Use of Fluidised bed chromatography for plasma fractionation. Karl McCann*, John Wu, Peter Gomme & Joseph Bertolini
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1 Use of Fluidised bed chromatography for plasma fractionation Karl McCann*, John Wu, Peter Gomme & Joseph Bertolini
2 Plasma Fractionation Industry Mature with well established processes Revenue growth - throughput - capital expenditure Competitive advantages can be gained from process innovations increased efficiency
3 Methods for Plasma fractionation Cohn (Ethanol precipitation) Chromatography
4 Cohn purification Advantages - High throughput - Lower cost - Low complexity Disadvantages - Lower purity - Lower yield - Ethanol usage (OHS) - Low temperatures
5 Chromatography purification Advantages - High purity - High yield - Better automation - More gentle on product Disadvantages - Low throughput - Higher cost - More complex - High buffer usage - Resin cleaning issues
6 Improving chromatography Improving efficiency = increased throughput & lower cost Increase efficiency by: - increasing flow rates - increasing binding capacity - increasing specificity Expanded bed adsorption chromatography?
7 Expanded bed adsorption (EBA) chromatography First used in late 80 s / early 90 s Biotechnology applications (capture of low abundant proteins from fermentation broths)
8 Packed Bed Expanded Bed
9
10 Traditional purification scheme Purification scheme using EBA Fermentation broth Fermentation broth Clarification Concentration EBA chromatography Capture Intermediate purification Intermediate purification Polishing Polishing
11 Advantages of EBA chromatography Higher flow rates Time savings Higher yields (fewer steps) Less capital costs Less proteolytic damage of valuable product (fermentations)
12 Incorporation of EBA chromatography into plasma fractionation process Isolation of Prothrombin complex concentrate (PCC) from Cohn SNI
13 Current manufacturing process 2500 kg SNI + 90 kg of Whatman DE32 resin (batch mode) Resin harvested by centrifugation Resin packed into column Washed to elute unbound proteins PCC eluted Column operated at 30 cm/h Column cleaned, regenerated and equilibrated
14 Issues with current method Cohn SNI contains residual fibrinogen packed bed chromatography is difficult Whatman DE32 cellulose based resin = poor rigidity resulting high compressibility and high back pressures Manual handling transfer of resin between centrifuge, column and tank
15 Experimental trials setup of EBA system Bed expansion trials for different resins Bed expansion trials in different solutions and protein feedstock BSA binding capacity packed bed versus expanded bed
16 Bed expansion factor (H/H o ) H H o Settled bed (zero flow) Expanded bed (flow applied)
17 Bed expansion of different resins Streamline DEAE Whatman DE32 DEAE Sepharose FF 3 Bed expansion (H/Ho) Optimum bed expansion Flow rate (cm/h)
18 Bed expansion in different solutions 3 Bed expansion (H/Ho) Water Buffer W Buffer E 1 M NaCl 1 M NaOH Cohn SNI Flow rate (cm/h) Streamline media
19 Viscosity and density of solutions Component Density (g/cm 3 ) Viscosity (cp) Water Buffer W Buffer E M NaCl M NaOH Cohn SNI
20 BSA binding capacity (EBA versus packed bed) BSA binding capacity (mg/ml) Resin type Flow rate Packed-bed Expanded-bed (cm/h) Whatman DE Streamline DEAE Streamline Q-XL
21 EBA method for PCC extraction from Cohn SNI Equilibration Load Cohn SNI Buffer W Flow through (Residual Factor II) Buffer E PCC Eluate 1 M NaCl 1 M NaCl wash Regeneration
22 Factor II levels in EBA flow through Starting level IU/mL Factor II content (IU/mL) Streamline DEAE Streamline Q-XL Volume loaded (ml)
23 Characterisation of current process % of total loaded Component PCC Eluate NaCl wash Factor II Factor IX Factor X ITI Protein S Protein C Plasminogen < 0.5 < 0.4 Ceruloplasmin Albumin < 0.1 < 0.1 IgG < 0.1 < 0.1
24 Characterisation of EBA process PCC Eluate % of total loaded Component Streamline DEAE Streamline Q-XL 1 M NaCl % of total loaded Streamline DEAE Streamline Q-XL Factor II Factor IX Factor X ITI Protein S Protein C Plasminogen Ceruloplasmin Albumin < IgG < 0.1 < < 0.1
25 Estimation of process times at Manufacturing-scale Column diameter Linear flow rate Volumetric flow Time (min) required to (mm) (cm/h) rate (kg/min) process kg batch (200 L) (110 L) (50 L)
26 Efficiency gains using EBA EBA Current process Loading 2.5 h Cohn SNI + resin & centrifugation 2 h Washing 0.5 h Washing 1 h PCC elution 0.5 h PCC elution 1 h Regeneration & Equilibration 2.5 h Regeneration & Equilibration 45 h
27 Summary EBA chromatography can be incorporated in to an existing plasma fractionation process EBA results in efficiency increase (30 cm/h vs cm/h) & reduces manual handling issues Feedstock differences High protein concentration (Higher density resins)
28 Future for EBA in plasma fractionation industry? Capture of low abundant proteins (long loading times) Processes where residence time is not critical (use of high flow rates)
29 Acknowledgements John Wu Peter Gomme Joseph Bertolini CSL Bioplasma
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