

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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

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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)

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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.

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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

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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

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BoNTE Hc Purification
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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.

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Credit Goes To

• Mehmet Inan (Molecular Biology)
• Vijay Jain (Molecular Biology/Fermentation)
• Wenhui Zhang (Fermentation)
• Mark Gouthro (Fermentation)
• Rick Barent (Purification)
• Joey Wu (Purification)

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Acknowledgements

• BoNT Hc work was funded by the United States Army
  Medical Research and Materiel Command.
• Contract No. DAMD17-02-C-0107