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Purification and Conformational Characterization of a Novel Interleukin-2 Mutein

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Abstract

Toxicity of high-dose IL-2-based therapies have motivated the development of the IL-2 mutein, which has low expansion properties for regulatory T lymphocytes. The development of two variants (A and B) for the IL-2 mutein purification as well as a conformational comparative study by Circular dichroism (CD) and fluorescence spectroscopy of these products were evaluated. For the first time, in our center, were used of DTT and 2% SDS in the solubilization step to decrease the aggregates on intermediate product, which favors that disulfide bridges are correctly formed during re-folding. A molecular weight of 18 kDa to the monomeric form and of 25–37 kDa to the oligomeric species were estimated by SDS–PAGE. IL-2 mutein showed similar far-UV CD spectral characteristic typical of cytokines with 41% of α-helix content. Batches obtained by Process B showed similar conformational features according near-UV CD and FS studies. However, those obtained by Process A differed in their folding. IL-2 mutein showed that conformational features by near-UV CD were affected by 2% SDS, no variations on secondary structure were observed. Melting temperature values by far-UV CD were higher than 95 °C, indicating a high thermal stability. Finally, the drug product obtained by Process B showed similar conformational characteristics by near-UV CD and FS, and higher biological activity values (7.0 × 103 ng/mL) in the cell proliferation assay with respect to Process A. Also, the recovery was 15% higher than in the Process A and exhibited a 78.48% of purity. Indeed, Process B was selected for the purification.

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Acknowledgements

The financial funds were provided by the Center of Molecular Immunology (Havana, Cuba).

Funding

The financial funds were provided by the Center of Molecular Immunology (Havana, Cuba).

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Authors and Affiliations

  1. Bioprocess R&D Department, Center of Molecular Immunology, 216 Street and 15 Avenue, Atabey, Playa, P.O. Box 16040, 11600, Havana, Cuba

    Leina Moro-Pérez, Sum Lai Lozada-Chang & Tammy Boggiano-Ayo

  2. Biology Faculty, Center for Protein Studies, University of Havana, 25 St. No. 455, Vedado, 10400, Havana, Cuba

    Carlos Álvarez & Laritza Rojas-Pérez

  3. Center of Molecular Immunology, Quality Control Direction, 216 Street and 15 Avenue, Atabey, Playa, P.O. Box 16040, 11600, Havana, Cuba

    Gabriela Rivas-García

  4. Center of Molecular Immunology, EPOVAC Facility, 216 Street and 15 Avenue, Atabey, Playa, P.O. Box 16040, 11600, Havana, Cuba

    Yamile González-González

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  1. Leina Moro-Pérez
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  2. Sum Lai Lozada-Chang
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Correspondence to Leina Moro-Pérez.

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Moro-Pérez, L., Lozada-Chang, S.L., Rivas-García, G. et al. Purification and Conformational Characterization of a Novel Interleukin-2 Mutein. Protein J 40, 917–928 (2021). https://doi.org/10.1007/s10930-021-10025-6

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  • DOI: https://doi.org/10.1007/s10930-021-10025-6

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