Introduction of Tangential Flow Filtration (TFF) Karen Chan 16 May 2017
What you will learn TFF principles and applications in mammalian cell processes TFF vocabulary definitions and key process parameters TFF membrane selection 2
Types of Filtration Normal Flow Filtration (NFF) Cartridge or Dead-ended Filtration Flow is perpendicular to the filter media All fluid passes through the media Particles are retained in/on filter 3
Normal Flow Filtration Fluid Flow FILTER SURFACE 4 Operate Tangential Flow Filtration Process Equipment rev 4 May 2016
Types of Filtration Normal Flow Filtration (NFF) Cartridge or Dead-ended Filtration Flow is perpendicular to the filter media All fluid passes through the media Particles are retained in/on filter Tangential Flow Filtration (TFF) Cross-flow Filtration Flow is tangential (parallel) to the filter surface A small percentage of the fluid flows through the filter media Retained particles are swept away from filter surface 5
Tangential Flow Filtration Bulk Concentration C b Liquid Tangential Flow Wall Concentration Cw MEMBRANE SURFACE 6 Operate Tangential Flow Filtration Process Equipment rev 4 May 2016
Applications of TFF in the BioPharm Industry Upstream Cell Harvesting (concentration) - Bacterial or yeast cells (product is intra-cellular) Cell Culture Clarification - Mammalian cell (product is extra-cellular) Downstream Protein Concentration Buffer Exchange (Diafiltration) 7
Example of a Mammalian Cell Process 8
Basic TFF Applications Clarification - product passes through the membrane - larger particles / molecules retained by membrane Concentration - product retained by the membrane - solvent (buffer) passes through the membrane Diafiltration (Buffer Exchange) - product retained by the membrane - solvent (buffer) passes through the membrane - new solvent added to product 9
What you will learn TFF principles and applications in mammalian cell processes TFF vocabulary definitions and key process parameters TFF membrane selection 10
Generic TFF System Buffer Tank Feed Tank V o, C o Air Supply L P R Transfer Pump Q T Recycle Tank V R, C b P F P f Q f Feed (Recycle) Pump Q F Product Recovery TFF Device 11
TFF Module Feed Pressure Retentate Pressure Feed Flow Membrane Permeate Pressure Permeate Channel Feed Channel (Screened or Open) Permeate Channel Permeate Flow Retentate Flow 12
Channel Flows (QF, QR, Qf) Feed Pressure Q F = Feed Flow (L/hr) Permeate Channel Feed Channel (Screened or Open) Permeate Channel Retentate Pressure Q R = Retentate Flow (L/hr) Membrane Permeate Pressure Q f = Permeate Flow (L/hr) 13
Pressure Drop P [bar or psi] = P F P R Difference in pressure along membrane feed channel Resistance to feed flow in the channel Function of viscosity, feed flowrate, channel geometry Feed Pressure (P F ) Retentate Pressure (P R ) Feed Channel (Screened or Open) 30 psi 20 psi Permeate Channel Permeate Pressure 2 psi P = 30 20 psi = 10 psi 14
Transmembrane Pressure (TMP) TMP [bar or psi] = [(P F + P R ) / 2 - P f ] Average driving force across the membrane Created by applying retentate pressure Feed Pressure (P F ) Retentate Pressure (P R ) Feed Channel (Screened or Open) 30 PSI Permeate Channel 20 PSI Permeate Pressure (P f ) 2 PSI TMP = (30 + 20)/2-2 = 23 psi 15
Flux (J) J [Lm -2 h -1 ]= Q f / Membrane Area (time) Flux (J): permeate flow rate normalized for area of membrane it passes through Production capacity of the membrane Inlet Pressure Retentate Pressure Feed Channel (Screened or Open) 30 psi Permeate Channel 20 psi Permeate Pressure 2 psi J J = 2 L/min per 2.5 m² = 48 LMH 16
Membrane Polarization Concentration gradient from Cb at center of feed channel to more concentrated protein at Cw Reversible (varies with operating conditions) membrane Q F C b Q R C w membrane TMP Q f C f Q F = feed flow rate [L h -1 ] Q f = filtrate flow rate [L h -1 ] P F = feed pressure [bar] P R = retentate pressure[bar] P f = filtrate pressure [bar] C b = protein concentration in bulk solution [g L -1 ] C w = protein concentration at membrane [g L -1 ] C f = protein concentration in filtrate [g L -1 ] 17
Membrane Polarization No Membrane Polarization Controlled Membrane Polarization Uncontrolled Membrane Polarization 18
19 Polarization Mechanism and Flux
Polarization, TMP and Feed Flow Key point: Maintain membrane performance by balancing polarization (particle deposition) and removal TMP is the driving force creating flux Crossflow provides a sweeping cleaning action across the membrane surface Also affected by temperature and concentration TMP Crossflow 20
