Tangential Flow Filtration (TFF) Optimization

Tangential Flow Filtration (TFF) is used extensively in biopharmaceutical industries for clarification of microbial cells/animal cells, inclusion bodies clarification, protein concentration and diafiltration, buffer exchange, viral clearance, etc. Multiple Centrifugation steps can be avoided by a well designed TFF system with an optimized protocol.

TFF Process Development
The first step in the development of a TFF process is to define the purpose of the process and the goals to be achieved with the process. A good knowledge of these things enables the selection of the appropriate equipment design and operating parameters.

Important process objectives to be considered are
– Final product concentration
– Process volumes
– Concentration factor
– Contaminant removal
– Process time

During the development phase, the combination of physical parameters and strategy of operation results in a process that meets the success criteria based on some targets. The most important targets to be considered for a successful tangential flow filtration process are as follows:

– Final yield of the product of interest
– Final Concentration of the product
– High level of purity of the product
– Final quality/activity of the product
– Level of Bioburden
– Process Economics
– Robustness
– Process time

The main objective of Process Development is to achieve high purity of the product, low solids content and to get reproducible results. Moreover, the process should be robust and easy to scale up and scale down. In addition, it should meet the validation requirements and also the economic issues.

Choosing the Equipment
During the development of a process, the appropriate equipment and relevant components should be chosen such that the process meets the requirements for success and robustness of the product yield. For choosing TFF equipment for a particular process, the following points should be considered

– Purpose of the process
– Membrane material, module format & pore size
– Membrane Molecular Weight Cutoff (MWCO)
– Flow channel height & configuration
– Holdup loss
– Cross Flow rate
– Membrane Area
– Transmembrane Pressure (TMP)
– Process Temperature
– Process volume

In the following section, selection of appropriate system components is discussed.

Process Objectives
The purpose of the process can be defined by the separation goals of the process – Diafiltration, Product Concentration or Fractionation of the product. Also the Concentration factor, Diafiltration type, Diavolume, Volume Concentration Factor (VCF) are the other factors to be considered.

Membrane Selection
The important components of a TFF process are the membrane material, module format and the pore size of the membrane. Membranes are made with different materials to suit a wide range of applications. Due to the complex interaction of various factors that determine the membrane performance, it is very difficult to identify which membrane type will perform best for a given process. The important criteria used for the selection of membrane type are the extent of fouling, level of protein adsorption, Product retention, ability to withstand extreme temperatures and pH.

Some of the common materials used for making membranes are
– Cellulose/Regenerated Cellulose (RC)
– Polyethersulfone (PES)
– Modified Polysulfone (PS)
– Ceramics
– Polypropylene (PP)
– Cellulose acetate

Out of these, Regenerated Cellulose and Polyethersulfone are the most commonly used materials for membranes. Polyethersulfone membranes tend to adsorb proteins as well as other biological materials leading to membrane fouling and thereby reducing the flux. These membranes can be operated over a wide range of temperatures and are stable at pH range from 1-14.
Regenerated cellulose membranes are very hydrophilic, exhibiting low fouling and protein adsorption. They are more compatible with organic solvents than the Polyethersulfone-based membranes, but are less tolerant to extreme pH.

Modules
TFF membranes are made into modules in several different formats. The most commonly used membrane modules are
– Hollow fibre
– Flat plate
– Spirally wound

Flat plate modules, also called cassettes with open channels are preferred for feed streams with high levels of suspended solids for clarification or for a shear-sensitive product while hollow fibre modules are mostly preferred for applications with very low or no suspended solids. In Spirally wound modules, screens are inserted into the feed/permeate channels to increase turbulence and thereby decrease membrane fouling.

Hollow fiber

Flat Plate

Spiral Wound

Membrane Pore size
The biomolecule of interest in the feed stream is called product. Filtration can be done by selecting a membrane which retains the product and allowing other low molecular weight/size molecules to pass through. Alternatively a membrane can be selected in such a way that it retains the higher molecular weight molecules and allows the product to pass through.
The general thumb rule for pore size selection is to select a membrane 3-6 times tighter than the molecule to be retained and 3-6 times greater than the molecule to allow them to pass through.
Membranes has to be selected with an average pore size which is significantly smaller than the size of the retained product thereby reducing the membrane fouling and ensuring that the product retained remains on the upstream side of the membrane. To select a membrane, different pore sizes have to be tested. The largest possible pore size has to be fixed that does not result in significant membrane fouling and does not allow the target molecules to pass through the membrane. Tighter membranes restrict the pass through of lower molecular weight/size contaminants through the membranes.

Membrane Molecular Weight Cutoff (MWCO)
The molecular weight cutoff (MWCO) of a membrane is its ability to retain a particular given percent of a target molecule in solution. To retain a target molecule, a membrane has to be selected with a MWCO that is 3 to 6 times lower than the molecular weight of the target molecule. If a Fractionation step is performed, where similar sized molecules to be selected, a membrane has to be chosen with MWCO that is lower than the molecular weight of the target molecule to be retained but higher than the molecular weight of the molecule which has to be passed through.

Flow Channel
The channel height should be selected such that the passage of the cells is sufficiently high. The objective to reduce the retentate channel height is to provide proper angle of attack and to prevent the excessive pressure drop. The retentate channel height increases with the increase in final product concentration. Lower channel height decreases the pump flow thereby increasing the pressure drop.

Flow Channel configuration
The concentration of the feed solution and its characteristics determine the configuration of the Flow Channel. There are three different channel configurations available.
Screen channel configuration is used for a clean, filtered solution (no particles or aggregates that can get trapped in the screen). A woven separator in the channel creates gentle turbulence along the membrane surface, thereby minimizing membrane fouling.

Suspended screen channel configuration has a more open structure in the retentate channel that provides better performance with highly viscous fluids or particle-laden solutions are being used. It can also be used to concentrate cells or clarify cell culture or fermentation broths.

Open channel configuration is used in the same applications as the suspended screen channel. It uses spacers instead of screen to define the channel height. Devices may be available in several channel heights. This structure minimizes cell lysis and increases the recovery of intact cells after concentration.

Holdup Loss
The holdup volume which can not be recovered in a process, accounts the product loss. Once filtration gets over, a small volume of the product remains in the membrane and the process piping. This product can be recovered by appropriate design of piping, development of a recovery step or by flushing with a buffer thereby minimizing the product loss.

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