Extraction and purification of metabolites by Centrifugal Partition Chromatography. Luc MARCHAL Sébastien CHOLLET, Jean-Hugues RENAULT
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1 Extraction and purification of metabolites by Centrifugal Partition Chromatography Luc MARCHAL Sébastien CHOLLET, Jean-Hugues RENAULT
2 PROCESS AND BIOPROCESS ENGINEERING Liquid-liquid separation processes and biorefinery
3 Experts in CPC process, from feasibility to industrialization
4 Summary CPC State of the Art I- Support-free chromatographic processes II- Purification of alkaloids III- Metabolites capture 4
5 State of the art C entrifugal P artition A separation process liquid-liquid C hromatography without solid support. 5
6 State of the art Ascending mode Cell PUMP Elution Descending mode Canal Cell Elution G Canal PUMP 6
7 State of the art CPC Column Biphasic System G Cell duct Disk 7
8 State of the art: outlines Testing facilities: 8
9 State of the art: outlines L 0.25 L 1 L 5 L 15 L
10 State of the art: outlines
11 Summary CPC State of the Art I- Support-free chromatographic processes II- Purification of alkaloids III- Metabolites capture 11
12 The support-free process dvt Equilibrium data Chromatographic system Target molecules Phases separation mechanism Hydrodynamics mass transfer Cell choice Visual CPC Geometry efficiency Flow, acceleration Process engineering Intensification Column sizing No yes Catalog column Workable? Custom column 12
13 Handling liq-liq biphasic systems From ATPS to nonaqueous systems, including DES, ScFluids. For applications from proteins purification to lipids 13
14 Hydrodynamics in CPC process
15 Columns designs
16 Summary CPC State of the Art I- Support-free chromatographic processes II- Purification of alkaloids III- Metabolites capture 16
17 ph-zone Refining for alkaloids Vindoline Catharanthine Madagascar periwinkle Purity (%) >70 >70 Recovery (%) >80 >85 3kg of crude extract per day
18 Process dvt methodology
19 Productivity claims CPC phzr separation time: 45 min/run, 1 day = 30 runs g 100 g of crude/run V inj = 300 ml
20 CPC model including reaction terms F= 250 ml/min, G= 310 g, Sf= 68%, koa= 0.12s cells are sufficient 1200mL CPC column
21 Experimental process scale FCPE cells 1400mL Sf=68%, koa = 0.16 s -1 Model vs Experimental productivity = 212 vs 199 mm.l -1.h g of crude extract per day is validated
22 Summary CPC State of the Art I- Support-free chromatographic processes II- Purification of alkaloids III- Metabolites capture 22
23 Protein capture at the PhotoBioReactor outlet B-Phycoerythrin from P cruentum ATPS are used for proteins extraction/purification High selectivity can be obtained by adjusting salt concentration, PEG mw and adding some cations Focus on protein capture and mass transfert
24 Capture of 240 kda protein The protein of interest is captured at the column inlet, concentrated 10 folds Capture kinetics is used for process modeling and column sizing koa= s -1 24
25 Capture of 240 kda protein Inlet purity factor
26 Metabolites capture: CPC for early stage in DSP Protein concentration x 10 Purity x 5 Full-scale production can be obtained with CPC columns The low cost of the stationary phase and the lowering of the handled volumes make the CPC process more competitive than conventional precipitation/resin columns processes 26
27 Wet route lipids extraction from N gaditana, P kessleri
28 Ph D : Emilie Angles, Valeria Montalescot Solvents Production Harvesting L-L Extraction Raffinate Extract Microalga Culture media interface Extracting solvent??? 28
