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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Figure 7 shows that over the course of the column studies, both the shape and the position of BSA breakthrough changed drastically as cycle number increased. An investigation using small-scale chromatography, dynamic light scattering, mass spectroscopy and Fourier transform infrared spectroscopy FTIRindicated that the most likely hypothesis was that resin was being fouled by a combination of product and host cell tmqe.
Visualizing two-component protein diffusion in porous adsorbents by confocal scanning laser microscopy. These results are all in agreement with the hypothesis that the foulant forms a layer on the surface of the resin and does not significantly penetrate into the particles.
Data arrived from Figure 7.
Tmaae 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.
The technique fractogfl a flowcell to measure changes at various stages of fouling in resin capacity and uptake kinetics, at a particle level. Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.
Bound BSA concentration as a function of time. Dractogel control experiment using clean resin at 0. However, a clear difference in the initial uptake rates was observed with clean resin having the fastest uptake, followed by partially fouled resin, then extensively fouled resin.
Control experiments investigating fluorophore bleaching upon repeated use were completed, and confirmed that bleaching could be neglected for the purposes of this work. An iterative procedure where a column 0. This is typically done by a simple linear profile evaluation through the central cross section of a scanned particle Hubbuch and Kula, The AEX resin has been successfully used as part of a two column platform process for the purification of numerous monoclonal antibodies in the past Kelley et al.
In contrast, when the resin was extensively fouled, onset of breakthrough was rapid, beginning after less than 1 CV, similar to what would be expected during operation in flow through mode with minimal protein binding.
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Scanning electron microscopy images of A: Results and Discussion The overall objective of this fractpgel was to determine the location of fouling on resin particles and the effect of this fouling on protein kinetics and resin capacity in an AEX polishing step from an industrial purification process.
Demonstration of the use of windows of operation to visualise the effects of fouling on the performance of a chromatographic step. J Chem Technol Biot. Confocal imaging of chromatographic fouling under flow conditions. Confocal laser scanning microscopy as an analytical tool in chromatographic research.
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 fractofel. XY image acquisition plane shown on diagram. The SEM imaging suggests a mechanism where the foulant blocks pore entrances but does not penetrate a significant distance into the particle, instead continuously growing outwards over successive cycles.
The large number of confocal images from the flowcell experiments were processed to generate a reliable set of radial gmae intensity profiles.
Once the breakthrough of BSA had been recorded at each flowrate, the column was returned to AEX chromatography cycling.
The column used in protein A chromatography was 1. The area of the particle under frcatogel was kept constant for each sample. Scale down cycling studies fmae fouled samples to measure BSA breakthrough over an extended range of flow rates. 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.
Very large scale monoclonal antibody purification: A mechanistic study of protein A chromatography resin lifetime. The knowledge gained from the use of these analytical methods increases process understanding, and thus provides for a better informed column development.
Processing of cell homogenates. Figure 8 illustrates the corresponding decrease in dynamic binding capacity DBCwhich for the normal operating flowrate of 0. Comparison of loading capacities of various proteins and peptides in culture medium and in pure state.