Development of novel polymer-protein conjugates and characterization of chromatographic supports

Abstract

Protein versatility has positioned them as a high-value biotechnological product. Among protein applications, its use as a therapeutic agent is highlighted. However, some therapeutic proteins need a modification process to increase their pharmacokinetic properties. During the protein modification process, the purification step, mainly performed by chromatography, represents a critical stage in ensuring the safety of modified therapeutics proteins. Being chromatography the main purification method, it is mandatory to fully understand the effect of all its operational variables on the separation performance. This work presents a deep analysis of chromatographic strategies used to purify modified therapeutic proteins commercially available. Furthermore, an alternative protein modification process is presented using N-(2-hydroxypropyl) methacrylamide (HPMA) polymer and its purification by chromatography. Besides, the development and characterization of PEGylated monoliths as an alternative to purify PEGylated proteins is performed. Finally, the characterization of core-shell particles being used as chromatographic support is developed. The results showed that the selection of purification strategies of commercial modified proteins depends on physicochemical properties of both protein and attached molecule and is highly dependent on the matrix where the protein is recovered. HPMA copolymers can be conjugated with Ribonuclease A (RNase A) under non-demanding conditions (PO4-3 buffer 50 mM pH 5.1 + 20 mM NaBH3CN). The new conjugates showed higher hydrophobic behavior than the native protein being this feature exploited by hydrophobic interaction chromatography (using 1.5 M (NH4)2SO4) to separate the conjugates from the unreacted protein. On the other hand, PEGylated monoliths can separate PEGylated RNase A and even isoforms when large polyethylene glycol molecules (>20 kDa) are attached to the monolith. Lastly, the CaptoTM Core 700 resin structural (50.4 nm pore size, 4.18 µm shell thickness, and 90.7 µm particle size) and adsorptive properties allowed modeling and adsorption prediction of two model proteins. In combination, all these results represent new knowledge in the polymer-protein technology and chromatography areas that proportionate guidelines to purify a wide range of molecules such as native, recombinant, and modified proteins efficiently.