Effects of chemical modifications in the partition behavior of proteins in aqueous two‐phase systems: A case study with RNase A

Abstract

Chemical modification of proteins is gaining importance due to the improvement in properties and the broader range of applications that these protein conjugates have. Once modified, several purification strategies need to be applied to isolate the conjugates of interest. Aqueous two-phase systems (ATPS) are an attractive alternative for the primary recovery of proteins and their conjugates. However, to better understand which biochemical parameters affect in greater degree the partition behavior of these modified proteins in ATPS, it becomes necessary to characterize the partition behavior of different species. In this work, ribonuclease A (RNase A) was selected as a model protein to address the partition behavior of chemically modified proteins in ATPS. Native, mono-PEGylated, Uniblue A, Dabsyl Chloride, and Direct Red 83 chemically modified RNase A's were partitioned in 16 different polyethylene glycol (PEG)-potassium phosphate ATPS. Results suggest that while the effects of system design parameters govern the partition of native RNase A, the behavior of the chemically modified species is more influenced by the physicochemical characteristics of the modifying molecules, that in most cases promote partition toward the top polymer-rich phase with recovery percentages as high as 86%. It has been found that both, the hydrophobicity and molecular weight of the modifying species play a preponderant role in conjugate partition behavior since as hydrophobicity increases partition is promoted towards the PEG-rich phase balancing the effect of the molecular weight of the modifying molecules that tends to shift partition towards the salt rich phase.

Partitioning in aqueous two‐phase systems: Analysis of strengths, weaknesses, opportunities and threats

“…One advantage of the recombinant approach is that relatively small MW affinity tags of a few amino acids can be constructed which provide for enhanced partition, as well as affinity, ion‐exchange or hydrophobic interaction chromatography performance [43]. A fourth affinity possibility is chemically modifying the target molecule to adjust its partition behavior [44, 45]. All of the above affinity approaches have advantages and disadvantages in regard to the need to produce generic (based on charge or hydrophobic interactions for example) or specific affinity ligands, and isolate affinity ligands from targets downstream.…”

Perspectives, Tendencies, and Guidelines in Affinity-Based Strategies for the Recovery and Purification of PEGylated Proteins

“…Affinity ATPS may be constructed for the purification of PEGylated proteins using the already known ligands for them. It should be mentioned that by themselves, some individual PEGylated proteins (mono-PEGylated RNase A and α-lactalbumin) have showed preference for the top-phase (or PEG-rich phase) in PEG-phosphate systems [15,53], but the use of affinity ligands may increase the recovery of these proteins in the PEG-rich phase. Incorporation of anti-PEGylated protein antibodies as ligands of PEG-modified proteins could favor the partitioning of these conjugates toward a determined phase.…”

Development and Characterization of PEGylated Chromatographic Monoliths as a Novel Platform for the Separation of PEGylated RNase a Isomers

“…In all cases, a small amount of Interestingly, when the 20.0 kDa PEGylated monolith was used to separate 40.0 kDa PEGylated RNase A, two peaks were obtained instead of one, which is usually expected (Figure 2). RNase A has a molecular weight of 13.68 kDa [27], therefore, the formation of a di-PEGylated conjugate is unlikely with a PEG of 40.0 kDa because of the produced steric hindrance. Once a PEG chain of this size is attached to the protein, it hinders other amino acid residues from reaction with other PEG molecules [1].…”