BOKU BILS 2016
•
0 likes•590 views
This document summarizes a presentation given by A. Dürauer at the 9th Annual BioInnovation Leaders Summit on February 11th, 2016. The presentation discusses how high-throughput screening platforms can be used to address key questions in early biopharmaceutical process development, specifically around cell disruption, inclusion body recovery and processing, solubilization, and refolding/oxidation. The document outlines experimental workflows that could be used at a microscale to rapidly screen conditions across these unit operations in parallel. It suggests this approach could significantly reduce the time and materials needed for early process development activities compared to traditional benchscale methods.
Recommended
Recommended
More Related Content
What's hot
What's hot (20)
Viewers also liked
Viewers also liked (11)
Similar to BOKU BILS 2016
Similar to BOKU BILS 2016 (20)
More from GBX Events
More from GBX Events (20)
Recently uploaded
Recently uploaded (20)
BOKU BILS 2016
- 1. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 1Feb 11th, 2016 HTS platform for process development – can we address essential questions at early stage? A. Dürauer February 10th, 2016 9th Annual BioInnovation Leaders Summit Berlin, Germany
- 2. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 2Feb 11th, 2016 Boehringer-Ingelheim RCV Biopharma Process Science Austria
- 3. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 3Feb 11th, 2016
- 4. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 4Feb 11th, 2016 Source: N. Ferrer-Miralles et al. (2009), Microbial Cell Factories Classification of Biopharmaceuticals in Accordance to their Production System
- 5. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 5Feb 11th, 2016 Overall Costs of Production for Biopharmaceuticals
- 6. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 6Feb 11th, 2016 Biosimilars Individual Medicine Limited access to available treatment options Top Challenges in Biopharmaceutical Production
- 7. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 7Feb 11th, 2016 Reduce Costs of Goods Fasten Time to Market Increase Flexibility Top Challenges in Biopharmaceutical Production
- 8. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 8Feb 11th, 2016 Change of perspective from Screening for API /APC Screening for High Titer Production System DSP Development
- 9. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 9Feb 11th, 2016 Change of Perspective to Screening for API /APC Screening for High Titer Production System DSP Development
- 10. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 10Feb 11th, 2016 Reduce Costs of Goods Fasten Time to Market Increase Flexibility Change of Perspective to Screening for API /APC Screening for High Titer Production System DSP Development
- 11. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 11Feb 11th, 2016 Vision Analytics Analytics Screening for High Titer Production System DSP Development
- 12. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 12Feb 11th, 2016 Screening for High Titer Production System Upstream & Bioprocess
- 13. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 13Feb 11th, 2016 Downstream Processing DSP Development Centrifugation Filtration - Ultra / diafiltration Chromatography Cell desintegration - Mechanical - Chemical - Enzymatic Extraction Precipitation Crystallization Flocculation IB solubilization Refolding
- 14. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 14Feb 11th, 2016 Generalized Workflow of DSP Fermentation Broth Cell separation Product intracellular Cell disintegration Product as IBs Solubilization Refolding/ Oxidation Product soluble Extraction Product membrane associated Extraction Product extracellular Capture Intermediate Purfication Polishing Formulation
- 15. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 15Feb 11th, 2016 Process development chainStrain development/ screening Screening cultivation conditions Fermentation Broth Cellseparation Product intracellular Cell disintegration IBRecovery& Processing IBSolubilization Refolding/ Oxidation Capture Intermediate Purification Polishing Screening for Host System or High Titer Defined DSP
- 16. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 16Feb 11th, 2016 Process development chainStrain development/ screening Screening cultivation conditions Fermentation Broth Cellseparation Product intracellular Cell disintegration IBRecovery& Processing IBSolubilization Refolding/ Oxidation Capture Intermediate Purification Polishing DSP Development Selected Bioprocess Conditions
- 17. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 17Feb 11th, 2016 Essential questions to addressStrain development/ screening Screening cultivation conditions Fermentation Broth Cellseparation Product intracellular Cell disintegration IBRecovery& Processing IBSolubilization Refolding/ Oxidation Capture Intermediate Purification Polishing
