011 PP Foundation Training TFF- new brand EU.pptx

TFF

1. Molsheim , 23 August 2017 Introduction to Tangential Flow Filtration (TFF) Karin Asbeck Technical Service Specialist GESA

2. What is Filtration?

3. Tangential Flow Filtration Normal Flow Filtration What is Filtration? Pressure driven separation process that uses (membrane) filters to separate components in a liquid (or suspension) based on their size Two types of filtration

4. Normal Flow Filtration (NFF) Feed Flow (in) Membrane surface Filtrate (out) Filtrate flux rate Volume filtered

5. Tangential Flow Filtration (TFF) Feed Flow (in) Membrane surface Filtrate (out) Filtrate flux rate Volume filtered Liquid Tangential Flow

6. Tangential Flow Filtration depends on the membrane used in the module Types of TFF 1 Microfiltration (typically 0.1 – 5µm) 2 Ultrafiltration (typically 1kD – 1,000kD MW) 3 Nanofiltration (typically 200 – 1,000D MW) 4 Reverse Osmosis (typically 40 – 200D MW) Pressure-driven membrane-based separation process in which particles and dissolved macromolecules larger than 0.1µm are rejected Pressure-driven membrane-based separation process in which particles and dissolved macromolecules smaller than 0.1um and larger than about 2nm are rejected Pressure-driven membrane-based separation process in which particles and dissolved molecules smaller than about 2nm are rejected Liquid-phase pressure-driven separation process in which applied transmembrane pressure causes selective movement of solvent against its osmotic pressure difference

7. Filtration Type vs. Typical Particle Size

8. Typical TFF Applications

9. Basic TFF Applications Clarification Product passes through the membrane Larger particles/molecules retained by membrane Concentration P roduct retained by the membrane Solvent (buffer) passes through the membrane 1 2 Diafiltration (Buffer Exchange) P roduct retained by the membrane Solvent (buffer) passes through the membrane New solvent added to product Small molecule clearance or impurity removal (purification) 3 Initial Feed Retentate Feed Tank Pump Filtrate Membrane Diafiltration Buffer Flush

10. Drug Manufacturing Template

11. TFF MF is a powerful separation tool, in one purification step Cell harvest/concentration Culture media removal and diafiltration Contaminant removal Typical Applications for MF (0.10 – 0.65µm), Open UF (300k – 1,000k MW) Membranes TFF MF Separation objectives by application Example Retentate Filtrate Clarification Expressed recombinant protein purification Bacteria expressing extra cellular re- combinant protein Lactococcus lactis 0.5 - 1.5µm Recombinant protein (5 – 500kd) Clarification Expressed MAb purification Mammalian cells expres- sing extra cellular Mab CHO cells 10 – 20µm Mab (IgG, 150kd) Harvest Virus purification Influenza virus egg or cell based purification Influenza virus 80 – 120nm Ovalbumin Host cell protein, DNA Harvest Washing and/or Concentration of Cells Bacteria, Yeast Neisseria meningitidis, 0.6 – 0.8µm E. Coli 0.5 – 2µm Cell culture media

12. Separate target molecules (e.g. proteins) from low molecular weight “contaminants” (e.g. smaller proteins, peptides, solutes, salts) Concentration Diafiltration or Buffer exchange Typical Applications for UF (1kD – 300kD) Membranes Application Separation objectives by application Example Retentate Filtrate Downstream Protein purification Albumin from plasma Albumin Small Contaminants, Salts Downstream Expressed high protein concentration Monoclonal antibody Monoclonal antibody Water – Salts

13. Reliable, reproducible performance Able to be validated No impurities added by filter system High yield/recovery of product Rapid processing Ease of scalability Sanitary design (cleanable, sanitizable in place) Cost effective process Vendor support (technical, applications, validation) Training BioPharmaceutical Customer Needs

14. Membranes used in TFF Modules

15. Ultrafiltration NMWL pore rating; 1kD – 1,000kD Separate proteins from small molecular weight contaminants E.g. Regenerated cellulose (Ultracel) & Modified polyethersulfone (Biomax) Microfiltration Micron pore size; 0.10 – 0.65µm Separate cells and cell debris from proteins E.g. PVDF (Durapore) Membranes Cross-section PVDF 0.2µm rated membrane (magnified 650×) Cross-section Regenerated Cellulose Composite 30kD membrane (magnified 2,000×)