29 Lipids are stored inside the cells as oleosomes i.e. «encapsulated» in the cytoplasm What is the mass transfer driving force? Solubility in the solvent; extraction phenomenon more close to coalescence at a liquid surface than to partitioning Mass transfer limitations? Cell membrane/wall (no exsudation) Meeting probability with the solvent (mixing, interfacial specific area-m 2 /m 3 ) Extracting capacity of the solvent 29
30 Cell disruption, a necessary step? Permeabilisation Slight disruption Desintegration Chloroplaste (pigments/lipides) Paroi cellulaire (carbohydrates) Cell disruption degree / Energy Lipides de réserve (TAG) Corps pigmenté (pigments/lipides) Membrane cellulaire (lipides)
31 Disruption Rate of N. oculata = f(p) by BHP No stress: N-repleted Stress: N-depleted + high light = Lipids accumulation 31
32 Cell disruption /LL-E coupling η E,N Cart AGT TFA Chl a Extraction controlled by disruption for selective solvent Potential selectivity of lipids with Heptane τ d,n 32
33 Solvent screening Solvent Molecular Formula s (g/l) d (g/cm -3 ) T eb ( C) Cyclohexane C 6 H 12 immiscible Heptane C 7 H Chloroform CHCl Toluene C 7 H MIBK C 6 H 12 O EtOAc C 4 H 8 O DMC C 3 H 6 O R-Limonen C 10 H 16 immiscible MeTHF C 5 H 10 O MtBE C 5 H 12 O CPME C 6 H 12 O EthylButylAmine C 6 H 15 N switchable dibutylamine C 8 H 19 N switchable
34 Solvent screening 3 extraction trends 34
35 Solvent screening Hansen Solubility Parameters (HSP) = (eq. Hildebrand) Water MeOH: Decomposition according to Hansen: E=Ed + EP + Eh δd2 + δp2 + δ h2 Or ΔHM = VM (δ1 - δ2)2 ρ1. ρ2 Δδ : Factor to minimize (δd1 - δd2)2 + (δp1 - δp2)2 + (δh1 - δh2)2 35
36 Extraction Kinetics with CPE Solvent DMC MTBE AcOEt Toluene DBA d (g/cm -3 ) T eb ( C) η (mpa.s) S olubility in water (g/l) different solvents : Extraction efficiency Polarity Solubility in water Density Disc and cells of CPE 36
37
38 38 Solvent (org) Feed (aq) CPE Extract (org) Raffinate (aq)
39 Power Concentration Solvent Pretreatment 39
40 Switchable Solvent A- Dibutylamine (DBA) : Solvent DBA Disrupted cells B- DBA : Switchable Solvent Acid Amine water Mixing Base ph= Pk a -1 ph= Pk a +1 Recovering Extract Residue Recovering Extract Residue Solvent + TFA Solvent + TFA 40
41 Mechanism of extraction with amine Amine droplets Disrupted cells Dibutylamine (DBA) improved extraction phenomena by switch phase: DBA action on disrupted cellular culture with nile red staining after switch (x1000) 1. Droplet formation close to cells debris 2. Solubilization of lipids 3. Coalescence of droplets enriched in lipids 4. Phase s separation 41
42 Mechanism of extraction with amine τ d = 30% DBA more efficient than toluene Switched DBA alone, has only improved the recovery of pigments 90% less of DBA increased extraction efficiency of toluene by 50% 42
43 Conclusions Cell disruption is a key and limiting step in the biorefinery scheme 1st investigate the metabolite production at the bioprocess step Selective release of metabolites is possible for finely tuned disruption process (modeling kinetics is necessary) Wet route extration of lipids is feasible and scale-up possible if extraction intensification is done : - Specific extraction modes (transitory, reactions ) - Solvent ratio lowering - Higher biomass concentration in the feed 43
44 Conclusions CPC has reached TRL7 : tools, knowledge, equipment ready for full-scale development Scale-up rules are known for elution and displacement mode : Invariants are capacity and mass transfer, depending on hydrodynamics CPC = intensified LL contactor Quite simple design, no solid support CPC especially promising for biomolecules
45 UMS ALGOSOLIS
46 L équipe ambiance!
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