- 18. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 18Feb 11th, 2016 Can we address essential questions at early stage? • Cell Disintegration • IB Recovery & Processing • IB Solubilization • Refolding /Oxidation
- 19. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 19Feb 11th, 2016 Cell disintegration Analytics: - Total protein release (UV, SDS-PAGE, 2D-DIGE) - Product release (flourescence, Bradford) - DNA release (picoGreen) - Endotoxin release (LAL)
- 20. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 20Feb 11th, 2016 Product release DNA release Cell disintegration Endotoxin release For process development: Bead mill is preferable to enzymatic digestion Product release DNA release Endotoxin release mgproduct/gBM
- 21. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 21Feb 11th, 2016 Can we address essential questions at early stage? • Cell Disintegration • IB Recovery & Processing • IB Solubilization • Refolding /Oxidation
- 22. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 22Feb 11th, 2016 Homogenization via bead mill Transfer of homogenate to new plate Separation via centrifugation Discard supernatant Resuspension ~1:10 in Wash buffer Separation via centrifugation Discard supernatant Transfer of IBs for analysis Analysis of supernatant (DNA, OD, SDS-PAGE) Analysis of supernatant /pellet (DNA, OD, SDS-PAGE) IB Recovery & Processing X time
- 23. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 23Feb 11th, 2016 IB Recovery & Processing Micro Scale Bench Scale Wash 1 Wash 2 IBs Wash 1 Wash 2 IBs
- 24. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 24Feb 11th, 2016 Wash 1 Wash 1 Wash 1 Wash 2 Wash 2 Wash 2 Wash 3 Wash 3 Wash 3 Homogenate IBs Homogenate IBs Homogenate IBs Overall Protein Overall Protein DNA content DNA content Product IB recovery & Processing Product ? Micro Scale Bench Scale
- 25. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 25Feb 11th, 2016 Can we address essential questions at early stage? • Cell Disintegration • IB Recovery & Processing • IB Solubilization • Refolding /Oxidation
- 26. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 26Feb 11th, 2016 A. Dürauer, S. Mayer, W. Sprinzl, A. Jungbauer, R. Hahn, Biotech. J. 4 (2009), 722 - 729 IB Solubilization
- 27. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 27Feb 11th, 2016 IB Solubilization DoE with screening of ! 3 pH values ! 3 urea concentrations ! Concentration of added GuHCl
- 28. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 28Feb 11th, 2016 Can we address essential questions at early stage? • Cell Disintegration • IB Recovery & Processing • IB Solubilization • Refolding /Oxidation
- 29. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 29Feb 11th, 2016 Indication of aggregation / precipitation = unfavored conditions Determine wavelength shift and scattering = Indication of right formation or aggregation Dilution of solubilized IBs into refolding buffer Turbidity measurement Intrinsic Flourescence Emission Scan Selective capture for refolded protein cGE analysis of eluted protein Refolding & Oxidation
- 30. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 30Feb 11th, 2016 Can we address essential questions at early stage? • Cell Disintegration • IB Recovery & Processing • IB Solubilization • Refolding /Oxidation
- 31. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 31Feb 11th, 2016 OutputStrain development/ screening Screening cultivation conditions Fermentation Broth Cellseparation Product intracellular Cell disintegration IBRecovery& Processing IBSolubilization Refolding/ Oxidation Capture Intermediate Purification Polishing Screening for Host System or High Titer Defined DSP Parallel screening of 32 fermentations Duration: 7 days
- 32. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 32Feb 11th, 2016 OutputStrain development/ screening Screening cultivation conditions Fermentation Broth Cellseparation Product intracellular Cell disintegration IBRecovery& Processing IBSolubilization Refolding/ Oxidation Capture Intermediate Purification Polishing DSP Development Selected Bioprocess Conditions Parallel screening 24 x solubilization, 96 x refolding and 24 x capture conditions Duration: 5 days
- 33. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 33Feb 11th, 2016 Benefits Protein [mg] Time Refolding screening Äkta Janus BioTx Tecan Bench 96 4 60 d 21 d 4 d ! Time and material consumption significantly reduced ! Broader knowledge of process
- 34. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 34Feb 11th, 2016 Can we address essential questions at early stage? Strain development/ screening Screening cultivation conditions Fermentation Broth Cellseparation Product intracellular Cell disintegration IBRecovery& Processing IBpreparation/ Solubilization Refolding/ Oxidation Capture Intermediate Purification Polishing Yes, we can !
