1 / 67

Multicolumn Continuous Countercurrent Chromatography

Multicolumn Continuous Countercurrent Chromatography. Massimo Morbidelli Institute for Chemical and Bioengineering, ETH Zurich, Switzerland. Integrated Continuous Biomanufacturing 2013, 20 th – 24 th Oct, Barcelona. Outline.

casey
Download Presentation

Multicolumn Continuous Countercurrent Chromatography

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Multicolumn Continuous Countercurrent Chromatography Massimo Morbidelli Institute for Chemical and Bioengineering, ETH Zurich, Switzerland Integrated Continuous Biomanufacturing 2013, 20th – 24th Oct, Barcelona

  2. Outline • Process evolution: from batch to multicolumn simulated moving bed chromatography • Countercurrent Chromatography for three stream purifications • Countercurrent Chromatography for highly selective stationary phases • Application examples Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  3. Batch Chromatography • Selective adsorption leads todifferent elution velocities: select switch times • Features: • Linear gradients • Three fraction separations fastcomponent chromatographic column liquid flow slow component Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  4. ? Continuous Countercurrent Chromatography Selective adsorption leads todifferent elution velocities: select solid speed liquid flow solid flow Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  5. Simulated Moving Bed Chromatography • The SMB scheme: Eluent Raffinate (early eluting) 4 4 1 3 1 3 2 2 Feed Extract(strongly adsorbing) Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  6. Batch versus SMB performance • Separation of a pharmaceutical intermediate racemate mixture on a chiral stationary phase (CSP)1 8x -80% Eluent need [L/g] Productivity [g/ kg/min] 1 J.Chrom A 1006 (1-2): 267-280, 2003 Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  7. Typical bio-purification problem Example: mAb purificationfromcellculturesupernatant typicalchromatogramfor mAb elution on cation-exchanger: mAb HCPs aggregates fragments Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  8. Purificationchallenge Genericpurificationproblem:separate into 3 fractions #2: mAb #3: late eluting impurities #1: early eluting impurities Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  9. Purificationchallenge in 3-fraction batchchromatography:intrinsictrade-off betweenyieldandpurity! high yield, low purity high purity, low yield Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  10. Purificationchallenge in 3-fraction batchchromatography:intrinsictrade-off betweenyieldandpurity! Alternatives:- Very Selective Stationary Phase (eg, Protein A)- Continuous Countercurrent Chromatography (MCSGP) process yield alternatives ? purity Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  11.  continuous feed  multi-fractionseparation  counter-current operation  linear solvent gradients  high efficiency Combining batch and SMB Batch chromatography: SMB: pulsedfeed lowefficiency  binary separation  step solvent gradients MCSGP (Multi-column Countercurrent Solvent Gradient Purification): Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  12. Principle 6 Column Purification unit • Load // elute light • elute overlapping product/light • elute product • elute overlapping heavy/product • elute heavy • Receive overlapping product/light 3 2 6 4 1 5 L P c H inerts t t t t tF Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  13. Animation 6 Column MCSGPunit Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  14. Contichrom® & MCSGP explained Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli 24

  15. Continuous Countercurrent Chromatography for three Stream PurificationsMCSGP Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  16. Applicationof MCSGP: productclasses Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  17. mAb charge isoform separation(Cation Exchange) Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  18. Example : varyingmAbprofiles (variable isoformcontent) (Contichrom-purified) Feed Product Avastin® (Bevacizumab) Herceptin® (Trastuzumab) Ref: T. Müller-Späth, M. Krättli, L. Aumann, G. Ströhlein, M. Morbidelli: IncreasingtheActivityofMonoclonalAntibodyTherapeuticsbyContinuousChromatography (MCSGP), Biotechnology and Bioengineering, Volume 107, Issue 4, pages 652-662, 1 November 2010 Erbitux® (Cetuximab) Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  19. Comparison of Batch and MCSGP chromatography • Herceptin: Yield-Purity trade-off: Inherent to batch chromatography, less important for MCSGP Prod: 0.12 g/L/h Prod: 0.12 g/L/h MCSGP Batch trade-off Prod: 0.03 g/L/h Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  20. MCSGP operation - stability Robustness of process against feed quality variations Feed spiked with mAbisoforms Product Feed Blue: Regular Feed Red: High W feed Feed Blue: Regular Feed Red: Spiked feed Blue: Regular Feed Red: Spiked feed Purified with same MCSGP process conditions MCSGP product purity: Not affected by change of feed. Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  21. Example: BiobettermAb «Herceptin» • Originator mAb product «Herceptin» contains 7 isoforms with different activities (10%-150%) • Using MCSGP, a homogeneous biobetter product has been isolated with high yield and purity, having 140% activity • Potential for a Biobetter „Herceptin“ with lower dosing and better safety profile shown • Isoformheterogeneityappliesto all therapeuticmAbs Activityof Herceptin isoforms 140% 100% 12-30% Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli 43

