Use of Fluidised bed chromatography for plasma fractionation Karl McCann*, John Wu, Peter Gomme & Joseph Bertolini
Plasma Fractionation Industry Mature with well established processes Revenue growth - throughput - capital expenditure Competitive advantages can be gained from process innovations increased efficiency
Methods for Plasma fractionation Cohn (Ethanol precipitation) Chromatography
Cohn purification Advantages - High throughput - Lower cost - Low complexity Disadvantages - Lower purity - Lower yield - Ethanol usage (OHS) - Low temperatures
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
Improving chromatography Improving efficiency = increased throughput & lower cost Increase efficiency by: - increasing flow rates - increasing binding capacity - increasing specificity Expanded bed adsorption chromatography?
Expanded bed adsorption (EBA) chromatography First used in late 80 s / early 90 s Biotechnology applications (capture of low abundant proteins from fermentation broths)
Packed Bed Expanded Bed
Traditional purification scheme Purification scheme using EBA Fermentation broth Fermentation broth Clarification Concentration EBA chromatography Capture Intermediate purification Intermediate purification Polishing Polishing
Advantages of EBA chromatography Higher flow rates Time savings Higher yields (fewer steps) Less capital costs Less proteolytic damage of valuable product (fermentations)
Incorporation of EBA chromatography into plasma fractionation process Isolation of Prothrombin complex concentrate (PCC) from Cohn SNI
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
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
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
Bed expansion factor (H/H o ) H H o Settled bed (zero flow) Expanded bed (flow applied)
Bed expansion of different resins 4 3.5 Streamline DEAE Whatman DE32 DEAE Sepharose FF 3 Bed expansion (H/Ho) 2.5 2 1.5 Optimum bed expansion 1 0.5 0 0 50 100 150 200 250 300 Flow rate (cm/h)
Bed expansion in different solutions 3 Bed expansion (H/Ho) 2.5 2 1.5 Water Buffer W Buffer E 1 M NaCl 1 M NaOH Cohn SNI 1 0 50 100 150 200 250 300 Flow rate (cm/h) Streamline media
Viscosity and density of solutions Component Density (g/cm 3 ) Viscosity (cp) Water 0.996 0.89 Buffer W 1.004 0.91 Buffer E 1.014 0.93 1 M NaCl 1.035 0.94 1 M NaOH 1.039 1.03 Cohn SNI 1.008 1.48
BSA binding capacity (EBA versus packed bed) BSA binding capacity (mg/ml) Resin type Flow rate Packed-bed Expanded-bed (cm/h) Whatman DE32 50 86 28 Streamline DEAE 300 57 52 Streamline Q-XL 300 176 170
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
Factor II levels in EBA flow through 0.7 0.6 Starting level - 0.589 IU/mL Factor II content (IU/mL) 0.5 0.4 0.3 0.2 Streamline DEAE Streamline Q-XL 0.1 0 0 500 1000 1500 2000 2500 3000 Volume loaded (ml)
Characterisation of current process % of total loaded Component PCC Eluate NaCl wash Factor II 57.5 5.0 Factor IX 54.2 7.5 Factor X 38.3 4.8 ITI 30.0 3.9 Protein S 5.4 0.4 Protein C 26.3 2.1 Plasminogen < 0.5 < 0.4 Ceruloplasmin 1.9 0.5 Albumin < 0.1 < 0.1 IgG < 0.1 < 0.1
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 19.5 13.0 Factor IX 18.6 6.2 Factor X 15.9 9.5 ITI 20.3 23.0 Protein S 10.0 17.7 Protein C 15.6 12.5 Plasminogen 2.4 1.7 Ceruloplasmin 5.7 14.3 Albumin < 0.1 0.1 IgG 0.4 0.1 42.4 50.1 19.9 33.4 8.3 61.2 24.7 26.1 8.2 11.6 5.6 13.7 4.0 0.8 1.0 10.3 < 0.1 < 0.1 0.1 < 0.1
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 2 500 kg batch 1200 150 28.3 88 (200 L) 900 150 16.0 156 (110 L) 600 150 7.1 354 (50 L)
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
Summary EBA chromatography can be incorporated in to an existing plasma fractionation process EBA results in efficiency increase (30 cm/h vs 150 300 cm/h) & reduces manual handling issues Feedstock differences High protein concentration (Higher density resins)
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)
Acknowledgements John Wu Peter Gomme Joseph Bertolini CSL Bioplasma