- 35. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 35Feb 11th, 201610.02.2016 35 Acknowledgements Lars Demmel Nicole Weiner Helga Zambiasi Marian Koglbauer Matthias Berkemeyer Cécile Brocard Georg Klima Boehringer-Ingelheim RCV Biopharma Process Science Austria BOKU Vienna Department of Biotechnology Cornelia Walther Martin Kellner
- 36. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 36Feb 11th, 2016 IB solubilization D H L F d h D H L F d h lF h d‘ d Scale Up Factor >500
- 37. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 37Feb 11th, 2016 Selected refolding conditions 1 (DoE 1) 2 (DoE 2) 3 (DoE 3) 4 (DoE 4) 4 M UREA 0.1 M Arginine 0.1 M Tris 0.18 mM Tween 20 2 mM Cysteine 2 mM cysteine pH9.3 3 M UREA 0.5 M Arginine 0.4 M Tris 18 mM CHAPS 2 mM Cysteine 2 mM cysteine pH 7.8 3.5 M UREA 0.1 M Arginine 0.5 M Tris 0.18 mM Tween 20 2 mM Cysteine 2 mM cysteine pH 8.7 4 M UREA 0.05 M Tris 2 mM Cysteine 2 mM cystine pH 9.5 DoE 4 buffer chosen because of ! Least CoG ! No high dilution of refold required to reach target conductivity before CIEX ! No additives which need to be removed later in DSP 1 (DoE 1) 2 (DoE 2) 3 (DoE 3) 4 (DoE 4) 4 M UREA 0.1 M Arginine 0.1 M Tris 0.18 mM Tween 20 2 mM Cysteine 2 mM cystine pH9.3 3 M UREA 0.5 M Arginine 0.4 M Tris 18 mM CHAPS 2 mM Cysteine 2 mM cystine pH 7.8 3.5 M UREA 0.1 M Arginine 0.5 M Tris 0.18 mM Tween 20 2 mM Cysteine 2 mM cystine pH 8.7 4 M UREA 0.05 M Tris 2 mM Cysteine 2 mM cystine pH 9.5 Yield in bench scale ~100% ~30% ~90% ~85% Refolding & Oxidation
- 38. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 38Feb 11th, 2016 Summary - Product titer IB Recovery & Processing Small Scale Processing is Reproducible g IB/g biomass mg Product/g IB wet Small Scale 1 Small Scale 2 Bench Scale Small Scale 1 Small Scale 2 Bench Scale Factor Bench/Small V554 0.408 0.33 0.136 17.6 17 126 7 V556 0.365 0.359 0.111 21.9 18.9 91.8 5 V561 0.259 0.279 0.124 19 21.3 81.4 4 V566 0.177 0.183 0.03 4.4 1.1 26.7 26 g IB/g biomass mg Product/g IB dry Small Scale 1 Small Scale 2 Bench Small Scale 1 Small Scale 2 Bench Factor Bench/Small V554 0.408 0.33 0.136 - 115.4 240.0 2 V556 0.365 0.359 0.111 - 199.9 266.2 1.4 V561 0.259 0.279 0.124 - 151.5 332.5 2 V566 0.177 0.183 0.03 - 7.65 92.4 11
- 39. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 39Feb 11th, 2016 Reject turbid samples for following analysis Refolding & Oxidation Turbidity
- 40. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 40Feb 11th, 2016 Combination of wavelength shift and scattering (RALLS) allows qualitative detection of successful refolding Determine aggregation Intrinsic fluorescence Refolding & Oxidation
- 41. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 41Feb 11th, 2016 Confirmation of capture to affinity resin SolubilizedIB Standard Standard Standard Refolding FT Eluate Refolding FT Eluate Refolding FT Eluate Refolding FT Eluate Refolding FT Eluate Refolded protein 90% of refolded protein binds to affinity resin, 60% of bound protein is eluted and can be used for cGE measurements Refolding & Oxidation
- 42. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 42Feb 11th, 2016 ! Fully automated screening of 96 refolding conditions ! Automated preparation of refolding buffers from stocks ! Variation of pH, salt concentration, stabilizing agents, redox system and additives ! DoE based ! Less than 4 mg of solubilized protein required per screening ! Result: Refolding yield & quality Refolding & Oxidation
- 43. University of Natural Resources and Life Sciences Department of Biotechnology 9th Annual BioInnovation Leaders Summit A. Dürauer 43Feb 11th, 2016 IB Solubilization ! Fully automated screening of 24 solubilization conditions ! Automated preparation of solubilization buffers from stocks ! Variation of pH, chaotrope concentration, reducing agents, additives ! DoE based ! Less than 4 mg of Ibs required per screening ! Result: Solubilization yield & kinetics