Tangential flow filtration- an overview

INTRODUCTION- In general, if we talk about filtration a picture becomes in our mind of separating particles or solid substrates from water. Natural separation can be through sedimentation or deposition of particles but if in-depth filtration is required then many mechanisms are present for the process. A simple thin barrier made of metal or plastic can be used to exclude large objects, for liquids like suspensions/ media that we use in Biotech or pharma industries needs much care. Biomolecules, cells, proteins, secondary intermediates have multiple sizes, and depending on their size different filtration and extraction methods are used. Microfiltration, Ultrafiltration are a few examples of the techniques used in laboratories and industries. For proper purification and dilution purposes, cross-flow filtration and Dead-end filtration are the mechanisms in use that separate particles based on their size and not charge.

DEAD- END FILTRATION- This technique is also known as Throughflow as the feed material is forced through the membrane. The flow is only in the direction perpendicular to the membrane. All the suspended solids in the feed end up on the membrane in a filter cake.

CROSSFLOW FILTRATION- Also known as TANGENTIAL FLOW FILTRATION is a technique where membranes are used to separate bio-molecules of different sizes. The feed material is allowed to flow parallel to the membrane, while the pressure gradient is across the membrane. The primary advantage of cross-flow filtration is that it allows the solids to be kept in suspension and minimizes the build-up of a filter cake to plug or foul the membrane. It is a quick and reliable method for the separation and purification of media in downstream processing.

Fig. shows the difference between TFF and dead-end filtration

COMPONENTS OF TFF- The material to be filtered is fed in by feed reservoir through peristaltic/ feed pump and goes to filter via a feed line. The material that does not pass through the membrane is called retentate and that passes through the membrane is called permeate. Retentate pumped back to the feed reservoir to again go to filter via feed line whereas, permeate tends to be collected separately in the collection vessel. For more concentration of the solution, the material is circulated again and again till the required concentration is not observed. If buffer needs to be exchanged with the original buffer, a new buffer is added to the feed reservoir. This filtration and simultaneous exchange of buffer are known as diafiltration- the principle of TFF.

• Feed reservoir

• Feed pump

• Filter

• Feedline

• Retentate line

• Permeate line

• Pressure gauge

• Collection vessel

MODULES OF TFF- This technique has many kinds of setup but the most common are hollow fiber and cassette modules.

1. Cassette set-up- In this style, layers of membranes are stacked inside of housing separated by small screens. Filtration occurs when feed containing small molecules pass through pores in adjacent membranes in permeate while molecules larger than the pores exit through retentate. Cassette modules have higher packing densities due to layers of membranes which allows linear scaling and turbulence promoted channels. In turbulence promoted channels, there is a higher chance of mass transfer at lower feed flow rates meaning higher fluxes are achieved with lower pumping requirements that reduce concentration polarization. Turbulence promoted feed channels are thus efficient as compared to the ones that do not have hollow fiber modules.

2. Hollow fiber set-up- It is a cylindrical set-up consisting of a membrane embedded as separate fibers. Filtration occurs as the fluid passes between the fibers. Their main feature is to have low binding modified polyethersulfone membranes that deliver consistently high process flux and produce yield. Polyethersulfone (PESU) polymer is stable over a broad pH and temperature range. Membrane regeneration and depyrogenation are accomplished by using (1N) NaOH at elevated temperatures as required. Set-up is generally made of high-class plastic, can be cleaned, stored, and used repeatedly. Ready to use- scalable design usually used in laboratories with batch volume from 10ml upwards. Assembly can be easily irradiated to reduce the risk of contamination.

Fig. Hollow fiber schematic set-up and description

PORE SIZE- Ultimately, separation is based on the pore size of the semi-permeable membranes used in industry. Each component or biomolecule is treated differently and separately.

1. Micro-filtration- Microfiltration membranes have pores ≥0.1 microns and they are used to separate cells, cellular debris, and other macromolecules like proteins and enzymes. When the material is supplied to a microfiltration membrane the smaller components pass through the membrane and larger ones are retained.

2. Ultra-filtration- ultra-filtration membranes having pores <0.1 microns are used for proteins of range 3kda to 1000kda. In this case, proteins larger than the pores are retained but smaller components like solutes and water go in permeate. This process is used to concentrate proteins or enzymes that is why Ultra-filtration is the principle of TFF and its working.

Fig. Difference between microfiltration and ultrafiltration.

APPLICATIONS OF TFF-

• Separation of large and small biomolecules, clarification, and/or removal of contaminants.

• Diafiltration (for buffer exchange, desalination, and removal of biomolecules)- Following filtration and concentration while the protein/enzyme solution is circulating, the new stored buffer solution is added to the feed. In fact, protein is been washed by the flow of new buffer solution in and all buffer solution out. As the diafiltration step proceeds, the new buffer solution added in the feed replaces the old buffer solution in which proteins and enzymes were originally present effectively removing any remaining salt or solutes. When this filtration step is completed, the protein/enzymes are again passed through 0.22micron filters and collected in appropriate containers usually bottles or bags.

• Concentration and desalination (of proteins, peptides, DNA/RNA, and oligonucleotide)

• Purification and/or recovery of recombinant proteins and antibodies from cell culture media.

• Purify and/or recover plasmid DNA from cell culture or cDNA from blood.

• Cell harvesting (bacteria, yeast, mammalian cells)

• Fractionate (mixtures of proteins/peptides)

• Clarify and desalt (whole cell lysates and homogenized tissue)

• Sample prep for chromatography.

• Reduce Bioburden and/or clean up water, solutions, buffers, and media (remove and/or reduce endotoxin load.)

• Recover and or remove virus particles and/or vaccines.

• Product fractionation

CONCLUSION- In downstream processes of bioprocess techniques, tangential flow filtration can be used to filter out proteins, enzymes, secondary intermediates, and other various biomolecules. whether the wanted molecules are intracellular or extracellular, this process can not only extract them from debris but also helps in concentrating the solution removal of endotoxins, salts, and solutes, and replace the original buffer by adding a new one for better down processing. But, before going to use this method the following things should be kept in mind:

Things to learn before TFF

• What molecular weight cut-off membranes have you evaluated?

• What membrane types (materials of construction) have you evaluated?

• What are your feed stream and buffer conditions (concentration, pH, and/or conductivity)?

• Do you have any process limitations (time, floor space, temperature, etc.) to consider?

• How will you define success (product quality, yield, etc)?

• What is your final concentration requirement?

• What is your final volume requirement?

• What are your final pH, conductivity, or buffer specifications?

REFERENCES: Smart water magazine

Protein Concentration, and Diafiltration by Tangential Flow Filtration, 2003

TangenX SIUS SINGLE-USE TFF CASSETTES

They manufactured hollow fibers by using an inversion of the wet-spinning process.

From: Biotextiles as Medical Implants, 2013

Molecular weight separation of collagen-base biomaterials by ultrafiltration