High yield process for the production of active human alpha-galactosidase a in suspension CHO-K1 cells

MARÍA CELESTE RODRÍGUEZ; NATALIA CEAGLIO; MARINA ETCHEVERRIGARAY; CLAUDIO PRIETO

Lausanne

Congreso; 25th Meeting of the European Society for Animal Cell Technology (ESACT); 2017

European Society for Animal Cell Technology (ESACT)

Background and novelty. Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α - Galactosidase A. Currently, two enzyme replacement therapies are commercially available. Recombinant human α-Galactosidase A (rhαGAL) has been successfully expressed in adherent CHO-K1 cells by a stable amplification method, achieving productivities up to 7.5 pg.cell‑1.day-1 [1]. We replaced the amplification strategy by lentiviral transduction of suspension cells, obtaining productivities up to 59 pg.cell-1.day-1.Experimental approach. Suspension CHO-K1 cells were serially transduced with third generation lentiviral vectors (LVs) containing rhαGAL sequence. Fifteen clones were obtained by limit dilution method. Two purification steps by anionic exchange and hydrophobic chromatography were developed and in vitro enzymatic properties were characterized. Monosaccharide compositions and 2-AB labeled glycans were analyzed. The properties of rhαGAL were compared to the commercial enzyme (Fabrazyme®).Results and discussion. LV transduction improved the global production process, as clones with high productivities (3.5 to 59.4 pg.cell.-1.day-1) and enzyme activities (2.9E103 to 4.1E104 IU.mg-1) were obtained. After two purification steps, the active enzyme was recovered (1.5E106 IU.mg-1) with 90% purity and 76% overall yield. Michaelis Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate (4-MUG) at a comparable rate to Fabrazyme® (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 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, maintaining the biochemical properties of the commercial product.