Recombinant Hyaluronic Acid Produced in Escherichia coli: Purification, Molecular-Size Profile and In Vitro Cellular Performance

Recombinant production platforms are promising approaches for producing hyaluronic acid (HA) for biomedical research. In this study recombinant HA produced in Escherichia coli (rHA) was purified and compared with commercial HA (cHA) through physicochemical, rheological, and preliminary cellular analyses. Purification reduced residual protein content (from 7.47 to 0.48 mg/L), nucleic-acid-associated absorbance and endotoxins level (from 0.315 to 0.0766 EU/mg) while preserving HA recovery. Electrophoresis and hyaluronidase digestion supported HA identity and a heterogeneous apparent molecular-size profile. Mass photometry provided an exploratory, solution-based estimate of molecular-size populations, identifying a predominant native-solution population centred at approximately 194 kDa. Fourier-transform infrared spectroscopy (FT-IR) revealed characteristic HA-associated functional groups, while thermogravimetric analysis (TGA) showed an initial mass loss attributed to water evaporation (30–150 °C), followed by polymer degradation occurring at a lower temperature for rHA (~150 °C) than for cHA (~250 °C). Both samples exhibited shear-thinning behaviour, whereas rHA showed lower viscosity and moduli than cHA. In C2C12 cells, rHA produced favourable relative sulforhodamine B (SRB) signals and scratch closure responses comparable to those of the two cHA (cHA_1 and cHA_2). These findings support rHA as a purified HA-compatible material with distinctive rheological properties and preliminary in vitro cellular performance.