FRACTOGEL TMAE PDF

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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Intra-particle mass transfer thus does not appear to be a limiting step. This causes the dramatic differences in uptake, but minimal differences in capacity which we saw between the resin samples. At this flowrate, Figure 7 shows that for clean resin, breakthrough begun after approximately 10 CV of material had been applied to the column.

A miniaturized flowcell was fabricated similar in design to that used by Hubbuch and Kula 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. Frits were placed at either end of the channel, which was then connected to a syringe pump. However, this method neglects the inhomogeneous nature of protein uptake due to effects such as particle contact points and fouling.

The conventional approach examines a range of performance indicators such as pressure drop profiles, dynamic capacity, and breakthrough curves where scaled down columns are repeatedly loaded with fouling material Boushaba et al.

Integrating the area underneath the radial light intensity profiles, and correcting for the spherical nature of resin particles, indicates the relative amount of BSA bound to the different resin samples throughout uptake Fig. 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.

Figure 3 shows that by the end of the experiment equilibrium had not been reached by any of the resin samples. Clarified condition media was then applied followed by a two column volumes CV wash of the equilibration buffer. Processing of cell homogenates.

These were generated by conducting multiple cycles of the AEX chromatography on a column 0.

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Data arrived from Figure 7. Finally, column studies are conducted fractogsl investigate the effect of the foulant on protein uptake and breakthrough performance of a column system. 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.

Effect of fouling on the capacity and breakthrough characteristics of a packed bed ion exchange chromatography column. The elution pool consisted of material collected from start in UV rise, to a total of 2.

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: However, it has been reported tmea 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.

Three resin samples were used in subsequent experimental studies to characterize the fouling. Competitive adsorption of labeled and native protein in confocal laser scanning microscopy.

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In the following, fracttogel first present the results from SEM imaging and batch uptake experiments which give initial indication of the foulant location and the progressive nature of the effect with cycle number. Effects of ionic strength on lysozyme uptake rates in cation exchangers. We found that using more than five particles gave negligible benefits in terms of the reliability of our data.

A linear equation with an R 2 value of 0. Support Center Support Center. Some tmaf would have expected, either localized or in general, between the different resin samples had foulant been irreversibly binding to intra-particular binding sites, but this was not the case.

These conditions would be expected to produce the desired product quality in large-scale manufacturing Iskra et al. This is typically done by a simple linear profile evaluation through the central cross section of a scanned particle Hubbuch and Kula, Breakthrough would have been expected to remain sharp if the mass transfer was not effected by fouling, which was not the case.

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Generation of Fouled Resin Samples Three resin samples were used in subsequent experimental studies to characterize the fouling. Author information Article notes Copyright and Fradtogel information Disclaimer. More fractoegl analysis of the data indicated that the initial uptake of BSA by clean resin was roughly twice as fast as uptake by extensively fouled resin, indicating fouling significantly impacts mass transfer.

After measuring the settled volume, the resin was fracctogel with ultra-pure Millipore water to remove the storage ethanol solution and then equilibrated with 0. An investigation using small-scale chromatography, dynamic light scattering, mass spectroscopy and Fourier transform infrared spectroscopy FTIRindicated that the frractogel likely hypothesis was that resin was being fouled by a combination of product and host cell proteins.

Visualising intraparticle protein transport in porous adsorbents by confocal microscopy. Confocal laser scanning microscopy as an analytical tool in chromatographic research.

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies. Further fracfogel 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. Finally, we present the results from column studies which include BSA breakthrough profiles and dynamic binding capacities over a range of flowrates with increasing cycle number and hence also increasing fouling.

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The radial profiles of each particle were then normalized by dividing the radial dimension by the appropriate particle diameter.

Protein adsorption and transport in cation exchangers with a rigid backbone matrix with and without polymeric surface extenders.

Discoloration of ceramic hydroxyapatite used for protein chromatography. Table I Experimental methodology for investigating clean, partially found, and extensively fouled resin samples. The case for conventional unit operations. Once the breakthrough of BSA had been recorded at each flowrate, the column was returned to AEX chromatography cycling.

Fouling of chromatographic resins over their operational lifetimes can be a significant problem for commercial bioseparations.