Maximizing the robustness of downstream protein purification processing can have a major impact on the economics and resource utilization of industrial-scale biopharmaceuticals production. This was one of the take-home messages at the recent Barr Enterprises’ “Prep-2007” symposium held in Baltimore.
Natraj Ram, Ph.D., and Alan Hunter, Ph.D., senior principal scientists in the downstream bioprocess R&D group at Pfizer (www.pfizer.com), in collaboration with Tim Pabst and Giorgio Carta from the University of Virginia, demonstrated that simple buffer systems can be used to create pH gradients on weak anion exchange resins for the scalable separation of protein isoforms.
Preparative-scale, high-resolution separation of protein isoforms can be used to generate material for characterization of impurities and to improve the homogeneity of a protein product. pH-based separation on ion-exchange resins takes advantage of the charge differences between protein isoforms, exploits positional or surface charges, and enables isolation of isoforms in their native state, according to Dr. Ram.
The researchers created an internal pH gradient by using appropriate buffer components and counter-ions that interact with the weak ion-exchange functional groups of the resin. The development of an internal pH gradient is based on more complicated theory and algorithms and a deeper understanding of ion equilibria than typical external salt gradients, but, according to Dr. Ram, “implementation could not be simpler,” and does not require complex proprietary buffer mixtures or equipment.
The Pfizer and University of Virginia group employed a convex hull algorithm to calculate the effective isotherm to enable calculation of pH transitions with complex buffer systems. Automation of optimization algorithms allowed the group to explore a large parameter space and derive the optimal operating point.
The strategy for developing and optimizing pH elutions was based on understanding the chemistry of the functional groups on the resins through titrations, as well as the isoelectric point, binding, and elution properties of the protein.
The group chose a target pH gradient range and selected three buffers to obtain that range on several anion exchangers. They simulated the pH gradients for a range of pHs using different proportions of each buffering species, maintaining the total buffer concentration, and rated the gradients based on desired characteristics.
They were able to conclude from the simulations that single component buffer systems were adequate to produce highly resolving (shallow or gradually changing) pH gradients and confirmed this through experimentation.
The model protein used in these experiments was apo-transferrin, which is sialated and has several glycoforms with charge differences. The target separation pH range was from 7 to 5. The separations were performed on DEAE resins. The researchers concluded that concave (versus linear) pH gradients were optimal for high-resolution separations and offered the best combination of strong binding conditions and the most shallow elution conditions. These gradients are easy to achieve with internal gradients, as they are chemistry-based rather than mechanically produced.
Ion-exchange chromatography using internal pH gradients is a “scalable protein characterization tool that allows for purification of protein in quantities needed for analysis, characterization, and determination of biological activity,” said Dr. Hunter.
“We have demonstrated scale-up of the process to a 100-mL column, with 5 g/L loading capacity, which would be sufficient for enriching desired product variants,” added Dr. Ram.