  22. Bispecific antibody separation(Cation Exchange) Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  23. Purificationchallenge (Representative analytical chromatogram (CIEX) of the clarified harvest) Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  24. MCSGP performance 2-column MCSGP: • delivershighpurity >99.5% • increasesyieldby 50%- batchyield: 37%- MCSGP yield: 87% batch +50% yield Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  25. α-1-Antitrypsin purificationfrom human plasma(Cationexchange) Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  26. α-1-Antitrypsin purificationfrom human plasma – A280 – %B HSA BufferPeaks AAT IgG Analytical AIEX chromatogram Analytical results confirmed by ELISA

  27. α-1-Antitrypsin purificationfrom human plasma

  28. α-1-Antitrypsin purificationfrom human plasma MCSGP Product(AAT) Weak(IgG, HSA) StrongImpurities

  29. PEGylated protein separation (Anion Exchange) Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  30. PurificationofPEGylatedproteins • Constraints: • Low yieldofdesiredspeciesat expensive productionstepusingbatchchromatography • MCSGPprovides 50% higher yield and purity with 5x higher throughput Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  31. PurificationofPEGylatedproteins • MCSGP provides 50% higheryieldwith 5x higherthroughput Analytical SEC offeedand MCSGPproduct MCSGP: +10% purity MCSGP: +30% yield Batch chromatography Prep. AIEX Batch elutionoffeed (load 4.3 g/L) Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  32. Peptide purification I(Reverse phase) Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  33. P Polypetidepurification Peptide, ca. 46% pure, hundreds of unknown impurities Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  34. Purification Result - Polypeptide Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  35. Purification Result - Polypeptide Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  36. Purification Result - Polypeptide Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  37. Purification Result - Productivity • Joint project with Novartis Pharma on Calcitonin: Productivity [g/L/h] factor 25 Yield for constant purity [%] Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  38. Peptide purification II(Reverse phase) Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  39. Feed and representative batch material • Comparison of feed and representative batch chromatography pool from BMS Feed material – red BMS batch chromatography pool – blue A215 Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  40. Comparison of Batch and MCSGP • Overview of results: Analytical chromatography Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  41. Comparison of Batch and MCSGP • Overview of results: Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  42. Comparison of Batch and MCSGP • Overview of results: Purity-Yield chart. Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  43. Fatty acid Ethyl Ester separation (Reverse phase) Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  44. MCSGP for -3 fatty acid ethyl ester production (EPA-EE) • Performanalytical RP-HPLC batchchromatography • Feed purity 74%, targetpurity >97%(genericfishoilfeedpurchasedfrom TCI Europe N.V.) • Main impurityDocosahexaeonicacidethylester (DHA-EE) EPA-EE DHA-EE Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  45. MCSGP for -3 fatty acid ethyl ester production (EPA-EE) • Result chromatograms Overlay of analytical reversed phase chromatograms of feed and fractions from MCSGP Feed: Ratio EPA/DHA= 4:1 Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  46. MCSGP for -3 fatty acid ethyl ester production (EPA-EE) Process for production of > 97% purity EPA-EE developed based on reverse phase chromatography with Ethanol as solvent Resin & solvent cost reduction of 80% with respect to batch chromatography Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  47. Multicolumn countercurrent chromatography withvery selective stationary phases (eg, Protein A)Objective: Improve Capacity Utilization

  48. Process Principle Batch Column Continuous Multicolumn feed unused resincapacity elution feed fullyloadedcolumn Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  49. Multicolumn Capture Processes: 4-col process • 4-column process (4C-PCC): Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

  50. Multicolumn Capture Processes • 3C-PCC principlepresentedbyGenzyme (June 2012): • Continuous feed with the same flow rate in all phases Biotechnology and Bioengineering, Vol. 109, No. 12, December, 2012 Integrated ContinuousBiomanufacturing 2013, Barcelona / Massimo Morbidelli

More Related