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

2. Introduction My thesis: the environment pressure pushes Continuous Chromatography to evolve like animals evolved according to Darwin’s law …. and for that, I will use shortcuts, approximations and manipulations …

3. Improving what ? System size Eluent consumption YieldOperations Comment Productivity kg/kg/day normally presented as the main objective function Minimizing solvent consumption (prior to recycling) is a must in all large scale applications. Can be a major cost contributor Control / Process Stability / Cleaning can be critical costs contributors. Mitigation large LP systems can be cheaper than smaller HP systems Biopharma industry still a bit less concerned Ancillary equipment is even more important than chromatography itself. Life is often a matter of compromize Continuous can help Counter-current can help

4. Continuous is NOT synonymous with counter-current A continuous but NOT counter-current animal

5. Be careful with comparisons Optimum particle size, eluent composition … are different for Batch and continuous/CC processes.

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

7. The first evidence of continuous chromatography

8. Environment pressure : 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 24 columns to reach total length significant bed length of 24 m (no pb because of low viscosity)

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 Add some degree of freedom by: – Connecting columns in different ways – Selecting different switching modes – Varying I/O lines composition and flows New processes are normally: – quasi-continuous instead of being truly continuous – taking advantage of «counter-current» contact

11. Fructose: further improvement Commercial solutions implemented: 4 or 6 columns Eluent consumption decreased by about 10-15 % compared to SMB Two sub periods «Cousin animals»: SSMB Novasep ISMB Mitsubishi A new animal

12. Environment pressure : optical isomers 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 N col … At large scale L/D < 0.1 …..

13. The first SMB animal for separating optical isomers (1992): Influence of petrochemical design is very visible …. and the modern version ….

14. Optical isomers: further improvement VariCol: Truly continuous Same hardware as SMB Typically saves one column out of 5-6. VariCol

15. Key successes For main successes, continuous chromatography is coupled with crystallization, isomerization and multiple effect evaporators

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

17. In Line Dilution is key

18. Environment pressure : need for multicomponent (bio)separations. Technically one can design a continuous three pure fractions TMB. I II III IV V VI VII VIII Imposes to work with two suboptimal systems ? The generic purification problem : reduces to a binary separation if one wants the green or the red product. SMB is perfectly adapted to these situations (ex.: desalting, xylenes, capture …), time W P S or change concept … An animal with no progeny ? Prefer two SMB in series : Paclitaxel, Cyclosporine, EPA …

19. Improving multicomponent separations S W P S W P I II III IV V VI P+S P+W A truly continuous gradient chromatographic process with 6-columns MCSGP (Multicolumn Countercurrent Solvent Gradient Purification) from ETH. Important features of the animal: Opening liquid loop Short-circuit Gradient Feed

20. S W P I II III IV V VI I II III IV V VI P+S P+W I II III IV V VI P+S P+W Can be done with three columns Not strictly continuous but counter current Two sub periods 3-column MCSGP “Cousin animal” from Novasep : GSSR Gradient (with) Steady-State Recycling

21. Environment pressure : need for capturing (biomolecules) S Very strongly retained W Non retained P Selectivity is such that a solvent change is mandatory Loading Washing Elution t Regeneration Equilibration t cycle

22. Bioseparations: the BioSc concept Use sequential feeding to reduce the number columns (as low as two). Regenerate Equilibrate Target Feed Elute Waste S W P Wash W Wash * Two sub periods Regenerate Equilibrate Target Elute Waste S W P Wash W “Cousin animals” from : Chromacon (Capture SMB) GE (3C PCC)

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

24. Lysine purification: switch from batch to continous (a decade ago) First step: Strong Cation Exchange Feed pH 3-4 so that Lys + (maximizes loading and separation) Then pH lowered to 1-2 so that Lys ++ (maximizes capture of small concentrations) Wash Elution by ammonia at pH 9 (Lys 0 ) Ammonia recycled through stripping  97% Lys with microbial byproducts and mineral cations Second step: Weak Cation Exchange Feed at pH 9 (Lys 0 goes through) Regeneration at pH with diluted sulphuric acid Wash Gain continuous (about 10-column systems) vs Batch: resin inventory and effluent divided by about 3

25. Biopharma Still looking for a first Biopharma industrial application of continuous chromatography ? Many proof of concept published: mAbs pegylated proteins peptides fatty acids …..

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

27. Thank you !

28. Process integration may lead to non intuitive processes … HFS 55 % Glu 95 % Isomerization Glu-Fru 58-42 SMBSMB Glu 90 % HFS 55 % Intuition SMBSMB Glu 95 % Isomerization Glu-Fru 58-42 Glu 90 % Fru 90 % Optimized