Structural and functional characteristics of virgin and fouled Protein A MabSelect resin cycled in a monoclonal antibody purification process

Abstract

The structural and functional characteristics of the Protein A MabSelect resin are determined for a virgin sample and for samples removed from a column that had been operated in an antibody capture process which had shown losses in product recovery over fewer than 20 cycles. Compared to the virgin resin, the cycled samples show reduced porosity and apparent pore size based on inverse size exclusion chromatography while transmission electron microscopy (TEM) shows accumulation of foulants on the cycled resin. Adsorption isotherms, batch adsorption kinetics, and batch desorption kinetics, obtained using the antibody in purified form, show that the cycled samples have about 10% lower binding capacity and slower mass transfer. Confocal scanning laser microscopy shows, however, that different degrees of fouling exist for different beads in the cycled samples, which may correspond to the existence of areas exposed to minimal or no flow in the process column. Replacing the standard cleaning procedure with an improved multi-step cleaning protocol prevented the accumulation of foulants in the resin beads, as evident from TEM, and resulted in a stable operation with high recovery.

Fluorescence based real time monitoring of fouling in process chromatography

“…TEM was performed after dehydrating the resin samples and washing them with increasing ethanol concentrations from 0 to 100% anhydrous ethanol. Sample was then embedded in LR-White resin and ultramicrotomed into sections and was viewed unstained11. It is observed that the fouled resin sample from the 100 th cycle exhibits deposition of foulants with a film thickness of 0.3 μm (Fig.…”

“…Each sample was then embedded in LR-White resin and ultramicrotomed into sections. For better contrast, sections of the virgin resin samples were stained with a mixture of uranyl acetate and lead citrate, while sections of the cycled samples were viewed unstained11.…”

Investigation of cathepsin D–mAb interactions using a combined experimental and computational tool set

“…Several studies have been carried out to confirm that mAb-HCP interactions were responsible for their lack of removal during protein A capture and subsequent polishing steps (Chollangi et al, 2015;Doneanu et al, 2012;Soderquist, Trumbo, Hart, Zhang, & Flynn, 2015;B. Zhang, Goetze, et al, 2016;S. Zhang, Goetze, et al, 2016;S.…”

Understanding the Chromatographic Processing of Recombinant Human Papillomavirus Vaccine Using Ceramic Hydroxypatite

“…CHT samples for TEM were prepared by immersing the particles in the acrylic resin LRWhite (London Resin Company, London, UK) and incubating them at 45°C overnight as discussed in reference [33][34] . The solid samples were then sectioned into 80 nm sections by ultramicrotome and viewed with a JEOL JEM-1230 transmission electron microscope.…”