Presentation1 tff

BRIEF DETAILS ON TFF

1. 1 Tangential Flow Filtration (TFF) SURAJ CHAUHAN

2. Filtration : Filtration is any of various mechanical, physical or biological operations that separate solids from fluids Types: Normal flow filtration (NFF) : Cartridge or Dead end filtration Flow is perpendicular to the filter media All fluid passes through the media Particles are retained in/on filter 2) Tangential flow filtration 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 2

3. 3 Advantages of TFF TFF is easy to set up and use TFF is fast and efficient Perform two steps with one system TFF can be scaled up and scaled down TFF is economical Basic TFF Application Clarification : Clarification is the process where unwanted things are removed mainly used to clarify the cell at harvest Concentration : Concentration is a simple process that involves removing fluid from a solution while retaining the solute molecules. Diafiltration or Buffer exchange : Diafiltration is the fractionation process that washes smaller molecules through a membrane and leaves larger molecules in the retentate without ultimately changing concentration Depyrogenation : Depyrogenation is the purification process in which pyrogen is removed by using selective membrane.

4. 4 TFF Module Feed flow Permeate d flow Retentate flow FEED CHANNEL Permeate channel Permeate channel Feed pressure Retentate pressure Permeate pressure F f Pf Pr R f Pp Pf

5. 5 Basic Terminology Pressure Drop ( Δ P= Pf – Pr ) Difference in pressure along membrane feed channel Resistance to feed flow in the channel Function of viscosity, feed flowrate, channel geometry 2. Transmembrane Pressure TMP = (Pf + Pr ) / 2 – Pp) Average driving force across the membrane Flux (J) L/m2/h Permeate flow rate normalized for area of membrane it passes through

6. 6 TMP adjustment and its effect on Flux By adjusting pump speed By controlling retantate valve By controlling permeate valve

7. 7 Membrane Polarization (Fouling) Accumulation of solute on membrane surface is called membrane polarization No Membrane polarization Controlled Membrane polarization Uncontrolled Membrane polarization

8. 8 Retention and passage Retentate Permeate %Passage %Retention Permeate %Passage = Retentate × 100 %Retention = 100 - % Passag Retention of protein = 100 – (1 g/L permeate/ 100 g/L retentate) × 100 = 99 % Retentate Membrane Permeate

9. 9 Volumetric Concentration Factor (VCF) Protein C oncentration Factor (CF) VCF = V initial V final CF = Final protein concentration Initial protein concentration Eg . 20L of feedstock ( 5g/L) are ultrafiltered until 18L (48 g/L) have passed through the filtrate VCF = 20L / 2L = 10L CF = 48 / 5 = 9.6

10. 10 Classification Microfiltration G enerally refers to the filtration of suspension particle such as cells and cellular fragments Microfiltration membranes with pore sizes typically between 0.1µ - 10µ Membrane chemistry : PVDF ( Durapore ), Polyethersulfone Ultrafiltration Ultrafiltration is the filtration of smaller molecules Ultrafiltration membranes with much smaller pore sizes between 0.001µ - 0.1µ Ultrafiltration membranes are typically classified by (MWCO) molecular weight cutoff rather than pore size Membrane chemistry : Low binding Polyethersulfone ( Biomax ) Composite regenerated Cellulose ( Ultracel ) UF membrane qualified by mixed Dextran test

11. 11 Basic Steps for TFF Operation A certain number of steps are necessary to perform successfully a TFF operation Flush Sanitization Integrity test NWP Buffer condition Process concentration Process Diafiltration Recover Product CIP/SIP Integrity test NWP Storage

12. 12 1. Flushing -Flushing is done to remove storage or cleaning solution -Flushing fluid (water) is directed to drain -Minimum feed flow rate and volume to be flushed from permeate and retentate is recommended by manufacturer Eg . For pellicon feed flowrate = 5L/min/m2 Typical retentate flush volume = 20 L/m2 Typical filtrate flush volume = 30 L/m2 at recommended TMP 2. Sanitization -Purpose : To monitor and control Bioburden, To remove any residual storage solution -Frequency : In case of new membrane and before each process -Sanitizing solution : NaOCl , NaOH , Peracetic acid, Formaldehyde, Henkel

