Upfront’s universal processing platform, Rhobust™, is a second-generation expanded bed technology that facilitates crude feedstock processing (Figure 1). The principle behind expanded bed adsorption (EBA) is to allow chromatography beads to fluidize in the feed stream, which is pumped at low pressure into the bottom of the column and out through the top. The expanded bed allows particulate impurities in the feed stream to pass freely—and at high flow rates—through the system without any clogging or pressure build up.
A key feature of the Rhobust platform is the use of tungsten carbide densified agarose beads, which provide greater operational flow rates for a given expansion of the adsorption unit compared to first-generation EBA materials. More importantly, tungsten carbide-agarose beads provide a greater operational buffer to process changes such as solution viscosity variation and bed disruption by particulates. Denser beads (3 g/mL) make for a more user-friendly operation.
There are two other critical innovations in second-generation EBA—dedicated hardware designs and mixed-mode (MM) ligand chemistry. It is the combination of all these developments that are the basis for the robust performance of EBA.
Processing unclarified mammalian cells, microbial lysates, or high cell density yeast feedstocks requires free movement of the particulates through the adsorbent and hardware. Upfront’s Rhobust platform provides a fluid distribution system that removes the requirement for column meshes, frits, or porous plates that would otherwise hinder the passage of particulates and subsequently cause cleaning issues and preferential flow issues.
The development of mixed-mode ligand chemistry has also enhanced the effectiveness of second-generation EBA—adsorbents with added selectivity limit any interactions with cell debris or sticky particulates. Mixed-mode ligands are small and stable compounds that interact with proteins with a combination of binding moieties. During the ligand development process, all ligands chosen for library screening have low intrinsic toxicity and are stable in NaOH solutions at high temperature. The selectivity of the adsorbent is optimized by adjusting the loading pH and conductivity of the separation.
This technology has now been adapted into small-scale disposable devices (Figure 2) for production of monoclonal antibodies for use in toxicity studies and Phase I to Phase III trials. Two ligand chemistries are available for Rhobust MabDirect adsorbents—protein A and a mixed-mode capture adsorbent. Thereafter, the same basic technology can be applied to full-scale manufacturing with fully cleanable and reusable process equipment.