Diafiltration for Rapid Buffer Exchange
Buffer exchange is a simple yet important technique in most macromolecule purification workflows. It can be used to remove unwanted substances, adjust sample conditions, or introduce components to a sample.
For example, efficient capture of a macromolecule in ion exchange or affinity chromatography is achieved under specific buffer conditions. Therefore, exchanging your sample into the optimal loading buffer prior to chromatography is usually recommended.
PRO TIP: Purifying his-tagged proteins from a clarified cell culture? Some culture media formulations include chelating agents, which can strip metal ions from your affinity chromatography matrix. Use buffer exchange to effectively remove these unwanted substances before loading your sample onto Sartobind® Lab IDA!
Common Applications for Buffer Exchange
- Removal of salt and detergent
- Adjustment of ionic strength and pH
- Removal of low-molecular weight contaminants
- Introduction of cofactors or inhibitors
- Addition of preservatives
There are three popular methods used for buffer exchange: dialysis, gel filtration and ultrafiltration (Table 1). Buffer exchange solutions from Sartorius use ultrafiltration as the method of choice, thanks to its flexibility, speed, and lower buffer consumption, when compared to other methods.
Dialysis | Gel Filtration | Ultrafiltration | |
|---|---|---|---|
Equipment Requirements | Low | High | Low-Medium |
Consumables | Single-Use | Single-Use or Reusable | Single-Use or Reusable |
Sample Throughput | Low | Low | High |
Process Time | Slow | Fast | Fast |
Buffer Waste | High | Low-Medium | Low |
Table 1: Comparison of methods used for buffer exchange.
Buffer Exchange Methods
Dialysis
In this method, the sample is placed into dialysis tubing or a cassette comprising a semipermeable membrane. This is surrounded by the exchange buffer (dialysate). Due to the concentration gradient between the sample and dialysate, buffer components and other low-molecular weight solutes pass through the membrane by diffusion (Figure 1).
Since dialysis only requires access to a magnetic stirrer and low-cost consumables, it is an economical technique. However, with diffusion as the driving force, this process takes many hours or even days to complete. Sample capacities are usually within the range of 0.1–100 mL, making this method suitable only in research applications. Furthermore, the single-use consumables and significant buffer consumption contribute to large quantities of waste. In some cases, an increase in the final sample volume may occur, causing dilution of the macromolecule and requiring a concentration step.
Figure 1: Dialysis is an economical but time-consuming method for buffer exchange.
Gel Filtration
This technique enables separation of macromolecules from the original buffer components by size exclusion on porous, resin-based chromatography columns. Macromolecules in the sample move through the column quickly, while the original buffer and low-molecular weight solutes are slowed down by entering the pores of the resin beads (Figure 2).
Gel filtration columns are available in various formats for buffer exchange under centrifugal force, gravity flow or on an FPLC system. Due to this broad range of handling methods, equipment costs are variable, but can be very high. Process time is also variable, although far faster than dialysis, especially with centrifugal or pump-driven consumables. In research laboratories, this is the most limited technique in terms of sample capacity, with a volume range of around 0.1–10 mL. Scale-up for biopharmaceutical manufacturing can be possible but here, the larger volumes contribute to high space, time, and buffer requirements.
Figure 2: Gel filtration enables faster buffer exchange, but with limited capacity for larger samples.
Diafiltration
This process involves retention of macromolecules inside a filter unit, while the buffer and low-molecular weight components pass through a semipermeable membrane. Therefore, buffer exchange occurs by progressive dilution and concentration of the retentate (Figure 3).
Like gel filtration, various designs of ultrafilters are available, which enable rapid diafiltration under centrifugal force, gas pressure, solvent absorption, or tangential flow filtration (TFF). The equipment needed for these is commonly available in most research laboratories (either a centrifuge, compressed gas source or peristaltic pump) and capacities from 0.1–500 mL and beyond suit the broadest range of process volumes.
Diafiltration Benefits:
- High Speed - Enjoy >90% time savings when compared with conventional methods
- Maximum Capacity - Single-step buffer exchange for sample volumes up to 500 mL.
- Plug and Play - Easy-to-use solutions with minimal equipment requirements.
