Enrique Carredano

Characterization of multimodal hydrophobic interaction chromatography media useful for isolation of green fluorescent proteins with small structural differences.

Hydrophobic interaction chromatography (HIC) has been developed as a powerful technique for separating and purifying proteins. In this study, we have characterized the ability of new multimodal pH‐HIC media to resolve proteins with only small differences in their primary structures. This was done by determining the retention times of different green fluorescent protein (GFP) mutants prepared from Escherichia coli extracts. The mutants, modified with single or double hydrophobic amino acid substitutions in two positions, N212 and T230, could be resolved successfully, up to 2.1 column volumes in retention difference for single substitutions and 2.6 column volumes for double substitutions, at two pH and on two media with varying ligand density. The retention times also correlated well with calculated theoretical retentions (R2 = 0.91) using a hydrophobic descriptor. This medium can therefore be very useful in a final polishing step during purification and the protein library prepared represents a good screening set in validating and characterizing new future media due to the accessible, but yet, extremely small differences in protein structure. Copyright

Simplification of Buffer Formulation and Improvement of Buffer Control with In-Line Conditioning (IC)

Abstract Buffer preparation for chromatography and filtration steps in bioprocessing is a challenge in terms of providing timely buffer delivery, reproducibility, and good process economics. Downstream processes also need to be intensified to match the advances in upstream productivity. In-line conditioning (IC) where the process buffers are formulated in-line from concentrated stock solutions of acids, bases, salts and additives and diluted with water has been presented as one possible solution providing reproducible automated buffer preparation processes. The control strategies of the IC system are designed to prepare buffers automatically where the critical process parameters can be controlled by feedback on conductivity, pH, flow, or combination of these. IC is aimed to facilitate buffer handling by reducing both buffer preparation time and volumes of buffers tanks and thereon operational costs. In this chapter the IC technology is described in the buffer theoretical and engineering contexts and its advantages are illustrated.