Cuantificación y caracterización del comportamiento de partición de la Ribonucleasa A y sus conjugados polímero - proteína en sistemas de dos fases acuosas polímero - sal-Edición Única

Abstract

Ribonuclease A from bovine pancreas and its PEGylated conjugates has proven to have several potential therapeutic applications. Aqueous Two-Phase Systems (ATPS) is a promising primary recovery strategy for the fractionation of proteins and their PEGylated conjugates. However, in order to characterize the partition behavior of these molecules in ATPS, an easy to implement method is needed to estimate protein concentration in each phase. Because RNase A quantification based on colorimetric methods usually renders poor sensitivity, this paper presents a methodology based on UV absorbance to quantify RNase A and its PEGylated conjugates on polymer (polyethylene glycol; PEG) and salt (potassium phosphate; PO4) rich environments, simulating conditions found on polymer – salt ATPS. The effect of PEG and PO4 concentrations and the effect of grafted PEG chains on RNase A upon UV absorbance were evaluated. Polymer and salt concentrations have a significant effect on RNase A absorbance, reflecting the need of specific standard curves in order to consider the impact of the chemical forming phase upon the extinction coefficient. The absorbance ratios at 280 nm between mono-PEGylated RNase A/native RNase A and diPEGylated RNase A/native RNase A in PEG and potassium phosphate free environments were found to be 0.32 and 0.46, respectively, demonstrating that the grafted PEG chains have also an important effect upon protein light absorbance. The method presented results in an easy-to-implement alternative, to chromatographic approaches, since just an UV-visible spectrophotometer or plate reader is required.