Noninvasive method for determination of immobilized protein A.

The pH transition method, developed for the determination of the ion-exchange group density on chromatographic stationary phase, was used for the quantification of immobilized protein A. Monolithic epoxy polyHIPE and particulate CNBr-Sepharose supports were used for immobilization. A lactate buffer was selected, having a buffer capacity peak approximately 0.5 pH units below the maximum buffer capacity of protein A. The pH transition measurements were performed at pH 4.3, where protein A exhibits maximum buffer capacity, with a lactate buffer concentration of 1 mM for protein A immobilized on polyHIPE monoliths and of 5 mM for protein A immobilized on CNBr-Sepharose. The pH transition height and full width at half maximum for the particulate support and the height for the polyHIPE matrix, showed a linear correlation with the amount of immobilized protein A determined from the absorbance difference before and after immobilization for both supports. The developed method allows a simple, non-invasive on-line determination of immobilized protein A using biological buffers, even for chromatographic columns with an amount of immobilized protein A as low as 0.25 mg. In addition, its sensitivity and duration can be easily adjusted by varying the buffer concentration and pH.

Non-invasive determination of ionizable ligand group density on high internal phase emulsion derived polymer.

Stepwise change between low and high salt concentration buffers of the same pH results in pH transition, the length of which was demonstrated to be proportional to the quantity of ion-exchange groups present on the matrix. In this work, we analyzed the effect of the ligand type, density, and buffer concentration on the pH transition shape for typical ion-exchange groups (QA, DEAE, SO3, and COOH) and ligands acting as metal-chelators, such as IDA, TAEA, and EDA. It was demonstrated that pH transition can occur either as a chromatographic or flat-top peak. pH transition peaks were evaluated by their length, height, and peak center parameters. While no parameter can describe the ligand density accurately with a single linear correlation for both peak types, all parameters can be used for the description of one peak type. Peak length and height exhibited the same accuracy, while their sensitivity depended on the pH transition shape: length being more sensitive for the flat-top peaks, while height for the chromatographic peaks. pH height can be obtained faster, at lower elution volume, and seems to be more suitable for the determination of low amounts of ligand, when typically chromatographic peak type pH transitions occur.

Flow-Through PolyHIPE Silver-Based Catalytic Reactor.

Catalytic reactors performing continuously are an important step towards more efficient and controllable processes compared to the batch operation mode. For this purpose, homogenous high internal phase emulsion polymer materials with an immobilized silver catalyst were prepared and used as a continuous plug flow reactor. Porous material with epoxide groups was functionalized to bear aldehyde groups which were used to reduce silver ions using Tollens reagent. Investigation of various parameters revealed that the mass of deposited silver depends on the aldehyde concentration as well as the composition of Tollens reagent. Nanoparticles formed on the pore surface showed high crystallinity with a cuboctahedra crystal shape and highly uniform surface coverage. The example of the 4-nitrophenol catalytic reduction in a continuous process was studied and demonstrated to be dependent on the mass of deposited silver. Furthermore, productivity increased with the volumetric silver density and flow rate, and it was preserved during prolonged usage and storage.