Behavior of host-cell-protein-rich aggregates in antibody capture and polishing chromatography.

Recent work has shown that aggregates in monoclonal antibody (mAb) solutions may be made up not just of mAb oligomers but can also harbor hundreds of host-cell proteins (HCPs), suggesting that aggregate persistence through downstream purification operations may be related to HCP clearance. We have examined this in a primary analysis of aggregate persistence through processing steps that are typically implemented for HCP reduction, demonstrating that the phenomenon is relevant to depth filtration, protein A chromatography and flow-through anion-exchange (AEX) polishing. Confocal laser scanning microscopy observations show that aggregates compete with the mAb to adsorb specifically in protein A chromatography and that this competitive interaction is integral to the efficacy of protein A washes.

Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions.

Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools.

Aggregates of α-chymotrypsinogen anneal to access more stable states.

Non-native protein aggregates present a variety of problems in fundamental and applied biochemistry and biotechnology, from quality and safety issues in pharmaceutical development to their association with a number of chronic diseases. The aggregated, often amyloid, protein state is often considered to be more thermodynamically and kinetically stable than (partially) unfolded or folded monomers except under highly denaturing conditions. However, evolution of the structure and stability of aggregated states has received much less attention.

Salting-in characteristics of globular proteins.

Protein solubility, and the formation of various solid phases, is of interest in both bioprocessing and the study of protein condensation diseases. Here we examine the the phase behavior of three proteins (chymosin B, β-lactoglobulin B, and pumpkin seed globulin) previously known to display salting-in behavior, and measure their solubility as a function of pH, ionic strength, and salt type. Although the phase behavior of the three proteins is quantitatively different, general trends emerge.