Protein adsorption on core‐shell resins for flow‐through purifications: Effect of protein molecular size, shape, and salt concentration

Abstract

This work addresses the functional properties of the core‐shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles. Protein binding and its trends with respect to salt concentration depend on the protein size and are different for the two agarose‐based multimodal resins. For the smaller proteins, the amount of protein bound over practical time scales is limited by the resin surface area and is larger for Capto Core 400 compared with Capto Core 700. For the larger proteins, diffusion is severely restricted in Capto Core 400, resulting in lower binding capacities than those observed for Capto Core 700 despite the larger surface area. Adding 500 mM NaCl reduces the local bound protein concentration and diffusional hindrance resulting in higher binding capacities for the large proteins in Capto Core 400 compared with low ionic strength conditions. The NPs are essentially completely excluded from the Capto Core 400 pores. However, 20 and 40 nm NPs bind significantly to Capto Core 700, further hindering protein diffusion. A model is provided to predict the dynamic binding capacities as a function of residence time.

Evaluation of restricted access media for the purification of cell culture‐derived Orf viruses

“…This is supported by the observation that a higher protein depletion was achieved by an increasing shell cut-off for all tested columns (Figure 1B). The larger pores in the shell facilitate a higher diffusion rate of the contaminating protein components into the particle, which was also previously observed for proteins and latex nanoparticles, when comparing CC700 and CC400 [28]. In this study, residence times of at least 2 min were suggested for loading a feed containing 1 mg protein mL −1 , whereas in the present experiments, residence times of 1 min were applied, using feed containing more than 2 mg mL −1 contaminating proteins in order to reduce the process time.…”

“…Following the evaluation of the general column suitability for the model virus purification, the selected RAM columns were cleaned (NaOH and H 2 O) and re‐equilibrated up to two times for an additional usage. Only the largest available cut‐offs were evaluated for each column, as they performed best in the previous study, and it can be assumed that these show the greatest regeneration potential due to a higher diffusion rate [28].…”

“…Hence, the ancillary hydrophobic interaction of the VP columns caused the additional product losses. However, irrespective of the ligands within the particle core, the ORFV (∼200 nm) is unlikely to enter the shell pores to bind to these ligands [28]. Nevertheless, the complex surface composition of viruses could enable the partial intrusion of viral surface proteins into the pores, thus leading to the retention of the whole virus particle.…”

“…Depending on the feed composition, the columns might be suitable for multiple re-use with an adequately optimized cleaning procedure for other viruses. Otherwise, a single-use application is clearly recommended, as was previously suggested elsewhere for the CC700 [23,28].…”

Purification processes of live virus vaccine candidates expressed in adherent Vero cell lines via multimodal chromatography in flowthrough mode

“…For example, its regeneration requires the use of solvents (Konstantinidis, Reinhart, et al, 2022), it represents a unique technology and, given its suitability to LVV purification, procuring it for commercial scale manufacturing may face supply chain limitations. Finally, its adsorption mechanism is characterized by irreversible binding of solutes diffusing into the pores of the resin (Carta et al, 2022;Mi et al, 2021Mi et al, , 2023. Hence, undesired solutes of large size may be co-purified with large target solutes, such as LVVs.…”