Jim Simmons is the Senior Director of Education at SpectrumLabs.com. He has over 30 years experience in membrane separations and spent 15 years as the Regional Sales Manager for the Central United States. Jim now conducts the Spectrum’s Training and Education Program.
Materials that will pass through the membrane can be washed away from particulates that are retained by the membrane (cells, particles, etc.) by passing a buffer through the solution. This is often taught using dialysis tubing.
When TFF is used to perform this task, the process is called Diafiltration. A hybrid of Dialysis and Filtration, diafiltration is the technique of using TFF to pass buffer through the membrane while retaining the sample. In this way, undesired species that will pass through the membrane are washed away.
Diafiltration is much faster than dialysis, requires less buffer, and is easily scalable.
Often preceded with a concentration process, diafiltration provides a method for the final complete purification of sample.
Some examples of diafiltration applications include:
- Washing away spent culture media from concentrated whole cells
- pH adjustment
- Moving the sample from one solution to another, such as in a specific buffer
- Removing excess unbound protein or drug from coated micro or nanoparticles
- Improving product yield in harvest applications
- Improving product purity in concentration applications
And a host of others.
The volume of the sample is equal to one diavolume. A wash is completed after a sufficient number of diavolumes of buffer have passed through the sample to completely remove the undesired species.
The number of diavolumes required to complete the wash depends on three main variables:
- The amount of the diavolume
A diafiltration process is generally preceded by a concentration step to reduce the volume of the sample. Strive to achieve the maximum concentration of the sample. Incomplete concentration results in a larger diavolume, requiring more time and buffer to complete the wash.
- The efficiency with which the buffer passes through the membrane
The job of buffer in a diafiltration process is to carry the undesired species through the membrane. Some buffers contain detergents or surfactants which inhibit passage of the buffer through the membrane, slowing the process and requiring more diavolumes and time to complete the wash.
- The efficiency with which the undesired species passes through the membrane
The undesired species may bind to the membrane surface or be inefficient in membrane passage in other ways, which adds to the required time and number of diavolumes needed to complete the wash.
Diafiltration may be performed using one of two methods:
- Discontinuous Diafiltration: Concentrating the volume in the feed reservoir and periodically adding buffer to regain the original volume. This method is recommended when doing process development studies to most accurately determine the point in the diafiltration process (number of diavolumes) when the wash is completed.
In this method, use a container with sufficient capacity to hold 2x the final concentrated volume of sample. Begin diafiltration by adding a volume of buffer equal to the volume of the sample and allowing it to mix. Then concentrate the sample back down to the original final concentration volume. Repeat this process until the wash has been achieved.
- Constant Volume Diafiltration: Adding buffer to the sample container at an equal rate as the rate of permeate passage through the membrane. The advantage to this method is it can be set up to run unattended.
In this method, a buffer line is fed into the sample container. Buffer may be added to the sample using one of two methods:
- Use a buffer addition pump controlled by the system controller to precisely match the permeate flow. This method is commonly used when using bags as processing containers.
- Using a sealed reservoir such as a bottle, attach the buffer feed line to the container’s vent fitting. Run this line to the buffer container.
- In both cases the concentrated sample volume does not change during the process. The buffer container must be open and allowed to empty.
In constant-volume diafiltration, mixing is of critical importance. Use a stirrer or platform rocker to assure that the buffer will properly mix with the sample, allowing the buffer to flush away the undesired species.
- For process development, it is generally best to characterize the process using discontinuous diafiltration, then switch to constant-volume diafiltration when passing the process along to manufacturing. Always verify the efficacy of the wash.
Diafiltration is one of the best ways to utilize the benefits of speed and efficiency that TFF brings to bioprocessing. When characterizing a TFF process, always seek the most efficient method. Your work on the bench may one day translate into immense gains in efficiency at the process scale later.
Next we will discuss the fundamentals of developing a TFF process.