Rodríguez María Celeste, Ceaglio Natalia, Antuña Sebastián, Tardivo María Belén, Etcheverrigaray Marina, Prieto Claudio
Universidad Nacional del Litoral, CONICET, School of Biochemistry and Biological Sciences, Cell Culture Laboratory, Ciudad Universitaria, Paraje El Pozo, C.C. 242, Santa Fe, S3000ZAA, Argentina.
Zelltek S.A., PTLC RN 168, Santa Fe, S3000ZAA, Argentina.
Biotechnol Prog. 2017 Sep;33(5):1334-1345. doi: 10.1002/btpr.2538. Epub 2017 Sep 4.
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.
法布里病是一种由溶酶体α-半乳糖苷酶A缺乏引起的X连锁隐性疾病。目前有两种酶替代疗法（ERT）。然而，孤儿药因其高昂的价格，其可及性仍然受限。选择合适的高表达系统仍是降低治疗成本的一项挑战。人们已经实施了几种策略，但成效各异，旨在优化重组人α-半乳糖苷酶A（rhαGAL）在中国仓鼠卵巢（CHO-K1）细胞中的生产过程。在此，我们首次描述了一种基于第三代慢病毒颗粒（LP）转导悬浮CHO-K1细胞的策略，以获得高产rhαGAL克隆（3.5至59.4 pg细胞天）。经过两步纯化后，回收了活性酶（2.4×10 U mg），纯度为98%，总产率为60%。米氏分析表明，rhαGAL能够以与法布赞（Fabrazyme®）相当的速率水解合成底物4MU-α-Gal，法布赞是目前用于法布里病的CHO来源的ERT。此外，rhαGAL的甘露糖-6-磷酸（M6P）含量与法布赞相同，酸性唾液酸含量高出约40%，免疫原性唾液酸（Neu5Gc）含量降低33%。与迄今报道的其他rhαGAL生产工艺相比，我们的方法在保持适当活性和糖基化模式的情况下实现了最高的rhαGAL生产率。更重要的是，考虑到rhαGAL改善的糖基化特性可能在药代动力学方面具有优势，我们的酶可被认为是法布里病治疗应用的一种有前景的替代物。©2017美国化学工程师学会生物技术进展，33:1334 - 1345，2017。