16. More about UF Membranes 2 1 Considerations Type of polymer Pore size/Nominal Molecular Weight Limit (NMWL) Cleaning conditions/Process solvents Chemical compatibility Interactions between membrane surface and feed stream solutes What is a UF membrane? Thin (semipermeable) barrier with ability to separate particles and/or molecules by size in solution

17. Selecting NMWL for a TFF Separation For a product that will be retained, use NMWL = 1/3 – 1/5 product size Target a product retention > 0.995 For a product passing to filtrate, use NMWL = 3 – 5× product size Target a product retention < 0.2 – 0.4 Retention is based on effective size in solution, not solute MW Size is a affected by of buffer chemistry & conditions (pH, ionic strength, concentration), protein-protein interactions, protein-contaminant interactions. To ensure acceptable retention or passage, trials must be conducted and the process parameters and fluid parameters optimized.

18. Membrane Selection Considerations Cellulosic Membrane (e.g. Ultracel) PES Membrane (e.g. Biomax) Membrane Composite regenerated cellulose (void free structure) Hydrophilic modified polyethersulfone (void free structure) Typical Available NMWL 5, 10, 30, 100, 300, 1,000kD 5, 8, 10, 30, 50, 100, 300, 500, 1,000kD Relative protein binding Ultra low (~0.1g/m 2 ) Use with any protein concentration (good with dilute) Low – medium (~0.2g/m 2 ) Use with solutions > 0.1mg/mL pH Stability 2 – 13 1 – 14 Comments Organic solvent resistance High yield Very hydrophilic – use with solutions containing hydrophobic components (i.e. antifoams or detergents) Resistant to rigorous cleaning regimes (strong bases and acids) High flux

19. TFF Devices

20. Module Types and their Uses 1 Differentiated by Channel Geometry Open Channel – higher solids capability Screened Channel – requires less pumping Path Length – longer gives different separation profile 2 Clarification Applications Typically Open Channel Hollow Fiber, Open Channel Cassettes 3 Ultrafiltration Applications Typically Screened (Turbulence Promoted) Cassettes (Flat sheet), Spiral wound

21. Spiral Wound Design Coarse screen in feed- retentate channel Require higher crossflow per unit surface area than cassettes Can be arranged in series to limit crossflow requirements

22. Cassette (Flatsheet) Design Retentate Feed Filtrate

23. Clarified protein solutions Clarified synthetic solutions Screened channel Viscous liquids High solids Open channel Screen Channel Geometry Open Suspended (e.g. V Screen) Coarse (e.g. C Screen) Fine (e.g. A Screen) 1 2 3 4

24. Ultracel PLC 10kD 30kD Biomax 10, 30, 50kD Ultracel PLC 5, 10, 30, 100, 300, 1,000kD PL 1, 3 kD Biomax 5, 8, 10, 30, 50, 100, 300, 500, 1,000kD Durapore 0.1, 0.22, 0.45, 0.65µm Example of Cassette Families, Area & Batch Volume Scale-up/Scale-down Screening/feasibility Pilot/production Pellicon 3 88cm 2 Pellicon 3 0.11m 2 Pellicon 3 0.57m 2 Pellicon 3 1.14m 2 50mL – 1L 2L and up 80mL – 10L 10L and up Pellicon XL 50 Pellicon 2 Mini 0.1m 2 Pellicon 2 0.5m 2 Pellicon 2 Maxi 2.5m 2 Pellicon 3 Pellicon 2

25. TFF Hardware

26. Cassette Holders

27. TFF systems Holder bottom plate Adapter Plate Tie rods Cassette Spacers, washers, nuts Holder top plate

28. TFF systems

29. TFF Basics

30. TFF has 2 driving forces Q F = Feed flow generated by pump TMP = Transmembrane pressure generated by the feed pump and retentate valve TFF Basics Retentate (Concentrate) P f P F Process Tank Diafiltrate MODULE Feed Pump Filtrate (Permeate ) Q D T Retentate Valve P R Membrane Q R Q F Q f or Q p

31. The TFF Balance TMP Crossflow Correct System Operation Balances Both Factors Maintain membrane performance and flux by balancing particle deposition and removal TMP is the driving force creating flux Deposits solutes onto membrane Crossflow provides a sweeping cleaning action across the membrane surface Reduces solutes on membrane

32. Where can I find more Information?

33. Merck Web Site http://1f29f0a3f7e56f02b107-3ba1f03abf09ad45f8c9658b81e76f9f.r19.cf1.rackcdn.com/Pellicon3v11/Pellicon_v11.html

34. Thank you. Any questions?