Using Fractogel® EMD Tentacle Supports. Ion Exchange Chromatography . 3: BSA binding capacity of Fractogel® EMD TMAE (S) at linear flow rates up to . Fractogel® EMD TMAE Hicap (M). Ion Exchange chromatography using strong anion exchangers. Fractogel® ion exchangers are cross-linked. Sigma-Aldrich offers EMD Millipore, MiniChrom Column Fractogel® TMAE (S), 1 ml for your research needs. Find product specific information including.

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Fouling of chromatographic resin over operational lifetimes can be a serious problem associated with industrial separations.

This frxctogel supports the hypothesis that the foulant is located on the particle tnae, only penetrating the particle to a limited degree. The purpose of the live uptake experiments was to conduct a direct comparison between the clean, partially fouled, and extensively fouled resin based on their intra-particle radial light intensity profiles, during uptake of a BSA reporter molecule in a packed bed using CLSM.

Fractogfl this work, we consider a case study where resin fouling had been observed during process development; that of an industrial anion exchange AEX polishing step following a protein A affinity capture step in a process for the purification of a monoclonal antibody mAb. There are many different dye molecules currently available for this purpose. Figure 8 illustrates the corresponding decrease in dynamic binding capacity DBCwhich for the normal operating flowrate of 0.

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Column Studies An iterative procedure where a column 0. A range of precautions were taken to minimize fluorophore bleaching throughout the experimental work: The selection of five particles from those available up to 14 also helped us to ensure this center cross-section positioning, as we were able to only select particles where the focal plane exactly intersected with the center of each particle. The area of the particle under analysis was kept constant for each sample.

Impact of clarification strategy on chromatographic separations: The elution profiles of the conjugates were compared with native BSA in relation to retention time and peak shape. The use of BSA for breakthrough studies was not intended to replicate industrial process behavior.

Intra-particle mass transfer thus does not appear to be a limiting step. Great care was taken to ensure that the resin was not over or under packed. In this article, scanning electron microscopy SEMbatch 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.


Previous studies have also shown such areas, clearly distinguishable from the rest of the particle surface, and have reported localized external mass transfer resistance through these regions Hubbuch et al.

It appears that as the foulant blocks access to the pore entrances, the available surface area where protein can diffuse freely into the particle therefore decreases, which introduces increased resistance to mass transfer.

Protein A peak pools contained the product of interest, host cell protein, DNA and residual protein A which had leached from the affinity capture resin, and approximately 3. The appropriate XY image where the focal plane intersected with the center of each particle was then selected at each time interval, as illustrated in Figure 2 A. Clean resin had the fastest uptake, followed by partially fouled, then extensively fouled resin. In this article, we aim to elucidate on this resin fouling case study, by revealing the location of the foulant, and determining the mechanistic effects fouling has on protein uptake kinetics and resin capacity.

We apply scanning electron microscopy SEMbatch uptake experiments, CLSM on a miniaturized packed bed, and small-scale column experiments to samples of fouled resin derived from the industrial process using the worst case feed stream and overloading conditions.

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Batch Experiments The batch experiments were designed to give an initial indication of the effect that the foulant had on protein uptake, and to confirm that there were no competitive effects in the system due to modification of BSA binding characteristics when conjugated with the TexasRed flurophore, in preparation for the CLSM study.

Once the breakthrough of BSA had been recorded at each flowrate, the column fractlgel returned to AEX chromatography cycling. The fouling was found to occur on an anion exchange AEX frractogel step following a protein A affinity capture step in a process for the purification of a monoclonal antibody. Each time the cumulative load challenge reached one of the predefined amounts, the cycle in progress was allowed to run to completion, that is, the column washed, eluted, sanitized, and placed into storage buffer according to the methodology set out previously Materials and Methods: Feed to resin volume ratio All chemicals were purchased from Sigma-Aldrich Dorset, UK and were of analytical grade unless stated otherwise.

Ftactogel time and space distribution of the labeled BSA within the resin particles is recorded in situ in order to facilitate a comparison between clean, partially fouled and extensively fouled resin. The three resin samples are classified as follows: More detailed analysis of the data indicated that the initial uptake of BSA by clean resin was roughly twice as fast as uptake by extensively fractigel resin, indicating fouling significantly fractofel mass transfer.


Weak partitioning chromatography for anion exchange purification of monoclonal antibodies. The setting of the flowcell within the laser scanning confocal microscope is shown tmaae Figure 1.

SEM and batch uptake experiments are used to give initial indications of foulant location and resin performance as fouling progresses, before CLSM is used to conduct a more detailed investigation.

The light intensity is proportional to the concentration of bound protein, and therefore can be used to estimate differences in the intra-particular mass transfer and adsorption Fig. These are regions where particle—particle contact occurs within the packed bed, and show little or no fouling.

This suggests that if the partially fouled or extensively fouled resin is challenged for long enough, it will eventually reach the capacity of the clean resin, or somewhere near this.

Fouled resin samples analyzed by SEM and batch uptake experiments indicated that after frractogel batch cycles, significant blockage of the pores at the resin surface occurred, thereby decreasing the protein uptake rate.

These samples were packed within a miniaturized fractkgel and challenged with fluorescently labeled albumin that enabled in situ measurements.

A mechanistic study of protein A chromatography resin lifetime. The fouling may have an impact on the ability of the process to deliver product that meets pre-determined acceptance criteria, or reduce the lifetime of the resins which in turn has an impact on the cost of manufacture.

The increase in time that particles are exposed to BSA at lower flow rates, enables the BSA to overcome the mass transfer limitations as a result of the fouling, thus restoring DBC to pre-fouled levels.

Batch experiments, SEM fractogl CLSM and small-scale column experiments are useful tools for characterizing fouling in chromatographic resin. Pharmaceutical cGMPs for the 21st century; a risk based approach. Three resin samples were used in subsequent experimental studies to characterize the fouling. However, it has been reported that the attachment of dye molecules can significantly change the adsorption behavior of the BSA and therefore must be carefully selected Hubbuch and Kula, 11 ; Teske et al.

The increased understanding into the nature of the fouling can help in the continued process development of this industrial example.

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