March 15, 2018 (Vol. 38, No. 6)
Payal Khandelwal Ph.D. Global Product Manager, Protein Purification Business Bio-Rad Laboratories
Product Designed to Achieve Excellent Throughput and High Target Recovery
Large biomolecules such as immunoglobulin M, immunoglobulin A, virus-like particles (VLPs), plasma proteins, and polyethylene glycol (PEG)-conjugated proteins are difficult to purify because of their size. Size-exclusion chromatography (SEC) is not an ideal solution. This purification technique has limited speed and specificity.
Ion-exchange (IEX) chromatography is often used to purify large biomolecules based on their charge separation. In traditional IEX resins, however, porosity or rigidity may not be optimized for large biomolecules. Such resins may prove disappointing in terms of purification productivity, to an extent determined by factors such as flow rate and dynamic binding capacity (DBC).
A resin incompatible with high flow rates (which could lead to reduced DBC, significant pressure increase, or complete column collapse, among other issues) will lead to a slow process, which is not ideal for process-scale purifications. Alternatively, a resin with a low DBC leads to poor process economics and therefore, also cannot be used for large-scale purifications.
- For process-scale purification of large biomolecules, the ideal resin should have the following features:
- High DBC and high flow rate compatibility—for ideal process economics
- Fast mass-transfer kinetics—to increase process efficiency
- Optimal pore size—for smooth large-biomolecule movement
- Ideal particle size and range—to maintain high DBC and reproducibility
- High target recovery—for ideal process economics
- High mechanical stability—for ease of use (to minimize column collapse at high flow rates)
- Broad chemical stability and compatibility—for ease of use
Bio-Rad has designed a strong, high-performance anion exchange (AEX) resin, Nuvia™ HP-Q, specifically for downstream purification of large molecules and plasma fractionation. It has all the required capabilities for process-scale purification. It is built on a rugged and hydrophilic base bead that provides fast mass-transfer kinetics and low nonspecific binding to increase productivity.
Stable Base Head
The stability of this base bead and its broad chemical compatibility contributes to a long lifecycle. The pore size of Nuvia HP-Q was developed for easy accessibility of large biomolecules, leading to better binding dynamics. Its internal spacer was designed to provide optimal binding of biomolecules even at high flow rates, thereby increasing process productivity.
The particle size of Nuvia HP-Q is optimized to offer high DBC at fast flow rates without excessive increase in backpressure, thereby delivering excellent process economics.
The Nuvia HP-Q resin is undergoing extensive and thorough evaluations. Initial studies have yielded encouraging results.
High Dynamic Binding Capacity for Ideal Process Economics
To test if the Nuvia HP-Q resin shows evidence of higher binding to large biomolecules than do some existing AEX resins, thyroglobulin (Mw: 670 kD) was chosen as a model protein. Three commercially available AEX resins were chosen, along with Nuvia HP-Q, based on their vendor-published DBCs and proposed application.
The 10% breakthrough (BT) curve—the protein (analyte) concentration at which 10% of the protein is seen in the effluent—was used in lieu of the DBC to indicate the amount of protein that could bind to each of the resins before column saturation. The results showed that Nuvia HP-Q demonstrated 10% BT at a significantly larger protein load as compared to the other AEX resins (Figure 1). This suggests that Nuvia HP-Q has the highest protein-binding capacity among the resins tested.
This higher binding capacity of Nuvia HP-Q is partly due to its unique structural chemistry and optimal particle size range of 38–53 µm. Its size range helps overcome the limitations of existing resins, which provide compromised DBCs (decreasing productivity) or flow rates (increasing the process time required).
In addition, Nuvia HP-Q has an optimized pore size and internal spacer. As a result, large biomolecules gain proper accessibility and are not excluded from the pores at fast flow rates. The ligand density in the beads is ideal for efficient binding of large biomolecules, minimizing the loss of DBC at high flow rates.
Excellent Pressure Flow Properties for High-Throughput Purification
Nuvia HP-Q resin is designed with an optimal bead size to achieve both laboratory- and process-scale purification of large biomolecules at high flow rates without being limited by column pressure. This could lead to an increase in productivity during protein purification and make Nuvia HP-Q compatible with high-throughput processes.
To test the flow rates at which Nuvia HP-Q–packed columns could be run without a significant pressure increase, the pressure flow curve of Nuvia HP-Q was tested in a 20 × 20 cm InPlace™ column packed using a compression method at 350 cm/hr to a compression factor of 1.20. The pressure developed in the column at different buffer flow rates ranging up to 800 cm/hr was studied. The column pressure remained below 1.5 bars at a linear velocity of 350 cm/hr (Figure 2).
The new Nuvia HP-Q AEX resin can help overcome the limitations in purifying large biomolecules. Initial studies show that it is superior to existing AEX resins due to its DBC for large biomolecules and the fact that it can operate at high flow rates without adverse pressure outcomes. These features are most critical for process-scale purifications.
Nuvia HP-Q Resin will be available in multiple user-friendly formats including prepacked Foresight™ columns and plates for purification condition screening and bulk bottles for pilot- to manufacturing-scale purifications.