The amazing ability of continuous chromatography to adapt to a moving environment.

1. The amazing ability of continuous chromatography to adapt to a moving environment. Roger-Marc Nicoud, Founder of Novasep Barcelona, October 2013

2. Introduction and for that, I will use shortcuts , approximations and manipulations … Mythesis: the environmentpressure pushesContinuousChromatography to evolvelikeanimalsevolvedaccording to Darwin’slaw ….

3. Improvingwhat ? Continuouscan help Counter-currentcan help Life isoften a matter of compromize

4. Continuousis NOT synonymouswithcounter-current A continuous but NOT counter-current animal

5. Be carefulwithcomparisons Productivitykg/kg/d Optimum particle size, eluent composition … are different for Batch and continuous/CC processes.

6. S.M.B.: the key evolution ….. Evolution lead to the first continuous AND counter-current animal solid liquid Minimizing E.C. Maximizeproductivity IV III Feed II I Eluent

7. The first evidence of continuouschromatography

8. Environmentpressure : Paraxylene • Scale (500,000 Mt/Y/Unit in 2010) • Chromatographic conditions: • Selectivity about 2 • Particle size about 1000 microns (inducing “poor” HETP ….) • Temperature about 180 °C (inducing low viscosity) • Technical constraints • Target purity: > 99.8 % • “individual bed length” : limited to 1 m due to mechanical constraints significant bed length of 24 m (no pb because of low viscosity) 24 columns to reach total length

9. Environment pressure : Fructose • Scale (50,000 Mt/Y/Unit in 2010): from Glucose-Fructose mixtures • Chromatographic conditions: • Selectivity about 1.5 • Particle size about 300-500 microns (inducing “poor” HETP) • Temperature: 60 °C (thus viscosity about 10 cp ) • Technical constraints • Target purity: about 90 % • Minimize energy consumption for evaporating Water total bed length of about 8-12 meters

10. Improving the classical SMB • Addsomedegree of freedomby: • Connectingcolumns in differentways • Selectingdifferentswitching modes • Varying I/O lines composition and flows • New processesare normally: • quasi-continuousinstead of beingtrulycontinuous • takingadvantage of «counter-current» contact

11. Fructose: furtherimprovement A new animal Twosubperiods • «Cousin animals»: • SSMB Novasep • ISMB Mitsubishi • Commercial solutions implemented: • 4 or 6 columns • Eluent consumptiondecreased by about 10-15 % compared to SMB

12. Environment pressure : opticalisomers • Scale (1-100 Mt/Y/Unit in 2010) • Chromatographic conditions: • Selectivity: typically between 1.2 and 2 • Little influence of particle size on packing cost • HETP depending on particle size … • Technical constraints • Target purity: 95-99 % • Minimize system size / CSP inventory • Use small particle size Total bed length about 0.5 m …. To be distributed between Ncol… At large scale L/D < 0.1 …..

13. The first SMB animal for separatingopticalisomers (1992): Influence of petrochemical design isvery visible …. and the modern version ….

14. Optical isomers: furtherimprovement VariCol • VariCol: • Trulycontinuous • Same hardware as SMB • Typicallysaves one column out of 5-6.

15. Key successes For main successes, continuouschromatographyiscoupledwithcrystallization, isomerization and multiple effectevaporators

16. Very large scaledoes not mean« second-class » performance

17. In Line Dilution is key

18. Environmentpressure : need for multicomponent (bio)separations. Technicallyone can design a continuousthree pure fractions TMB. The generic purification problem : An animal with no progeny ? VIII Imposes to workwithtwosuboptimalsystems ? VII time W P S VI V Prefertwo SMB in series : Paclitaxel, Cyclosporine, EPA … reduces to a binaryseparation if one wants the green or the redproduct. SMB isperfectlyadapted to these situations (ex.: desalting, xylenes, capture …), IV III II or change concept … I

19. Improvingmulticomponentseparations MCSGP (MulticolumnCountercurrentSolvent Gradient Purification) from ETH. VI W P+W • Important features of the animal: • Openingliquidloop • Short-circuit • Gradient V Feed P S W IV P P+S A trulycontinuous gradient chromatographicprocesswith 6-columns III II S I

20. 3-column MCSGP Twosubperiods VI VI VI P+W P+W W V V V IV IV IV Can bedonewiththreecolumns Not strictlycontinuous but countercurrent “Cousin animal” from Novasep : GSSR Gradient (with) Steady-State Recycling P III III P+S P+S III II II II S I I I

21. Loading t Washing Elution Regeneration Equilibration tcycle Environmentpressure : need for capturing (biomolecules) Selectivityissuchthat a solvent change ismandatory S Verystronglyretained P W Non retained

22. Bioseparations: the BioSc concept Twosubperiods W W Wash * Feed W • “Cousin animals” from : • Chromacon (Capture SMB) • GE (3C PCC) W Wash Wash Use sequentialfeeding to reduce the numbercolumns (as low as two). P P Target Target Elute Elute S S Regenerate Regenerate Waste Waste Equilibrate Equilibrate

23. Lysine production main steps FERMENTATION MEMBRANE BIOMASS FILTRATION ION EXCHANGE PURIFICATION About 50,000 MT/year/unit ACIDIFICATION EVAPORATION CRISTALLISATION CRUDE L LYSINE HCl

24. Lysine purification: switch from batch to continous (a decadeago) • First step: Strong Cation Exchange • Feed pH 3-4 sothat Lys+ (maximizesloading and separation) • Then pH lowered to 1-2 sothat Lys++ (maximizes capture of small concentrations) • Wash • Elution by ammoniaat pH 9 (Lys0) • Ammoniarecycledthrough stripping • 97% Lys withmicrobialbyproducts and mineral cations • Second step: Weak Cation Exchange • Feedat pH 9 (Lys0goesthrough) • Regenerationat pH withdilutedsulphuricacid • Wash • Gain continuous (about 10-column systems) vs Batch: resininventoryand effluent divided by about 3

25. Biopharma Stilllooking for a first Biopharmaindustrial application of continuouschromatography ? • Many proof of concept published: • mAbs • pegylatedproteins • peptides • fattyacids …..

26. Evolution …. Continuouscharacter Xylene 100 % Sugars 12 6 4 24 2 I-SMB S-SMB Number of columns 50 % MCSGP GSSR BioSc 3C PCC VariCol MCSGP 1960 1980 2000 2010 2020 ?

27. Thankyou !

28. Processintegrationmay lead to non intuitive processes … Intuition S M B Glu 90 % Isomerization Glu-Fru58-42 Glu 95 % HFS 55 % Optimized S M B Glu 90 % Isomerization Glu-Fru58-42 Glu 95 % HFS 55 % Fru 90 %