Ribonuclease A modification with poly[N-(2-hydroxypropyl)methacrylamide] copolymers: new route of synthesis and purification
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© 2020 Society of Chemical IndustryBACKGROUND: N-(2-Hydroxypropyl)methacrylamide (HPMA), a synthetic polymer, has biocompatible features that allow it to be used as a drug carrier and used in drug delivery systems; however, its potential as a protein drug carrier has not been completely exploited. This study provides a new conjugation pathway to obtain HPMA¿protein conjugates and, the use of hydrophobic interaction chromatography (HIC) to separate polymer¿protein conjugates exploiting the hydrophobicity change on the protein surface due to the conjugation. In this work, Ribonuclease A (RNase A, EC 18.104.22.168), with recognized therapeutic properties, was used as a model protein. RESULTS: HPMA was copolymerized with N-(3-aminopropyl)methacrylamide (APMA) and then activated with glutaraldehyde. The activation process raises copolymer hydrophobicity. Additionally, the HPMA¿RNase A conjugation reaction was carried out at pH 5.1 through reductive amination. At higher copolymer molecular weight (> 50 kDa), the conjugation does not occur whereas molecular weights between 30 and 50 kDa promote the conjugation reaction. The native protein was separated from modified protein using HIC and conjugates with a narrow molecular weight were obtained. CONCLUSIONS: RNase A can be conjugated with HPMA¿APMA copolymer, activated with glutaraldehyde, through reductive amination. This represents an alternative to previous conjugation reactions. Moreover, the molecular size of the copolymer impacts strongly in the conjugation reaction. The HPMA¿RNase A conjugates can be separated employing HIC and, it is possible to obtain conjugates with a narrow molecular weight dispersion. This work proposes a new conjugation pathway and purification process in route to developing chromatographic alternatives to efficiently separate HPMA¿protein conjugates. © 2020 Society of Chemical Industry.