Introduction to PALL s State-of-the-Art chromatography sorbents, columns and membrane adsorbers

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1 Life Sciences Introduction to PALL s State-of-the-Art chromatography sorbents, columns and membrane adsorbers by Björn Hammarberg, ABD Life Sciences (HK) Filtration. Separation. Solution. SM

2 Outline 1. PALL BioSepra Chromatography Resins Gel-in-a-Shell 2. Mustang Membrane chromatography 3. PKL-system from Lab to Pilot 4. PALL Euroflow Resolute columns 5. PK-systems

3 This happens when you increase the flow rate with conventional gels Low linear flow Spherical bead Good capacity High linear flow Bead compresses Loss of capacity!

4 Evolution of Beads - Soft to Rigid Soft Bead High Capacity Compressible Trisacryl, Ultrogel Rigid Bead Non-compressible Lower capacity Spherosil, Spherodex Hybrid Bead Non-compressible High capacity HyperD, HyperZ

5 Gel in a Shell Pall BioSepra Ceramic HyperD (HyperDiffusion) Porous, non-compressible ceramic bead >0.2 µm (2000 Å) pores filled by hydrogel containing the functional groups Unique design, patented (1993) Several FDA approved processes include HyperD CM, Q, S, DEAE IEX sorbents Monomer intrusion In situ polymerization

6 Dynamic Binding Capacity (DBC) of a HyperD vs. older conventional IEX DBC@10% breakthrough (mg/ml) CL Agarose 90 µm Human 0.5g/L in 50 mm acetate, ph 4.6. Column : 0.2 cm ID x 15 cm. S Ceramic HyperD Polymeric 30 µm 0 1,000 2,000 3,000 4,000 Linear Velocity (cm/h)

7 Ceramic HYPERD IEX Sorbent Gel in a shell Structure Charge Density meq/ml sorbent (packed bed) meq/ml hydrogel (in situ) CM >250 >860 Q >200 >690 DEAE >180 >620 S >150 >515 Pores of ceramic shell are completely filled with hydrogel Å spacing between adjacent polymer chains ~15 Å spacing between adjacent charged groups High charge density creates a molecular pump which drives the protein entry into the sorbent

8 Enhanced Diffusion in Gel-Filled Pore Also referred to as Hyperdiffusion

9 HyperZ for Expanded Bed Adsorption Extension of HyperD technology Based on Zirconium oxide (density 3.2 g/ml) High speed, high biomass compatible Outstanding chemical stability IEX In situ polymerization

10 Commercial-scale Antibody Capture Some strategic and operational goals Reduce sorbent costs Maintain antibody integrity Minimize upstream operations No Dilution / Concentration Allow stringent clean-in-place (1M NaOH) Eliminate considerations associated with leached Protein-A Combine efficiently with subsequent steps in the scheme Reproducible and robust Bind immunoglobulin that are poorly retained on Protein-A (e.g. rat, goat, human IgG 3, human IgD, etc)

11 The introduction of a New class of Chromatography Sorbents Hydrophobic Charge Induction Chromatography (HCIC) - MEP HyperCel for mab Mixed-Mode Chromatography MBI HyperCel for primarily polyclonal Ab and some acid sensitive mab

12 Interaction of 4-MEP Ligand with Adsorption at near-neutral ph Antibody S pka = 4.8 N Hydrophobic interaction

13 Interaction of 4-MEP Ligand with Adsorption at near-neutral ph Antibody S pka = 4.8 N Hydrophobic interaction Desorption at ph S + N H Electrostatic Repulsion ph % in (+) Form % %

14 MEP HyperCel Antibodies How It Is Used Load clarified cell culture media on column without preliminary adjustment of ph or ionic strength. MEP HyperCel is antibody-selective. Binding is independent of IgG subclass or species. Elute the mab by changing to ph4, which is milder conditions than Protein A (ph ) Capacity for mab:s similar to Protein A MEP HyperCel is base-stable (1M NaOH). No need to monitor or remove leached Protein-A.

