Fouling of an anion exchange chromatography operation in a monoclonal antibody process: Visualization and kinetic studies

Abstract

Fouling of chromatographic resins over their operational lifetimes can be a significant problem for commercial bioseparations. In this article, scanning electron microscopy (SEM), batch uptake experiments, confocal laser scanning microscopy (CLSM) and small-scale column studies were applied to characterize a case study where fouling had been observed during process development. The fouling was found to occur on an anion exchange (AEX) polishing step following a protein A affinity capture step in a process for the purification of a monoclonal antibody. Fouled resin samples analyzed by SEM and batch uptake experiments indicated that after successive batch cycles, significant blockage of the pores at the resin surface occurred, thereby decreasing the protein uptake rate. Further studies were performed using CLSM to allow temporal and spatial measurements of protein adsorption within the resin, for clean, partially fouled and extensively fouled resin samples. These samples were packed within a miniaturized flowcell and challenged with fluorescently labeled albumin that enabled in situ measurements. The results indicated that the foulant has a significant impact on the kinetics of adsorption, severely decreasing the protein uptake rate, but only results in a minimal decrease in saturation capacity. The impact of the foulant on the kinetics of adsorption was further investigated by loading BSA onto fouled resin over an extended range of flow rates. By decreasing the flow rate during BSA loading, the capacity of the resin was recovered. These data support the hypothesis that the foulant is located on the particle surface, only penetrating the particle to a limited degree. The increased understanding into the nature of the fouling can help in the continued process development of this industrial example.Scanning electron microscopy (SEM), batch uptake experiments, confocal laser scanning microscopy (CLSM) and small-scale column experiments were applied to characterize a case study where fouling had been observed on an anion exchange chromatography in a monoclonal antibody process. The results suggest the foulant is located on the particle surface, resulting in a minimal decrease in saturation capacity, but having a significant impact on the kinetics of adsorption, severely decreasing protein uptake rate.

In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

“…Unfortunately, mobile phase analysis does not reveal bound fouling contaminants while preserving the resin intact. Transmission and scanning electron microscopy of fouled resin beads clearly showed irreversible containment accumulation 6 14 31 43 . Although these studies were very informative, they were performed on dried resin beads, bearing little resemblance to the hydrated gel 44 .…”

“…To achieve high protein purity, the culture fluid first undergoes depth filtration before successive preparative chromatography steps 10 11 . The first step affinity chromatography can clear over 98% of HCP and inactive protein fragments in a single step with a ligand designed to bind only the appropriately folded full-length mAb product 2 12 13 , prior to anion and cation exchange chromatography steps which remove most remaining impurities 10 14 15 16 .…”

“…Protein fouling can occur during mAb capture or following low pH elution. The low pH employed during elution promotes aggregation of mAbs 30 which could then become trapped in the resin pores 10 14 26 29 . Moreover, hydrophobic HCPs such as histone 8 and antibody fragments can bind to the mAb product during capture to form mixed protein aggregates 29 .…”

Lifetime and Aging of Chromatography Resins during Biopharmaceutical Manufacture

“…Several researchers have investigated the contribution of fouling to the decline in column performance and proposed techniques to observe this phenomenon [4], [5]. Commonly reported techniques include the use of different microscopy and spectroscopy procedures [6], [7] (Figure 1). However, only a few studies have employed these techniques in-situ or in real time.…”

High throughput determination of cleaning solutions to prevent the fouling of an anion exchange resin

“…Characterization of resin foulants and investigation of cleaning procedures often requires multiple experimental and analytical methods, which can be difficult to employ in a high throughput (HT) manner (Close et al, ; Siu et al, ). Recently, miniaturized microliter scale chromatography columns have been shown to be an effective model for HT screening (HTS) of resin‐binding capacity (Chollangi et al, ) and step gradient elution criteria (Weindahl et al, ).…”