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

Abstract

Effective cleaning of chromatography resin is required to prevent fouling and maximize the number of processing cycles which can be achieved. Optimization of resin cleaning procedures, however, can lead to prohibitive material, labor, and time requirements, even when using milliliter scale chromatography columns. In this work, high throughput (HT) techniques were used to evaluate cleaning agents for a monoclonal antibody (mAb) polishing step utilizing Fractogel(®) EMD TMAE HiCap (M) anion exchange (AEX) resin. For this particular mAb feed stream, the AEX resin could not be fully restored with traditional NaCl and NaOH cleaning solutions, resulting in a loss of impurity capacity with resin cycling. Miniaturized microliter scale chromatography columns and an automated liquid handling system (LHS) were employed to evaluate various experimental cleaning conditions. Cleaning agents were monitored for their ability to maintain resin impurity capacity over multiple processing cycles by analyzing the flowthrough material for turbidity and high molecular weight (HMW) content. HT experiments indicated that a 167 mM acetic acid strip solution followed by a 0.5 M NaOH, 2 M NaCl sanitization provided approximately 90% cleaning improvement over solutions containing solely NaCl and/or NaOH. Results from the microliter scale HT experiments were confirmed in subsequent evaluations at the milliliter scale. These results identify cleaning agents which may restore resin performance for applications involving fouling species in ion exchange systems. In addition, this work demonstrates the use of miniaturized columns operated with an automated LHS for HT evaluation of chromatographic cleaning procedures, effectively decreasing material requirements while simultaneously increasing throughput. Biotechnol. Bioeng. 2016;113: 1251-1259. © 2015 Wiley Periodicals, Inc.

Chromatography process development aided by a dye‐based assay

“…It is essential to establish and validate an effective cleaning protocol for these operations during process development, which is generally achieved in several phases. The first phase involves screening a large number of cleaning protocols whilst running them in parallel using a scale‐down high‐throughput approach . The next phase involves identification of the most effective protocols from the initial phase and testing them at a larger scale before a manufacturing run, or choosing one protocol to validate straight at the manufacturing scale .…”

“…The use of standard gel electrophoresis techniques has advanced into miniaturized devices (lab‐on‐a‐chip), which can determine the amount of DNA, RNA and protein . Turbidity measurements taken at 320 nm can be used to analyse the clearance of high molecular weight (HMW) species and host cell impurities, whereas absorbance measurements at 280 nm can indicate the presence of the desired protein in the flow‐through, elution and regeneration steps . The amount of host cell DNA and protein detected indicates the effectiveness of the cleaning‐in‐place (CIP) and by extension the degree of fouling.…”

“…To understand the impact of repeated cycles on a chromatography resin, microscopy‐based techniques can be used to produce highly detailed images, providing insight into the extent and location of residual impurities. Scanning and transmission electron microscopes, for example, can reveal morphological changes to the bead surface under poor cleaning conditions . However, dry samples are a prerequisite for most forms of electron microscopy imaging, which can compromise the bead structure .…”

Development of pseudo‐linear gradient elution for high‐throughput resin selectivity screening in RoboColumn® Format

“…The range of possible applications for MCCs is broad and mostly includes sample preparation, dynamic binding capacity determination, establishing cleaning regimens, and optimization of step elution . Academic groups have proven early on that also complex separations are possible with this format employing pseudo‐linear gradients, and industry is recently following suit .…”

“…1 The range of possible applications for MCCs is broad and mostly includes sample preparation, dynamic binding capacity determination, establishing cleaning regimens, and optimization of step elution. [2][3][4][5] Academic groups have proven early on that also complex separations are possible with this format employing pseudo-linear gradients, [6][7][8] and industry is recently following suit. 9 Common high-throughput technologies were extended towards the characterization of a multicolumn chromatographic purification, and the technique was found to make an appropriate scale-down tool for the formal characterization of biotherapeutic processes.…”