15 Influence of Ionic Strength on Binding Capacity Binding of Polyclonal h-igg on MEP HyperCel (5mg/mL) DBC (mg/ml) at 10% BT ph 7 Conductivity: 5 to 90 ms/cm NaCl Concentration (M)

16 Influence of ph on Binding Capacity Binding of Polyclonal h-igg on MEP HyperCel (5mg/mL, conductivity 5mS/cm) DBC (mg/ml) at 10% BT ph

17 Optimization of Elution-pH on MEP HyperCel Typical step-elution sequence for initial evaluation ph 5.5 ph 5.2 Elution of relatively basic proteins. Elution of relatively hydrophilic proteins. Elution of basic / hydrophilic impurities. ph 4.9 ph 4.6 Elution of target antibody. ph 4.3 ph 4.0 ph 3.0 Elution of relatively acidic proteins. Elution of relatively hydrophobic proteins. Elution of acidic / hydrophobic impurities.

18 Purification of Rat IgG. 2a on MEP HyperCel Initial isolation from AIEC-purified fraction. No ph optimization M Flowthru & Wash I 1 Flowthru 2 Wash II hc lc 3 ph 4 4 ph 4 5 ph 4 6 ph 3 7 ph 3 8 ph 3 9 Ref. IgG ph 4.0 ph 3.0 Elution ph Studies by J. Ford and D. Conrad, Virginia Commonwealth University

19 Purification of Rat IgG2a on MEP HyperCel Isolation from clarified CCS containing 15% FBS. With ph step elution M hc 1 Feedstock 2 Flowthru 3 Wash lc 4 ph ph ph ph % Pure 8 ph 3.0 Studies by J. Ford and D. Conrad, Virginia Commonwealth University Elution ph

20 Purification of Rat IgG. 2a on MEP HyperCel Initial isolation from AIEC-purified fraction. No ph optimization Flowthru & Wash I ph 4.0 Elution I Column: 1.6 cm ID x 4.0 cm Flowrate: 2.0 ml/min OD (280) H 2 0 Wash II ph 3.0 Elution II Equil.: 50 mm Tris HCl, ph 8.0 Wash I: 50 mm Tris HCl, ph 8.0 Wash II: Pure water Elution I: 50 mm NaOAc, ph 4.0 Elution II: 50 mm NaOAc, ph Fraction No. Studies by J. Ford and D. Conrad, Virginia Commonwealth University

21 IgG capture by MEP HyperCel (HCIC) Purification n 1 Purification n 2 Absorbance at 280 nm Loading Washing Elution Scale up x 60 Loading Washing Elution MEP HYPERCEL Column volume : 10 ml (11 mm ID x 100 mm) 1 liter of hybridoma CCS (135 mg IgG 1 ) elution sodium acetate or citrate, ph 4.0 Cleaning NaOH M MEP HYPERCEL Column volume : 600 ml (90 mm ID x 95 mm) 32 liters of hybridoma CCS (4,320 g IgG 1 )

22 MEP HyperCel Viral, Endotoxin & DNA Clearance Viral clearance (MVM or MuLV) 3 to 4 log reduction Endotoxin clearance 6500-fold reduction DNA removal Substantial reduction

23 MEP HyperCel: mab Summary and Conclusions Facilitates development of simplified, robust purification schemes Without adjustment of ph or ionic strength of the feedstock Compatible with stringent CIP procedures (1M NaOH) Eliminates need to monitor & remove leached Protein-A High binding capacity, regardless of IgG subclass or species Preserves antibody integrity Provides significant better production economy Longer service-life Approximately 4x economical compared with typical Protein-A sorbents Commercial Success Very fast adoption, 7 drugs in Phase I; 3 in Phase II More than 300 biopharmaceutical sites have selected MEP HyperCel for trials

24 MEP HyperCel as HIC HCIC can serve as an effective alternative to traditional HIC. Facilitate binding at reduced salt concentration. Accomplish binding using NaCl in place of costly lyotropic salts. Facilitate enhanced product recovery. Recover product in relatively dilute buffer. Control chromatography based on both ph and salt concentration.

