(629d) Reactive Aqueous Two-Phase Systems for the Production and Purification of Pegylated Proteins

Bioprocess intensification depends heavily in the ability of combining different stages of a production process in single unitary operation. In this sense, Aqueous Two-Phase Systems (ATPS) represent a potentially adaptable stage to combine two or more operations with this purpose. As known, ATPS are a liquid-liquid fractionation technique where the product of interest partitions towards one of the phases according to its partition coefficient (K_P) which is directly related to the physicochemical properties of the compound and the phase forming chemicals. ATPS have been successfully used in the separation of different biomolecules and their use as a primary recovery stage is widely accepted. However, little work has been made in using these systems for the production and primary purification of different biomolecules.

For instance, PEGylated proteins are chemically modified proteins where at least one chain of poly (ethylene glycol) (PEG) is covalently attached to its structure without having the protein losing its properties. The advantages conferred by this modification like a lower kidney clearance, longer blood circulation times and protection from antibodies are of great importance in pharmaceutical proteins. The production and purification of PEGylated proteins however, is not a trivial task. The modified proteins have to be separated according to their number of attached PEG chains and their positional isomerism since not all of these isomers posses the same desired attributes. Previous work in our group has demonstrated that the use of PEG – phosphate salt ATPS can potentially fractionate the modified proteins from the unreacted species in a PEGylation reaction. With this line of thought, and since PEG is used in both the production and purification of these proteins a Reactive Aqueous Two-Phase System (Rx-ATPS) strategy is proposed for the in situ production and primary recovery of PEGylated proteins combining the reaction and primary recovery operations in a single stage.

Binodal curves were made with reactive monomethoxy-PEG (mPEG) and phosphate salts at pH 5.1 for polymers with molecular weights of 5.0 and 20.0 kDa. Rx-ATPS systems were then constructed with tie-line lengths (TLL) of 15, 25, 35 and 45% w/w and volume ratios (V_R) of 0.33, 1.0 and 3.0. Ribonuclease A (RNase A) was used as a model protein and was used at a concentration of 2 mg/mL across all of the constructed systems. After 17 hours of agitation at 4 °C samples were centrifuged and the phases were separated for their analysis by Size Exclusion Chromatography (SEC). As expected, reactions took place and the products fractionated mainly to the top PEG-rich phase while the unreacted protein fractionated towards the phosphate salt-rich phase. However it was found that Rx-ATPS design parameters have an important role in the behavior of each system. For instance, in systems where 5.0 kDa mPEG was used, the production of the PEGylated proteins reduced as TLL and V_R increased. In systems were 20.0 kDa mPEG was used, production of the conjugates increased as TLL and V_R values increased. Although some protein degradation was observed, reaction/recovery yields varied from 20% to 78% depending on the Rx-ATPS selected. It should be noted that reaction conditions in these systems vary heavily from those optimized in regular PEGylation reactions giving different results. There are still many variables to be explored but this is the first report of the use of Rx-ATPS for the production and purification of PEGylated proteins and the optimization of these systems will be addressed.