Application note. Purification of PEGlyated proteins with Contichrom
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1 Application note Purification of PEGlyated proteins with Contichrom ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
2 Summary The purification of PEGylated proteins from the API and undesired multipegylated proteins is a challenging separation In the presented application example, batch chromatography achieves for the desired pegylated product at 93% purity only yields of 56% With Contichrom equipment and the MCSGP process, the yield can be increased to 83% Yield of PEGylated protein 100% 83% 50% 56% Batch Contichrom ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
3 Purification of PEGylated proteins Model system: Separate PEGylated forms of alpha-lactalbumin with anion-exchange chromatography a) With batch chromatography b) With Contichrom in MCSGP chromatography mode ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
4 mpeg-spa: Structure and Reactivity Lysine residues attach to the activated PEG to form the PEGylated protein Hydrolysis half-life at ph 8, 25 C, of mpeg-spa is 16.5 min a Nearly 80 % of the mpeg-spa has reacted to give PEGylated -Lactalbumin. The missing 20 % mpeg-spa may be breakdown product hydrolysis Roberts, M. J.; Bentley, M. D.; Harris, J. M. Advanced Drug Delivery Reviews 2002, 54, 459. ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
5 -Lactalbumin: Structure and Reactivity Molecular weight 14.2 kda 122 amino acids 12 lysine residues a Isoelectric point b Hydrodynamic radius 2.0 nm c a) Viaene, A.; Volckaert, G.; Joniau, M.; De Baetselier, A.; Van Cauwelaert, F. European Journal of Biochemistry 1991, 202, 471. b) Browne, W. J.; North, A. C. T.; Phillips, D. C.; Brew, K.; Vanaman, T. C.; Hill, R. L. Journal of Molecular Biology 1969, 42, 65. c) Smith M.; Handbook of Biochemistry, 2nd edn, Cleveland: The Chemical Rubber, p. C3 C292 Figure: PDB ID 1F6S ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
6 SEC chromatogram of PEGylated protein Analytical HP-SEC chromatogram (Superdex /300, A280) from batch PEGylation of - lactalbumin with mpeg-spa 5 kda in PBS (25 mm Phosphate, ph 7.4) ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
7 SEC chromatogram of PEGylated protein Analytical HP-SEC chromatogram from batch PEGylation Product definition: The mono-pegylated form P1 is the desired species A: Aggregates P3: tri-pegylated P2: di-pegyated P1: mono-pegylated P0: native protein M: mpeg-spa breakdown product A P3 P2 P1 P0 M ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
8 Non-PEG Mono-PEG Di-PEG Tri-PEG -Lactalb Feed SDS-PAGE of SEC Fractions 160 kda 110 kda 80 kda 60 kda 50 kda 40 kda Non-reducing conditions a Silver staining of purest PEGamer fractions isolated from HP-SEC Protein standards MW do not correlate with PEGylated protein Hydrodynamic radii 30 kda a) Zheng, C. Y.; Ma, G.; Su, Z. ELECTROPHORESIS 2007, 28, kda 15 kda 10 kda 3.5 kda Lane 1: F25 P0: native -Lactalbumin Lane 2: F20 P1: mono-pegylated Lane 3: F15 P2: di-pegylated Lane 4: F13 P3: tri-pegylated Lane 5: -Lactalbumin (MW 14.2 kda) Lane 6: concentrated Feed Lane 7: Protein Standards ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
9 conc [g/l], cond [ms/cm]/20, ph/10, A280 [mau] scaled purity Preparative AIEX Separation batch Linear gradient elution, single column, load 4.3 g/l Run # 0613A P1 A280 P2 cond 100% 90% 0.40 purity 80% % % % % % % % % time [min] Pool fraction ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
10 Preparative AIEX Separation MCSGP Use Contichrom software wizard to automatically convert batch chromatography with linear gradient elution into MCSGP operating parameters screenshot Contichrom wizard Equilibration, loading & washing Recycle P2/P1 target pool P1 recycle P1/P0 ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May P0
11 UV-signal (A280) recorded at P-outlet Preparative AIEX Separation MCSGP Operating point generated by wizard provides high purity & yield Start-up Steady-state with stable performance ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
12 Preparative AIEX Separation comparison For the same purity, significantly higher yield is achieved with MCSGP Blue: Feed (PEGylated protein mixture) Desired Product P1 Red: MCSGP product pool (corresponds to 83% P1 yield) Green: Single column product pool (corresponds to 56% P1 yield) ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
13 Preparative AIEX Separation comparison Batch chromatography +10% purity - 50% impurities MCSGP +30% yield Batch chromatography ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
14 Summary Separation of mono-pegylated protein from multiand un-pegylated protein is very challenging Batch chromatography achieves only 56% yield Batch can be automatically converted into MCSGP operation using Contichrom wizard Performance improvements of MCSGP with respect to batch: + 30% yield -50% buffer consumption ChromaCon AG // Purification of PEGylated proteins with Contichrom // // ver. May
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