25 Mixed-Mode Chromatography on MBI Binding at ph 5 6 HyperCel OH Binding Repulsion, no binding. Albumin pi ~4.9 Heterocyclic molecule contribute for mild ionic net charges. Thiophilicity by S enhances ability to capture antibodies MBI Benzimidazole ring and sulfonic acid is critical Carries a net negative charge over ph 3-9

26 Mixed-Mode Chromatography on MBI HyperCel Desorption at ph OH Charge Repulsion

27 Membrane Vs. Resin 10,000 Å

28 Flow Dynamics of Resins Resins: Low flow rates (diffusive flow), low binding capacities

29 Flow Dynamics of Membranes Membranes: High flow rates (convective flow), high binding capacities

30 Contaminant Removal Applications for Membranes DNA Virus* HCP Endotoxin

31 Capture/Purification Applications for Membranes R O S P O - O R Viral vectors DNA vectors Antibodies & Proteins Anti-sense Oligo s

32 Mustang Membrane Chromatography Features Unique open pleat design, the Ultipleat technology proven in PALL s Supor filters For Capture or Polishing High flow rate Membrane Vol/min, 20-80x faster than Columns Singe-pass High binding capacity Autoclavable Single-Use Cartridge/Capsule format No packing Ready to use Scalable Sizes from Coin 5mL to 5L (Mustang XT5000 for up to 50L/min, stackable Economical No Cleaning and packing Validation Lower Capital Investment Lower Operating cost

33 Mustang is Scalable Q, S and SDR Volume: 0.35 ml ml ml 5 15L Capacity: <20 mg 200 mg - 3 g 5 g - 35 g 90 g g

34 Resolute Columns

35 Columns Resolute Columns by PALL Euroflow Sanitary design Fully flushed flow path and adjuster seal for clean-in-place (CIP) Minimum dead space fixed cell seal arrangement True linear scalability Standard column diameters mm Standard bed heights mm Patented nozzle valve (Pack-In-Place) All column functions required for packing, unpacking and running of the column in a closed system. The packing process is controlled by a slurry packing system Completely reproducible column packing methods for PALL BioSepra chromatography media.

36 Resolute Three-position Nozzle Valve Pack Mobile Phase

37 Resolute Three-position Nozzle Valve Mobile Phase Process Mobile Phase

38 Resolute Three-position Nozzle Valve Unpack Mobile Phase

39 Nozzle Valve Benefits Pack and unpack in place Reproducible packs (vs traditional packing methods) No column disassembly Contained process Fewer operators Quicker Mobile phase wetted parts: polypropylene, EPDM, Teflon, PEEK - eliminates risk of corrosion within valve Slurry inlet port wetted parts: stainless steel and PEEK Column clean-in-place (CIP) capability

40 Pressure Versus Flow Curves Columns filled with water Competitor Resolute

41 Dynamic Axial Packing Columns (DAP/M ) Flow Packing Principle Inlet Pressure Piston Clarified head space Formed bed Hydraulic Cylinders Hydraulic cylinders move piston at set rate equal to the set packing pressure

42 DAP/M Column Adjuster Cell Ejected for Maintenance

43 Pall Chromatography System

44 Standard PK System Range PK10 PK25 PK50 PK L/h L/h L/h L/h ¼ Tri-Clamp ½ Tri-clamp ¾ Tri-Clamp 1½ Tri-Clamp 6 Bar 6 Bar 6 Bar 6 Bar 316L Stainless Steel 0.4µ Ra + Electro-polish 8 Inlets 4 Outlets Gradient or Isocratic Conductivity, Flow, Pressure, ph, UV, Temperature

45 Selected Resolute Column references Amgen * (Reference with approval) Avecia Bayer (2 x 800mm with PALL BioSepra HyperCEL media) Baxter Centacor * (Ireland) Genentech HGS (mab) Lonza * (2000 mm & 1800 mm etc) MedImmune (vaccines) Several hundred columns mm delivered

46 Chromatography Process Positioning 100L 1,000L 10,000L Capture Purify Polish

47 Pall s Chromatography Product Range Mustang Euroflow PK systems PKL systems Process Proteomics Sorbents