- Less Waste - With low exchange volumes, reduce buffer consumption by >98%.
Figure 3: Diafiltration enables rapid and efficient buffer exchange of virtually any sample volume, using ultrafilters.
PRO TIP: Is your target molecule at the concentration you need? Macromolecules can be retained by ultrafiltration membranes, while the sample volume is reduced. Try Vivaspin® Turbo or Vivaflow® to rapidly increase the concentration of your macromolecule, either before or after buffer exchange.
Diafiltration Techniques
Discontinuous Diafiltration
This is an iterative process, which involves removal of the original buffer, followed by addition of exchange buffer (or vice versa) (Video 1). These steps are repeated several times, resulting in highly controllable buffer exchange. This can be particularly useful when assessing molecule stability as factors such as salt concentration or pH are gradually adjusted.
Video 1: Discontinuous diafiltration in Vivaspin®.
Continuous Diafiltration
In this technique, exchange buffer is added to the sample at the same rate as the original buffer is removed (Video 2). This single-step process results in increased speed and efficiency of buffer exchange, but may be complicated to set up, with increased equipment requirements.
Video 2: Continuous diafiltration with Vivaspin® 20 diafiltration cups.
Combining Ultrafiltration with Diafiltration
Ultrafiltration is also a useful tool for sample volume reduction and concentration. Therefore, both ultrafiltration and diafiltration can be performed sequentially, often using the same ultrafilter. The benefit here is that the number of sample manipulation and transfer steps can be reduced, which could otherwise contribute to a reduction in target molecule recovery. UF/DF. It is a win/win.
Selected Buffer Exchange Resources
Frequently Asked Questions
To achieve ≥99% buffer exchange by diafiltration, use 3–5X volumes of exchange buffer, relative to the sample volume.
For comparison, in dialysis, an exchange buffer (or dialysate) which is 200–500X the sample volume is usually recommended. Furthermore, to improve the efficiency of buffer exchange by this method, the dialysate is usually replaced at least once. This leads to large volumes of buffer waste.
Start by choosing a more efficient buffer exchange technique, such as diafiltration. The already low level of buffer consumption in this method can be limited further, by using your chosen ultrafilter to reduce the sample volume before starting diafiltration - a so-called ultrafiltration/diafiltration (UF/DF) process.
With Vivaspin® 20 Diafiltration Cups, up to 2 mL of sample can be added to the ultrafilter. For samples up to 20 mL in volume, these should be concentrated to ≤2 mL before diafiltration.
For larger sample volumes, TFF may offer increased process efficiency. The Vivaflow® Diafiltration Reservoir has a 500 mL capacity. To minimize buffer consumption, we recommend concentrating sample volumes larger than this (up to 5,000 mL) down to 500 mL or less, before performing diafiltration.
Diafiltration offers >90% time savings over alternative methods, such as dialysis. A broad range of ultrafilter designs enable diafiltration with the equipment already available in your laboratory, so there is typically no requirement for additional investment in capital equipment.
Furthermore, the higher capacities of ultrafilters in comparison to dialysis bags or gel filtration columns means that buffer exchange can be performed on the entire sample, in most cases avoiding the need to pre-concentrate or process your sample in multiple, smaller aliquots.
Membrane fouling or polarization can sometimes occur, contributing to increased processing times (though still much faster than dialysis, for example). However, fouling and polarization are less common with modern ultrafilters such as Vivaspin® and Vivaflow®, which utilize a crossflow-like technology and highly optimized flow paths.
Selected Products for Buffer Exchange
Vivaspin® 20 Diafiltration Cups
Single-step buffer exchange. For sample volumes up to 2 mL, the diafiltration cup enables simple, continuous diafiltration.
Vivaflow® Diafiltration Reservoir
Plug-and-play diafiltration for the lab. Macromolecule concentration and buffer exchange by TFF has never been easier.
Complete Your Workflow
Cell Culture Clarification
Avoid centrifugation. Sartoclear Dynamics® Lab kits support rapid cell removal in a single step.
Chromatography
Purification as simple as filtration. Discover Sartobind® Lab for ion exchange or affinity chromatography in a fraction of the time possible with resins.