Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems

Characterization of partitioning behaviors of immunoglobulin G in polymer-salt aqueous two-phase systems

“…The generated results were used to support the linear relationship between the natural logarithm of partition ratio, ln G, and the PEG concentration difference between the top phase and the bottom phases, D[PEG], as proposed by Chow et al (18). Moreover, a linear correlation, as proposed by Chow et al (19) for describing the protein partitioning behavior of biomolecules as the functions of phase composition and pH, was further extended in this work to account for the influence of the concentration of NaCl on the partitioning behavior of IgG. Other parameters of ATPS such as molecular weight of PEG, volume ratio (V R ) and system pH were evaluated for the optimum recovery of IgG in the PEGephosphate ATPS.…”

“…Based on the previous work (19), the natural logarithm of the partition coefficient (ln K) of a charged protein can be expressed as a function of non-electrostatic term and electrostatic term as follows:…”

Evaluation of Driving Forces for Protein Partition in PEG-Salt Aqueous Two- Phase Systems and Optimization by Design of Experiments

“…In contrast, the bottom phase becomes more positively charged, thus distributing more hydrophilic and negatively charged proteins (e.g., BSA/OVA at pH 7) preferentially into the more hydrophilic salt-rich bottom phase in PEG-salt ATPS by addition of already small amounts of NaCl up to 2 wt.%, and consequently decreasing K as well as the protein hydrophobicity with a larger positive charge [7,8,70,82]. Hence, up to 2 wt.% NaCl the partition behavior of BSA/OVA is depending primary on the surface net charge, and to a lesser extent of the hydrophobicity effect [2,70,82].…”

“…Furthermore, above 2 wt.% NaCl the partition behavior of BSA/OVA is highly dependent on increasing hydrophobic protein-PEG interactions and totally independent of the surface net charge, thus increasing K [1,70,82]. Additionally, the protein shift from the bottom into the top phase occurs due to an increasing salting-out effect in the salt-rich bottom phase as well as a rising shielding of the proteins' surface charges caused by Na + and Cl − ions [13,84].…”

“…In general, negatively charged proteins (e.g., BSA/OVA at pH 5-9 as well α-CT at pH 9) selectively partition into the PEG-rich top phase, thus increasing K, while positively charged proteins (e.g., LYZ at pH 5-9 and α-CT at pH 5-8) prefer a partitioning into the salt-rich bottom phase, thus resulting in a decreasing value of K due to electrostatic interactions between protein and PEG units as a result of charge distribution [1,6,7,13,44,51,62,66,74,88]. Hence, within the requirement of electroneutrality at the interface, an increasing value of K with rising pH can be explained by considering the protein surface charge compared to its pI and the fact that negatively charged proteins, such as BSA/OVA at pH 5-9 as well as α-CT at pH 9, become more negatively charged with increasing pH due to stronger electrostatic interactions between each protein and the PEG-rich top phase which has a higher positive charge density, thus increasing K [7,44,51,62,66,82,89]. However, the investigated positively charged proteins (e.g., LYZ at pH 5-9 and α-CT at pH 5-8) tend to partition predominantly into the PEG-rich top phase with rising pH, thus increasing K. Thereby, an enhanced affinity of the positively charged α-CT/LYZ for the top phase and rising K occurs with increasing pH based on a rising salting-out effect in the bottom phase as well as enhanced hydrophobic interactions between α-CT/LYZ and PEG in the top phase due to an increased ratio of phase-forming salt ions and high surface hydrophobicity.…”