High yield process for the production of active human α‐galactosidase a in CHO‐K1 cells through lentivirus transgenesis

Abstract

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 price. 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 Chinese hamster ovary (CHO-K1) cells. Herein, we describe for the first time the application of a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells to obtain high-producing rhαGAL clones (3.5 to 59.4 pg cell d ). After two purification steps, the active enzyme was recovered (2.4 × 10 U mg ) with 98% 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. In addition, rhαGAL presented the same mannose-6-phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved 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. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1334-1345, 2017.

Comprehensive Characterization of Biotherapeutics by Selective Capturing of Highly Acidic Glycans Using Stepwise PGC-SPE and LC/MS/MS

“…We could confirm that the position of the fucose is the outer, not the core, by a diagnostic ion at m / z 512.190 [HexHexNAcFuc + H] + . The presence of NeuGc in therapeutic lysosomal enzymes has been reported in previous studies, , but the detailed glycan structure was first revealed in this study.…”

Development and biological activity of long-acting recombinant human interferon-α2b

“…The increasing incidence of certain cancers and viral hepatitis has increased the demand for rhIFN-α2b, suggesting that rhIFN-α2b must be optimized in all the expression systems used for its production [22]. Although many mammalian expression systems can undergo posttranscriptional modification, Chinese hamster ovary (CHO) expression system was considered as the preferred production platform for the expression of proteins that require posttranslational modification, because it has the following advantages: the expressed proteins are closest to natural proteins in terms of molecular structure, physicochemical properties and biological functions; CHO cells can grow at high density in suspension culture under serum-free culture conditions; protein products can be secreted extracellularly, facilitating downstream protein purification [25]. For the above reasons, we chose the CHO system to express IFN.…”