Tangential Flow Filtration (TFF)

INTRODUCTION TO TANGENTIAL FLOW FILTRATION

Filtration: Filtration is any of various mechanical, physical or biological operations that separate solids from fluids. Filtration divided in two types,

1)      Normal flow filtration (NFF): Also, called cartridge or dead end filtration, in this flow is perpendicular to the filter media, Particles retained in/on filter. 

      2)   Tangential flow filtration (TFF): Also called Cross flow filtration in this flow is tangential (parallel) to the filter surface, a small percentage of the fluid flows through the filter media and retained particles are swept away from filter surface.

Basic Terminology

• Pressure Drop (ΔP= Pf – Pr ): Difference in pressure along membrane feed channel, it can be determined by using equation ΔP= Pf – Pr  where Pf = feed pressure and Pr = retentate pressure.
• Transmembrane Pressure TMP = (P_feed + P_retantate) / 2 – P_permeate): It is the average driving force across the membrane
• Flux (J) L/m2/h: Permeate flow rate normalized for area of membrane it passes through
• Cross flow rate (CF) / Retentate flow rate: CF is the volumetric flow rate at the retentate point of feed solution and measure in litre/minute.
• Cross flow flux rate (CFF): The recirculating flow rate of feed solution at retentate point per unit time per unit membrane area and measure as litre/minute/m2.
• Membrane Polarization (Fouling): Accumulation of solute on membrane surface is called membrane polarization. 
• Retention and passage: % Passage = Permeate/Retentate * 100   and   % Retention = 100 - % Passage              Retention of protein = 100 – (1 g/L permeate/ 100 g/L retentate) × 100                                                                      = 99 %
• Volumetric Concentration Factor (VCF):   VCF = Initial volume / Final volume
• Protein Concentration Factor (CF)
        CF = Final protein concentration / Initial protein concentration
        Eg. 40L of feedstock (5g/L) are ultra-filtered and concentrated up to 10L (48 g/L)
        VCF = 40L / 10L
                 = 4L
        CF = 48 / 5
        = 9.6   
•        Molecular weight cut-off (MWCO)
  MWCO is define as the lowest molecular weight of the solute retained by the membrane. eg. 30KD membrane having ability to retain 90% of 30KD size solute from the solution. 

Classification

Microfiltration:

Generally refers to the filtration of suspension particle such as cells and cellular fragments. In TFF system Microfiltration membranes with pore sizes typically between 0.1µ - 10µ, some places KD size membrane also used for microfiltration. Membrane chemistry: PVDF (Durapore), Polyether sulfone

Ultrafiltration (UF):

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) regenerated Cellulose (Ultracel).   

Basic Steps for TFF Operation

A certain number of steps are necessary to perform successfully a TFF operation

1. Flush: Flushing is done to remove storage or cleaning solution and generally water is used for flushing. Minimum feed flow rate and volume to be flushed from permeate and retentate is recommended by manufacturer
2. Sanitization: Sanitization carried out for new membrane and before each process. The main purpose of sanitization is to remove any residual of storage solution, monitor and control the bioburden. Sanitizing solution used as recommended by manufacturer. Sanitizing solution : NaOCl, NaOH, Peracetic acid, Formaldehyde, Henkel.
3. Integrity test: Integrity testing is carried out pre and post use of membrane to check the system set up and cassettes are integral.
4. Normalized water permeability (NWP): Measure the passage of clean water through the membrane under standard pressure and temperature conditions. Water flux divided by the TMP is the normal water permeability (L/m2/h/bar). Always measure NWP at same Feed flow rate, TMP, Conductivity and Temperature. Recommendation take two measurement under the same condition and use the average.

NWP = Flow rate (L/h) * TCF (Temperature correction factor) / TMP *  Membrane Area

Measure benchmark NWP (NWP after initial sanitization), recommended NWP for reproducibility is 50% from benchmark and ± 20% from run to run.

            5.    Buffer condition: Main purpose of buffer conditioning is to remove cleaning and storage        solution and wetting the membrane with a buffer that is compatible with the feed solution

6. Process: TFF system used for two types of process, 1) Concentration and 2) Diafiltration and Clarification

Concentration

Concentration is simple process in which fluid remove from the solution while retaining the solute molecules.

To concentrate choose a Ultra filtration 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.

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. Normally 5 diafiltration volumes will reduce the ionic strength by ̴ 99%.

Discontinuous Diafiltration

Discontinuous diafiltration carried out in two ways.

1) Add diafiltration volume of buffer to the sample and performed diafiltration, 2) First concentrate the sample and then add diafiltration volume. Normally in discontinuous diafiltration 5 diafiltration volumes will reduce the ionic strength by ̴ 96%.

7. Recover Product: In this step system and cassette hold up is taken out. It can be done by     Buffer rinse washes product through membrane, Gravity drain of permeate line, Pressure blow-down of permeate line. Yield can be calculated by using following formula.

Theoretical Yield [%] = 100 x e ^{(Retention – 1) (N + lnX)}

Where N = number of diavolumes and X = concentration factor

Actual Yield [%] = 100 x (V_retentate [L] x C_retentate [g/L]) / (V_initial [L] x C_initial [g/L])

Mass Balance [%] = 100 x {(V_retentate [L] x C_retentate [g/L]) + (V_permeate [L] x C_permeate [g/L]) + (V_rinse [L] x C_rinse [g/L])} / (V_initial [L] x C_initial [g/L])

8. CIP/SIP: Manufacturer recommended cleaning agent should be used for Cassette cleaning. Main purpose of cleaning to remove product residue and prevent cross contamination, remove bioburden, remove endotoxin, restore process performance, achieve long device lifetime.

Some 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

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

09.  Integrity test
      10. NWP
      11. Storage: Cassette should be store as recommended by manufacturer. Some cassette can store in dry condition while others are store in storing agent, main purpose is to keep the membrane wet, prevent growth of bacteria, mold, fungi etc. when the system is not in use.

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

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

Steps for selection of TFF system 

STEP-1 Purpose of TFF process

•      Concentration

•      Diafiltration / Fractionation

STEP-2 Select membrane size and membrane material as per application

•      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. 6 times 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

STEP-3 Memrane configuration

Based on sample nature determine the type of memrane configuration required for the application

Ø  1- Screen channel configuration : used for clear solution having less or no suspended particles or aggregates. There is a woven separator in the channel that creates gentle turbulence and prevent fouling on the membrane surfaces.  

Ø  2-Suspended channel configuration : used for viscous materials and particles loaded sample due to having open structure in the retentate channel that provides the better performance. It can be used to concentrate cells or clarify cells or fermentation broth.

Ø  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  

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)

Example

What TFF system should I use to concentrate 20L to 400ml in 2.0 hours?

Assume the average filtrate flux rate is 50L/m2/h

Volumetric throughput  (volume of filtrate) = 20L-0.4L = 19.6L

 A =V / J*T

Where

A =Membrane area (m2), V = Volume of filtrate generated (L), J = Flux (l/m2/h), T = Time (h)

A = 19.6/50*2.0

    = 0.196m2

To perform a scale-down process simulation, the same volume to area ratio is used