13. 13 3. Integrity Testing Purpose If the system (holder, gasket, module) is not integral, TFF performance may be affected and product may be lost Is recommended by regulatory agencies as risk reduction Increasingly required to satisfy GMPs Principle Wet the membrane completely with water Measure air flow through the wetted membrane at low pressure Test Diffusion test -We can not use bubble point test with UF membranes as it either compresses or rupture the membrane -Water bubble point exceed filter and system capabilities Frequency Before use and after process and cleaning

14. 14 Steps for integrity testing Record cassette lot No Place gasket then cassette then gasket on holder Tighten nuts, Check torque Membrane wetting / Flushing Troubleshooting failed test Improper pre-use wetting / flushing procedure Improper storage condition Incorrect diffusion specification/ test air pressure Incorrect temperature Improper installation (gasket miss/incorrect use, insufficient holder compression)

15. 15 4. Buffer Conditioning What is the buffer conditioning? Wetting the membrane with a buffer that is compatible with the feed solution Removing cleaning and storage solution How do I condition the membrane? Recirculate in total mode an appropriate buffer through the membranes -Retentate valve open -Recommended feed flowrate = 5 L/min2, 5min

16. 16 5. Processing Types of processing Concentration Diafiltration / Clarification Procedure Fill tank with process fluid Start pump and adjust system to recommended flow / pressure Concentrate and Diafiltrate Concentration Concentration is simple process in which fluid remove from the solution while retaining the solute molecules To concentrate choose a UF membrane with a MWCO that is substantially lower than the MW of solute to be retained A good general rule is to select a membrane with a MWCO that is 3-6 times lower the MW of the molecules to be retained If only concentrate then 3 times lower is sufficient if significant diafiltration will also be applied to sample then an even lower i.e. 6times lower is advisable

17. 17 Diafiltration Diafiltration is the fractionation process that washes smaller molecules through a membrane and leaves larger molecules in the retentate It can be used to remove salts or exchange buffers There are two types of diafiltration Continuous Diafiltration In continuous diafiltration the diafiltration solution is added to the sample feed reservoir at the same rate as filtrate is generated In this way the volume in the sample reservoir remains constant but the small molecules that can permeate through the membrane are washed away Using 5 diafiltration volumes will reduce the ionic strength by ̴ 99% Discontinuous D iafiltration Discontinuous diafiltration can be carried out by two ways -Add diafiltration volume to the sample without concentrating the sample -First concentrate the sample and then add diafiltration volume -Using 5 diafiltration volumes will reduce the ionic strength by ̴ 96%

18. 18 6. Product Recovery Buffer rinse washes product through membrane Gravity drain of permeate line Pressure blow-down of permeate line

19. 19 7. Cleaning Why cleaning? To remove product residue from the system, prevents cross contamination To remove bioburden (bacteria, viruses, mold) To remove endotoxin To restore process performances, reproducible filtration from run to run Achieve the longest device lifetime How do we get there? Develop the cleaning protocol Document the procedure Validate the protocol Consistently apply the procedure

20. 20 Examples of cleaning agents Degradation Hydrolytic agent : NaOH , Acid, Proteolytic enzymes Oxidants : Bleach( NaOCl ), Chlorine dioxide Dissolution Warm buffers 6M urea or 7M guanidine for protein Detergency Surfactants : Tween, Tergazyme Choosing a cleaning agent Must be able to removal of cleaning agent and its byproducts from system after cleaning is completed Able to remove process remnant Not react destructively with materials of construction of membrane/module/system Able to develop analytical method for agent needed

21. 21 Most Frequently Used Cleaning Agents NaOH Hydrolyzes proteins and saponifies fats Less effective on polysaccharides and cell debris Easy to validate removal ( Ph , conductivity) Concentration 0.1-1 N, Temperature 30-50 ͦC, Time 30-60 min Drawbacks Incomplete action to remove complex polysaccharides Ineffective with some cellular debris Reacts slowly with and ages some filter materials Concentrate solution may cause changes in cellulose membrane

22. 22 NaOCl (bleach) at pH 9-10 Oxidizes nearly all organic compounds and cellular debris Efficient against spore formers Easy to validate removal (colorimetric and other test) Concentration 200-300ppm free CL, Temperature 30-50 ͦ C, Time 30-60 mins Drawbacks React with and ages some filter materials pH must be controlled >9 CL corrosive for SS Degrades all cellulosic membranes and some PES membrane

