Lentivirus transgenesis for high yield production of human alpha-Galactosidase A in suspension CHO-K1 cells

MARÍA CELESTE RODRÍGUEZ; NATALIA CEAGLIO; SEBASTIÁN ANTUÑA; MARÍA BELÉN TARDIVO; MARINA ETCHEVERRIGARAY; CLAUDIO PRIETO

Valencia

Conferencia; Protein Expression in Animal Cells Conference (PEACe); 2017

Protein Expression in Animal Cells

Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α-Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high cost. Selection of adequate high-expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α-Galactosidase-A (rhαGAL) in CHO cells. Herein, we have replaced the methotrexate amplification strategy (through which the highest rhαGAL producer clone described until this moment was developed) by a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells. LP transduction improved the global production process, as clones with productivities higher that the ones previously reported (3.5 to 59.4 pg.cell-1.d-1) with high specific enzyme activities were obtained. After two purification steps, the active enzyme was recovered (2.4 x 106 U.mg-1) with 100% purity and 60% overall yield. Michaelis-Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU-α-Gal at a comparable rate to Fabrazyme®, the current CHO-derived ERT available for Fabry disease (Vmax 60.1 ± 12.4 nM.min-1 and 63.2 ± 25.3 nM.min-1; Km 1.6 ± 0.6 mM and 1.2 ± 0.2 mM; Kcat 20.04 ± 4.14 and 21.1 ± 8.4 s-1 for rhαGAL and Fabrazyme®, respectively). Although glycosylation pattern and in vitro stability in plasma of both molecules were similar, rhαGAL contained 40% higher level of sialic acid. In summary, our process achieves the highest rhαGAL productivity reported to date (8-fold higher), maintaining the biochemical properties of the commercial product. Even more, considering the glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease.