TY - JOUR
T1 - Full-length human CCBE1 production and purification
T2 - leveraging bioprocess development for high quality glycosylation attributes and functionality
AU - Silva, Marta M.
AU - Gomes-Alves, Patrícia
AU - Rosa, Sara
AU - Simão, Daniel
AU - Inácio, José M.
AU - Peixoto, Cristina
AU - Serra, Margarida
AU - Belo, José A.
AU - Alves, Paula M.
PY - 2018/11/10
Y1 - 2018/11/10
N2 - 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.
AB - 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.
KW - Glycosylation
KW - Proteomics
KW - Purification
KW - Recombinant CCBE1 protein
KW - Transient transfection
UR - http://www.scopus.com/inward/record.url?scp=85052661842&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2018.08.015
DO - 10.1016/j.jbiotec.2018.08.015
M3 - Article
C2 - 30165116
AN - SCOPUS:85052661842
VL - 285
SP - 6
EP - 14
JO - Journal of Biotechnology
JF - Journal of Biotechnology
SN - 0168-1656
ER -