23. 23 Acids : phosphoric, citric acid Hydrolyze nucleic acids and solubilize inorganic salts Must remove biological solutes first Easy to validate removal (pH) Concentration 0.1N, Temperature 30-50 ͦ C, Time 30-60mins Drawbacks Beware of corrosion of metals Concentrated solution may cause changes in cellulose membranes

24. 24 8. Storage Objectives Keep the membrane wet If recommended to be stored in dry condition then cleaning agent is not required Prevent growth of bacteria, mold, fungi etc. when the system is not in use Effective storage agents Prevent growth of organism Do not attack any material of construction in membrane, device or system Stable for long periods of time Easily wet all surfaces Do not absorb to system materials irreversibly Easily flus out validatably Best storage recommendation In the vendor’s product literature Recommended agents Concentration Time limits Storage condition (Temperature)

25. 25 Measuring Cleaning Effectiveness Key elements to monitor when assessing cleaning effectiveness include Normalized water permeability (NWP) Flush water residuals Process reproducibility Physical analysis of contamination through destructive techniques

26. 26 Normalized water permeability (NWP) Measure the passage of clean water through the membrane under standard pressure and temperature conditions The rate of clean water flux through the membrane is measured as liters per membrane area per hour Water flux divided by the TMP is the normal water permeability (L/m2/h/bar) How NWP is measured Cleaning and storage agent removal with pure water, flush membrane through the pure water Always measure NWP at same Feed floe rate, TMP, Conductivity, Temperature Recommendation take 2 measurement under the same condition and use the average NWP = Flow rate(L/h) * TCF (Temperature correction factor) / TMP * Membrane Area How use from run to run Measure benchmark NWP (NWP after initial sanitization) Recommended NWP for reproducibility 50% from benchmark and ± 20% from run to run

27. 27 NWP is affected by Membrane Device System Membrane area NWP Troubleshooting NWP unusually </>20% of precedent value Check if system pipe has changed Maintenance was made on the pump Check if permeate valve is fully opened Check if modules have not been over torqued Check water conductivity and temperature Check cleaning condition

28. 28 Available methods for cleaning assessments Methods Can be used Main advantage Main drawback Run to run For cleaning development NWP yes yes Ease to use System dependent TOC yes yes Reliability Need extra equipment SEM No yes Membrane visible Dextructive , Expensive FTIR No yes Contaminant identification Dextructive , Expensive Autopsy No yes Device and membrane visible Dextructive

29. 29 Single pass TFF Traditional TFF is operate in batch mode where the feed/ retentate is recirculated through the filter assembly In single pass TFF cassettes operate in parallel or serial configuration to achieve the desired concentration Feed Tank Feed pump Cassetee Retentate Permeate Batch Feed Tank Feed pump Cassetee Retentate Permeate Single pass Feed Permeate Retentate Serial configuration Retentate Parallel configuration Feed

30. 30 Advantages of single pass Single pass TFF runs at constant operating condition throughout the process Higher product recovery Reduces the risk of product damage associated with recirculating TFF operation Reduces the working volume limitation

31. 31 How to choose the proper TFF system for your application STEP-1 Define the purpose of TFF process Concentration Diafiltration / Fractionation STEP-2 Choose the membrane molecular weight cutoff and membrane material MWCO is defined by its ability to retain a given percent of a molecule in solution A good general rule is to select a membrane with a MWCO that is 3-6 times lower the MW of the molecules to be retained If only concentrate then 3 times lower is sufficient if significant diafiltration will also be applied to sample then an even lower i.e. 6times lower is advisable Membrane material : cellulosic ( Ultracel RC) – low binding, easy cip , moderate NaOH resistance :Polyether sulfone ( Biomax PES) – Higher binding, More difficult to clean, High pH resistance

32. 32 STEP-3 Choose the flow channel configuration Depending on sample concentration and solution characteristics (viscosity, particulates etc.) determine the type of channel configuration required for the application 1- Screen channel configuration : Used for clean filtered solution, having no particles or aggregates that can get trapped in the screen A woven separator in the channel create gentle turbulence along the membrane surface , minimizing the membrane fouling 2-Suspended channel configuration : Open structure in the retentate channel that provides the better performance when highly viscous fluids or particle laden solution are being used. It can be used to concentrate cells or clarify cells or fermentation broth 3 -Open channel configuration : There is no screen in the feed channel, instead it uses spacer to define the channel height. Typically a channel height between 0.5-1.0 mm is used for cell harvest applications This structure minimizes cell disruption and maximizes recovery of intact cells after concentration

