Full-length human CCBE1 production and purification: leveraging bioprocess development for high quality glycosylation attributes and functionality

Marta M. Silva, Patrícia Gomes-Alves, Sara Rosa, Daniel Simão, José M. Inácio, Cristina Peixoto, Margarida Serra, José A. Belo, Paula M. Alves

Research output: Contribution to journalArticle

Abstract

Collagen and calcium-binding EGF domain-1 (CCBE1) is a secreted protein critical for lymphatic/cardiac vascular development and regeneration. However, the low efficient production of the recombinant full-length CCBE1 (rCCBE1) has been a setback for functional studies and therapeutic applications using this protein. The main goal of this work was to implement a robust bioprocess for efficient production of glycosylated rCCBE1. Different bioprocess strategies were combined with proteomic tools for process/product characterization, evaluating the impact of process parameters on cell performance, rCCBE1 production and quality. We have shown that rCCBE1 volumetric yield was positively correlated with higher cell density at transfection (HDT), and under these conditions the secreted protein presented a mature glycosylated profile (complex N-glycans). Mild hypothermia was also applied to HDT condition that resulted in enhanced cell viability; however an enrichment of immature rCCBE1 variants was detected. Mass spectrometry-based tools allowed the identification of rCCBE1 peptides confirming protein identity in the affinity chromatography enriched product. rCCBE1 biological activity was validated by in vitro angiogenesis assay, where enhanced vessel formation was observed. Herein, we report a step forward in the production and characterization of human glycosylated rCCBE1, amenable for in vitro and in vivo studies to explore its regenerative therapeutic potential.

Original languageEnglish
Pages (from-to)6-14
Number of pages9
JournalJournal of Biotechnology
Volume285
DOIs
Publication statusPublished - 10 Nov 2018

Keywords

  • Glycosylation
  • Proteomics
  • Purification
  • Recombinant CCBE1 protein
  • Transient transfection

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