Novel bioengineering strategies for the recovery and purification of PEGylated lysozyme conjugates: in situ ATPS and affinity chromatography
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PEGylation is the modification of therapeutic proteins with polyethylene glycol (PEG) with the goal of improving their bioavailability and effectivity in the organism. During the PEGylation process, proteins with different degrees of PEGylation and positional isomers are generated. Numerous chromatographic and non-chromatographic techniques have been used for the purification of the adequate or most active conjugate. However, the obtained yields are still low, representing an interesting engineering challenge to address. Therefore, novel techniques to achieve this must be devised. In the present work, two strategies were explored: in situ aqueous two-phase systems (ATPS) and affinity chromatography. Aqueous two-phase systems (ATPS) are a promising alternative for recovering modified proteins but this technique has not been tested with complete PEGylation reactions. In this work, lysozyme PEGylation reactions were used as part of the phase-forming chemicals to form in situ ATPS. This was best achieved by adding a 4M ammonium sulphate in a 20 mM Tris-HCl pH 7.0 solution. The phases were separated and analyzed by monolithic chromatography and SDS-PAGE. Results indicate that PEGylated lysozymes (mono- and di-) are mainly fractionated to the top phase (56% and 100% respectively) while native lysozyme was found in the bottom phase (97.7%). On the other hand, PEG-modified and native lysozyme adsorption to Heparin Sepharose was described by Langmuir isotherms. The affinity of the conjugates decreased with the PEGylation degree with no significant binding of the reactive 20 kDa mPEG to the resin. A method in Heparin Affinity Chromatography (HAC) eluting with NaCl gradient was developed and optimized through Response Surface Methodology for the purification of mono-PEGylated lysozyme with a better yield, purity and productivity than other reported chromatographic modes. A formulated rate model could model and simulate the separation of mono-PEGylated and native lysozymes in HAC. Diverse mass transfer data were obtained from this simulation. Finally, as an antecedent to the generation of immunosorbents to purify PEGylated proteins, the immobilization conditions of antibodies on NHS Sepharose 4 Fast Flow were viii optimized with a factorial design. The antibody isotype showed effects on the coupling efficiency, being improved when isotype G was used. In conclusion, in situ ATPS and heparin affinity chromatography combined with optimization tools such as design of experiments, modelling and simulation represent new and never reported before techniques in the recovery and purification of PEGylated lysozyme with several advantages with respect to currently used methods.