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Bench to Clinic of Biotherapeutic Molecules: Issues to Consider

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. Objective of the Presentation.

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Bench to Clinic of Biotherapeutic Molecules: Issues to Consider

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  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)

  6. 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.

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

  8. 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.

  9. 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

  10. 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.

  11. USAMRIIDBotulinum 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 N C Light Chain (50 kd) Heavy Chain (100 kd) Vaccine

  12. Effect of Gene Copy Number on Cell Growth During MeOH Induction

  13. Effect of Gene Copy Number on Methanol Consumption

  14. 1.4 1.2 1 unit/unit of BoNTC(Hc) 0.8 293-1 0.6 299-2 300-3 0.4 301-4 0.2 0 0 10 20 30 40 50 Induction time (hr) Effect of Gene Copy Number on BoNTC(Hc) Production

  15. Gene Copy Number Specific MeOH Growth Rate (hr-1) 1 0.0544 2 0.0231 3 0.0054 4 Not Constant 1.20 1.00 WB (old) 0.0166 0.80 BoNTC(Hc) maximum unit/unit of 0.60 0.40 0.20 0.00 0 1 2 3 4 5 Gene copy number Effect of Gene Copy Number on MeOH Growth Rate and Production of BoNTC Hc

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  21. 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)

  22. 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.

  23. 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

  24. 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

  25. BoNTE Hc Purification

  26. 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.

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

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

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