33. 33 STEP-4 Determine the required membrane area for the application Choosing of an appropriate cassette depends on the total sample volume, required process time and desired final sample volume Following equation useful to calculate the membrane area required for processing a sample in a specified time A =V / J*T Where A =Membrane area (m2), V = Volume of filtrate generated (L), J = Flux (l/m2/h), T = Time (h)

34. 34 The following basic experiments should be considered during development of processing methodology : 1) Optimization : Impact of transmembrane pressure (TMP) and feed flow on process flux and retention Impact of product concentration and buffer conditions on process flux and retention Impact of diavolumes on buffer exchange and contaminant removal 2) Paper design and full process simulation with chosen processing parameters

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36. 36 Part 1. TMP Optimization at Initial Concentration

37. 37 Part 2. Concentration DF Optimization Parameter = Concentration [g/L] x Flux [LMH]

38. 38 Part 3. TMP Excursion at Final Concentration Part 4. Diafiltration Remaining Contaminant [%] = 100 x e (Retention – 1) x N where N is the number of diavolumes Part 5. Product Recovery Theoretical Yield [%] = 100 x e (Retention – 1)(N + lnX ) where N = number of diavolumes and X = concentration factor Actual Yield [%] = 100 x ( Vretentate [L] x Cretentate [g/L]) / ( Vinitial [L] x Cinitial [g/L]) Mass Balance [%] = 100 x {( Vretentate [L] x Cretentate [g/L]) + ( Vpermeate [L] x Cpermeate [g/L]) + ( Vrinse [L] x Crinse [g/L])} / ( Vinitial [L] x Cinitial [g/L])

39. 39 Assume an example process scenario (this would have been determined by optimization data, DF parameter, etc .): -2.9X Concentration: 10 g/L to 29 g/L; flux decreases from 150 LMH to 80 LMH -7X Diafiltration : 29 g/L; flux increases from 80 LMH to 85 LMH -3.4X Concentration: 29 g/L to 100 g/L; flux decreases from 85 LMH to 20 LMH -Desired process time is 4 hours Manufacturing scale volumes as determined by the customer: Feed volume = 5000 L Retentate volume at end of 2.9X concentration = 5000 L/2.9 = 1724 L Permeate volume removed during 2.9X concentration = 5000 L – 1724 L = 3276 L 7X Diafiltration buffer volume = 7 x 1724 L = 12,068 L Retentate volume at end of 3.4X Concentration = 1724 L/3.4 = 507 L Permeate volume removed during 3.4X concentration = 1724 L – 507 L = 1217 L

40. 40 Average process flux for concentration step: J avg = J final + 0.33 ( J initial – J final ) = J initial x 0.33 + J final x 0.67 For 2.9X concentration: J avg = 150 LMH x 0.33 + 80 LMH x 0.67 = 103 LMH For 3.4X concentration: J avg = 85 LMH x 0.33 + 20 LMH x 0.67 = 41 LMH Average process flux for diafiltration step: For diafiltration the average flux can be estimated as the initial and final process flux during the diafiltration step. Required area: Area = [(Permeate volume/Average flux) Concentration + (Permeate volume/Average flux) Diafiltration + … ] / Time In this example: Area = [(3,276 L/103 LMH) + (12,068 L/83 LMH) + (1,217 L/41 LMH)] / 4 hours = 51.6 m2 Add 20% safety factor: Area = 62 m2

41. 41 To perform a scale-down process simulation, the same volume to area ratio is used For example, if the process is to be performed on one Pellicon ® 2 Mini cassette (with an area of 0.1 m2), then the required feed volume will be: Scale-down feed volume = 0.1 m2 x (5000 L/62 m2) = 8 L Instead, if there is a specific volume of feedstock to process (example: 25 L), then the required membrane area will be: Scale-down membrane area = 25 L x (62 m2/5000 L) = 0.3 m2

42. 42 Example What TFF system should I use to concentrate 10L to 200ml in 2.5h hours? Assume the average filtrate flux rate of 50L/m2/h Volumetric throughput (volume of filtrate) = 10L-0.2L = 9.8L A =V / J*T Where A =Membrane area (m2), V = Volume of filtrate generated (L), J = Flux (l/m2/h), T = Time (h) A = 9.8/50*2.5 = 0.08m2 Recommended system : Ultrasette lab TFF device, area of 0.084m2 or centramate holder with 1 membrane cassette area of 0.093m2

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