Introduction of Tangential Flow Filtration (TFF) Karen Chan 16 May 2017
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1 Introduction of Tangential Flow Filtration (TFF) Karen Chan 16 May 2017
2 What you will learn TFF principles and applications in mammalian cell processes TFF vocabulary definitions and key process parameters TFF membrane selection 2
3 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
4 Normal Flow Filtration Fluid Flow FILTER SURFACE 4 Operate Tangential Flow Filtration Process Equipment rev 4 May 2016
5 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
6 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
7 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
8 Example of a Mammalian Cell Process 8
9 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
10 What you will learn TFF principles and applications in mammalian cell processes TFF vocabulary definitions and key process parameters TFF membrane selection 10
11 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
12 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
13 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
14 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 = psi = 10 psi 14
15 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 = ( )/2-2 = 23 psi 15
16 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
17 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
18 Membrane Polarization No Membrane Polarization Controlled Membrane Polarization Uncontrolled Membrane Polarization 18
19 19 Polarization Mechanism and Flux
20 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
21 Retention & Passage Retentate Permeate % Retention % Passage % Passage = [ ] permeate [ ] retentate x 100 % Retention = % Passage Retention of protein X = (1 g/l permeate/100 g/l retentate) x 100 = 99 % 21
22 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
23 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
24 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
25 Yield Yield = product recovered at end of process loss to permeate adsorption to membrane unrecoverable product due to hardware limitations V final Finish 25
26 What you will learn TFF principles and applications in mammalian cell processes TFF vocabulary definitions and key process parameters TFF membrane selection 26
27 Microfiltration Membrane Structure Micron pore size µm NMWL kd kd Separate cells and cell debris from proteins Durapore (PVDF) Durapore GVVP 0.2um magnified 650x 27
28 Ultrafiltration Membrane Structure Micron pore size µm NMWL - 1 kd kd Separate proteins from low molecular weight contaminants Ultracel (regenerated cellulose) Biomax (polyethersulfone) Biomax 30 kd magnified 1000x 28
29 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
30 Ultrafiltration Membrane Structure Classical membrane with voids Biomax membrane with homogenous microporous support 30
31 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
32 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
33 Choices in Selecting a Filter Module type Channel turbulence promoter (screen) Membrane pore size Membrane material 33
34 TFF Filter Types 34
35 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
36 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
37 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 > Example: 150kD MAb should use 30kD membrane For a product passing to filtrate, use: NMWL = 3-5X product size target a product retention <
38 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
39 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
40 Summary We have reviewed TFF principles and applications in mammalian cell processes TFF vocabulary definitions and key process parameters TFF membrane selection 40
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