Retention & Passage Retentate Permeate % Retention % Passage % Passage = [ ] permeate [ ] retentate x 100 % Retention = 100 - % Passage Retention of protein X = 100 - (1 g/l permeate/100 g/l retentate) x 100 = 99 % 21
Volumetric Concentration Factor (VCF) VCF = V initial V final V initial 20 L of feedstock are ultrafiltered until 18 L have passed through the filtrate VCF = 20 L/ 2 L = 10 fold concentration V final Start Finish 22
Protein Concentration Factor (CF) CF = Final Protein concentration Initial Protein concentration V initial 20 L of feedstock are ultrafiltered until 18 L have passed through the filtrate VCF = 20 L/ 2 L = 10 fold concentration Start This 20 L feedstock contained 5 g/l of product and a concentration of 48 g/l was found in the retentate CF = 48 / 5 = 9.6 V final Finish 23
Diafiltration after concentration wash retentate with buffer PURIFICATION when is product YIELD ENHANCEMENT when is contaminant Example : Product is albumin and Contaminant is NaCl 24
Yield Yield = product recovered at end of process loss to permeate adsorption to membrane unrecoverable product due to hardware limitations V final Finish 25
What you will learn TFF principles and applications in mammalian cell processes TFF vocabulary definitions and key process parameters TFF membrane selection 26
Microfiltration Membrane Structure Micron pore size - 0.10-0.65 µm NMWL - 500 kd - 3000 kd Separate cells and cell debris from proteins Durapore (PVDF) Durapore GVVP 0.2um magnified 650x 27
Ultrafiltration Membrane Structure Micron pore size - 0.001-0.10 µm NMWL - 1 kd - 500 kd Separate proteins from low molecular weight contaminants Ultracel (regenerated cellulose) Biomax (polyethersulfone) Biomax 30 kd magnified 1000x 28
TFF Membranes - Chemistry Membrane Chemistry Microfiltration - PVDF (Durapore ) - Polyethersulfone Ultrafiltration - Low Binding Polyethersulfone (Biomax ) - Composite Regenerated Cellulose (Ultracel ) Durapore GVVP 0.2um magnified 650x 29
Ultrafiltration Membrane Structure Classical membrane with voids Biomax membrane with homogenous microporous support 30
Destructive Test : UF Membrane Qualification UF membrane are labelled by "Nominal Molecular Weight Limit" (NMWL) Does it specify the membrane? NO! Why? - There is no standard definition for NMWL among manufacturers Typically suppliers uses Protein markers 31
Destructive Test : UF Membrane Qualification : Mixed Dextran Mixed Dextran testing uses a polydisperse mixture of Dextran molecules and maps the entire retention profile of the membrane allowing better reproducibility batch-to-batch tighter specifications meaningful QA/QC release But, there is NO correlation between retention and non-destructive integrity test results 32
Choices in Selecting a Filter Module type Channel turbulence promoter (screen) Membrane pore size Membrane material 33
TFF Filter Types 34
Filter Module Selection Flat-Plate (Cassette) - most prevalent for bioprocessing, especially MAbs - efficient packing density and mass transfer - wide choice of membranes and screens - simple linear scale-up and scale-down Spiral - good lower-cost option for very large operations Hollow Fiber - requires high pumping rates for effective mass transfer Stirred Cell - useful for screening studies only 35
TFF Device Characteristics Pellicon 2 and Pellicon 3 Cassettes Flat Sheet Membrane Device Linear Scalability Screened Retentate Flow Channel Highest Efficiency - highest flux at given cross-flow velocity in feed channel Smallest Working Volume and Hold-up Volume Minimal Floor Space 36
Filter Pore Size Rules of Thumb For a product that will be retained, use: NMWL = 1/3-1/5 product size target a product retention > 0.995 Example: 150kD MAb should use 30kD membrane For a product passing to filtrate, use: NMWL = 3-5X product size target a product retention < 0.2-0.4 37
Selecting a Membrane for a Separation Understand the solvent ph Ionic strength Viscosity Processing Conditions Prefiltration / Pretreatment Temperature Time / Frequency Hardware restrictions Cleaning / Sanitizing chemical selection Personnel Restrictions 38
Some Membrane Selection Questions Will it be UF / MF? Solute size / characteristics Membrane performance What is the required solvent compatibility? Solvent type / concentration Are there any issues with cleaning chemicals Compatibility / Exposure / Validation What are the membrane binding / adsorption characteristics Initial & final protein concentration is important What are the yield / retention issues? Remember UF membranes are not totally retentive Establish acceptable standards first 39
Summary We have reviewed TFF principles and applications in mammalian cell processes TFF vocabulary definitions and key process parameters TFF membrane selection 40