Advances in Protein A Chromatography Resins

Aaron Moulin, field application scientist, Purolite (North America).

Aaron Moulin spoke about Purolite’s patented jetting technology, highlighting the performance of Praesto Jetted A50 and Praesto Jetted A50 HipH protein A affinity resins.

He began by describing the traditional way to produce agarose beads through batch emulsification, which takes six to eight weeks. Manufacturers perform solubilization, emulsification (with a nonpolar atrophic solvent, usually toluene), crosslinking, screening (sieving), functionalizing, and final testing for quality attributes. The sieving step alone takes three to four weeks.

Jetting works through a similar emulsification process, although it uses food-grade mineral oil instead of a solvent. But the beads are formed differently through use of a “membrane can” instead of an impeller. About half the size of a soup can, the apparatus has thousands of laser-edged holes. Beads are formed through its design in coordination with the flow velocity, viscosity, surface tension, and the frequency of the nozzle (the specific oscillation of the can). Agarose extrudes through the can when the motion cuts off the beads. The spherical beads then are hardened by cooling and collected in a vessel. With neither an impeller nor a sieving step, beads are formed of appropriate size and distribution just by their jetting through the can. The process could be essentially continuous. Moulin noted that “as long as you keep supplying agarose and mineral oil, you will keep making beads.” The current process is limited by the size of the collection vessels, in which the company collects about 600 L of beads/batch.

The main difference between the two methods lies in their resulting particle size distributions. A typical batch-emulsified resin has a relatively flat distribution of small, large, and mean-sized beads, whereas those formed through jetting have a much narrower distribution with a larger centroid peak. Having fewer smaller particles enables higher flow velocities, which reduces fouling in chromatography operations, improves column packing efficiency, and increases efficiency of mass transfer.

Moulin described the flagship Praesto Jetted A50 product to emphasize how a jetted resin offers faster uptake and plateauing because of the better mass-transfer properties of a more uniform bead size. He detailed information related to pressure flow and other strategies designed to take advantage of the mass transfer properties of jetted beads. A series of slides provided detailed examples.

The Jetted A50 HipH resin was developed in response to the increasing complexity of antibody purification as the types of antibodies and antibody-like constructs have increased. Many are problematic at the low pH levels usually required for protein A resins. They are prone to aggregation and can be acid labile. The need to elute from them at relatively higher pHs and mitigate some of those issues resulted in development of the high-pH ligand. Purolite developed the product in partnership with Repligen and Navigo Proteins using a library of shuffled monomers, a mixture of “not A domain, not B domain, not Z domain, but a little bit of all of them.” The monomers are tested against desired criteria, and the desired properties are modified and enhanced.

Moulin offered a few comparisons of the two jetting products, then moved on to optimization steps. The HipH product, he said, provides a much wider range of elution possibilities to explore during development of purification strategies. His slides showed details of those comparisons.

His remaining points touched upon issues of column volumes, protein A leakage, resin lifetime, and host-cell protein (HCP) removal. He summarized an Alvotech case study of a resin-screening study for purification of a crystallizable fragment (Fc) fusion protein, applying the Praesto Jetted A50 HipH product at multiple different pHs. The purity was excellent across all pH levels tested; the HCP clearance was good, although it showed a potential for improvement at high pH levels; and DNA content “took a nose-dive” at pH 4.0. The resin seems to show good downstream benefits toward subsequent removal of HCP and DNA.

Moulin responded to audience questions regarding comparative data with other protein A resin’s capacity and impurity profiles, concerns about downstream processes for certain monoclonal antibodies and fusion proteins, and control (monitoring) of pore size.

Fill out the form below to view the full presentation now.