Sample concentration and buffer exchange are integral to any preparative scale protein production, whether for structural biology or general biochemistry needs. These steps are required to ensure specific chromatographic requirements in between the purification steps. Even after purification, the protein must be formulated in a specific buffer at appropriate concentrations to ensure its structural integrity and effectiveness for downstream applications. Similarly, buffer exchange may also be required for in vitro refolding of the solubilized inclusion bodies by controlled denaturant removal.
Given the importance of these steps, factors such as gentleness, time efficiency, scalability, and cost-effectiveness must be taken into account while selecting a method for protein processing. Tables 1 and 2 outline the typical methods employed for protein concentration and buffer exchange, respectively:
| Considerations |
Column Chromatography | Lyophilization | Evaporation | Absorption |
Ultrafiltration Dead-end TFF |
|---|---|---|---|---|---|
| Gentleness | ✔ | ✔ | ✔ ✔ | ||
| Scalability | ✔ | ✔ | ✔ | ✔ | ✔ |
| Time Efficiency | ✔ | ||||
| Cost Efficiency | ✔ | ✔ | ✔ ✔ | ||
| Automation | ✔ |
Table 1. Methods for protein concentration
Ultrafiltration addresses all the concerns for protein concentration as well as buffer exchange compared to conventional methods. However, dead-end ultrafiltration creates a concentration gradient at the membrane as the sample is not recirculated. This slows down the filtration process and may result in material loss due to aggregation. The only way to monitor the concentration progress or to mix the sample, is by periodic interruption of the centrifugation, making it a hands-on method and adding further to the processing time. Lastly, the dead-end centrifugal devices lack scalability and can process only limited volumes.
These concerns are effectively addressed by tangential flow filtration (TFF), an alternative form of ultrafiltration that involves constant sample recirculation, thus avoiding the filter cake formation and ensuring high filtration rates. TFF also offers scalability and advantage of simultaneous buffer exchange and sample concentration. However, traditional TFF systems have a large footprint with hold-up volumes in tens to hundreds of milliliters, making them unsuitable for lab-scale applications.
| Considerations |
Column Chromatography | Dilution |
Equilibrium Dialysis |
Ultrafiltration Dead-end TFF |
|---|---|---|---|---|
| Gentleness | ✔ | ✔ | ✔ | ✔ ✔ |
| Scalability | ✔ | ✔ | ✔ | |
| Time Efficiency | ✔ | |||
| Cost Efficiency | ✔ ✔ | |||
| Automation | ✔ |
Table 2. Methods for buffer exchange
Protein Processing using the µPulse - TFF System
The Formulatrix µPulse is a fully automated and miniaturized TFF system, explicitly designed for sample concentration and diafiltration at lab scale. The entire fluid path is miniaturized on the filter chip by combining microfluidic pumping technology with TFF. This has reduced the hold-up volume to just 0.65 mL, ensuring maximum sample recovery.
The µPulse filter chips are currently available with modified polyethersulfone (mPES) and regenerated cellulose (RC) membranes that exhibit low fouling characteristics and are compatible with a variety of sample types. The µPulse offers customization for the operating pressures to make the process gentle, ensuring the structural integrity of proteins and other biomolecules. Furthermore, the µPulse processes samples 4x faster compared to dead-end centrifugal filters, and in a walk-away approach.
Webinars
Learn a fast, gentle, and automated approach for harvesting extracellular VLPs as well as refolding, and formulating various proteins using the µPulse.
Experience the single-step and scalable purification of ADCs and other macromolecules modified with small molecules using the µPulse.
Application Notes
Uncover the ease of use, scalability, and reusability of µPulse for lab-scale formulation of recombinant L-asparaginase and other biomolecules.
Learn about the time efficiency, cost effectiveness and gentleness of µPulse for refolding denatured proteins compared to equilibrium dialysis.
Publications
Structural and functional analyses of Pcal_0917, an α-glucosidase from hyperthermophilic archaeon Pyrobaculum calidifontis
Genome analysis of Pyrobaculum calidifontis revealed the presence of α-glucosidase (Pcal_0917) gene. Structural analysis affirmed the presence of signature sequences of Type II α-glucosidases in Pcal_0917. We have heterologously expressed the gene and produced recombinant Pcal_0917 in Escherichia coli. Biochemical characteristics of the recombinant enzyme resembled to that of Type I α-glucosidases, instead of Type II. More... | Related Solution: µPulse - TFF System