Abstract
Chromatography is a widely used separation process for purification of biopharmaceuticals that is able to obtain high purities and concentrations. The phenomena that occur during separation, mass transfer and adsorption are quite complex. To better understand these phenomena and their mechanisms, multi-component adsorption isotherms must be investigated. High-throughput methodologies are a very powerful tool to determine adsorption isotherms and they waste very small amounts of sample and chemicals, but the quantification of component concentrations is a real bottleneck in multi-component isotherm determination. The behavior of bovine serum albumin, Corynebacterium diphtheriae CRM197 protein and lysozyme, selected as model proteins in binary mixtures with hydrophobic resin, is investigated here. In this work we propose a new method for determining multi-component adsorption isotherms using high-throughput experiments with filter plates, by exploiting microfluidic capillary electrophoresis. The precision and accuracy of the microfluidic capillary electrophoresis platform were evaluated in order to assess the procedure; they were both found to be high and the procedure is thus reliable in determining adsorption isotherms for binary mixtures. Multi-component adsorption isotherms were determined with a totally high-throughput procedure that turned out to be a very fast and powerful tool. The same procedure can be applied to every kind of high-throughput screening.
Highlights
In the downstream processing of a biopharmaceutical product, chromatography represents the most used technique for the separation and purification of mixtures of proteins [1]
We propose the application of a different analytical method to determine multi-component adsorption isotherms, which are a powerful tool to better understand process dynamics, especially in the process development step
Adsorption isotherms determination allows us to more closely investigate the adsorption mechanisms of chromatography, in which the separation of components is mainly affected by these mechanisms
Summary
In the downstream processing of a biopharmaceutical product, chromatography represents the most used technique for the separation and purification of mixtures of proteins [1]. Chromatography is the most effective and widely used technique to separate a protein, it represents a bottleneck in the downstream processing. It allows us to obtain very high purity and concentration values of the product, but it is a very complex process in which a large number of operating parameters must be taken into account (e.g., type of resin, eluents type and concentration, protein concentration, dimensions of the column, flow rate, etc.) [2]. High-throughput experimental methodologies allow the investigation of several conditions at the same time, using a very low amount of sample and chemicals.
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