Xuemei He, Ph.D., senior staff scientist, process chromatography division, Bio-Rad Laboratories, described the use of Nuvia™ cPrime™, a hydrophobic cation exchange media, for mixed-mode chromatography for the purification of a variety of recombinant proteins expressed by prokaryotic or mammalian cell lines. Dr. He cites the main advantages of Nuvia cPrime as high selectivity, tolerance of load conductivity, and gentle purification conditions.
Nuvia cPrime is composed of a functional ligand on a base matrix of macroporous hydrophilic polymers. Potential interaction modes for protein binding include weak cationic exchange, hydrophobic interaction, and hydrogen bonding. Using lysozyme isolation from an E. coli lysate as an example, Dr. He presented two different purification method development schemes and associated optimization strategies.
In the first example, she investigated the effect of buffer salt concentration and pH on the purity and yield of final product via a design of experiment (DoE) approach. The “sweet spots” for optimal yield and purity of lysozyme were identified using a set of 11 experiments that covered the pH range of 4–8 and NaCl concentration range of 10–400 mM for binding and 10–1,000 mM for elution.
In all of these experiments spin columns containing 50 µL of Nuvia cPrime media were used. Purification conditions obtained from these DoE screening tests were experimentally confirmed as being effective for host cell protein and double-stranded DNA removal, and were suitable for use in production scaleup.
In the second purification method development strategy, Dr. He described an approach based on creating different types of buffer gradients on a traditional chromatographic column—pH, conductivity, or elution with additives. Each of these methods was tested for yield and purity of target protein. They were then refined and converted to a step elution protocol for larger scale production.
Mixed-mode chromatography offers additional advantages for protein separation and purification, according to Dr. He. With options for efficient target protein elution and tolerance of a range of feed conductivity, this technique often allows easy transition between chromatography unit operations, thus eliminating the need for dilution and extensive feed manipulation.
“Despite the fact that multiple functionalities may be employed for selective target protein binding, chromatographic methods can still be developed in a straightforward fashion by looking into the effects of buffer pH and conductivity on purification performance,” she said.
Dr. He also presented case studies on a diverse set of therapeutic proteins, including highly glycosylated recombinant viral antigens and monoclonal IgGs and IgMs, which demonstrated the efficient removal of process and product-related impurities such as degradation fragments and high molecular weight aggregates.
James Woo, from Rensselaer Polytechnic Institute, presented a study comparing the retention of a library of common proteins on a variety of multimodal cation-exchange ligands, including Bio-Rad’s Nuvia cPrime resin. The study explored the effect on protein retention of changes in the ligand chemistry and geometrical presentation of the functional groups—the effects of weak vs. strong charges at pH 5 in 20 mM acetate solvent, and the effects of secondary groups and hydrogen bonding.
The results indicated that protein retention tended to decrease with increased charge and was affected by even subtle changes in ligand chemistry. Certain hydrophobic proteins, such as serum albumins, were selectively excluded from the resin, suggesting that by elucidating the role of different functional groups and modulating the design and hydrophobic nature of a multimodal ligand it is possible to cover an expanded design space and target specific selectivities for a given multimodal process step.