Proteomic analysis of a human lyophilized 3D scaffold free tissue engineered product for bone reconstruction

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20m
ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków

Speaker

Theys, Nicolas (Novadip Biosciences)

Description

Introduction: Bone tissue engineering is a promising therapeutic approach for bone reconstruction, which has been successfully applied in clinical trials. NVDX3 is an allogenic and off-the-shelf candidate advanced therapy intended for use as bone void filling materials. It is a lyophilized, terminally sterilized powder derived from a 3-D cell product containing extracellular matrix and osteodifferentiated cells associated with hydroxyapatite/beta-tricalcium phosphate (HA/βTCP) particles. Based on in vitro and in vivo data, we showed that NVDX3 leads to bone formation enabling induction of endochondral ossification and inhibition of osteoclasia.
Methodology: To address the protein composition of NVDX3 product, deep proteomic profiling was performed on samples derived from different stages of the manufacturing process of three independent batches. For the unbiased quantification of all detectable peptides and proteins, HRM™ (Hyper Reaction Monitoring) mass spectrometry was applied on the tested samples. HRM mass spectrometric data were analyzed using Spectronaut software. Corresponding proteomic networks were analyzed via the UniProt Consortium and STRING consortium databases.
Results: More than 5500 proteins were identified in the drug substance (DS) and the final product (DP) of NVDX3, among which 233 were consistently upregulated during the maturation of the 3-D cell product (CP) and maintained in the final product. Untargeted analysis on the most abundant proteins in the product or the most upregulated ones during the manufacturing process showed a strong correlation with collagen-containing extracellular matrix (ECM), blood vessel development and bone growth. To identify potential key players for the activity of the product, a targeted proteomic analysis approach was also performed revealing >170 proteins implicated in extracellular matrix composition, >30 proteins involved in bone development, including markers of endochondral bone formation, and >10 factors with angiogenic or antiresorptive properties.
Conclusion: NVDX3 product composition is characterized by the presence of collagen-containing ECM components which have been produced during the manufacturing process and it includes growth factors known to have an osteogenic, angiogenic and anti-resorptive activity. The deep proteomic analysis allows to identify key attributes involved in the mode of action of the product and to better understand the mechanism of bone formation on tissue explant from in vivo studies.

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