Poster W17 - Stability of rhGALNS Enzyme in Buffers for Sustained Release Applications

Introduction: Mucopolysaccharidosis IVA (MPS IVA, Morquio A) is a lysosomal storage disease characterized by deficiency in active N-acetylgalactosamine-6-sulfate sulfatase (GALNS) and systemic build-up of glycosaminoglycans (GAG) keratan sulfate and chondroitin-6-sulfate. Recombinant human GALNS (rhGALNS) is currently administered by intravenous infusion weekly, but the treatment is costly, time-consuming, and inefficient. Patient quality of life may be improved by a sustained release device for rhGALNS, but enzymatic activity must be maintained throughout the release window up to cell uptake. Polyethylene glycol (PEG) hydrogels may be employed as a hydrophilic, tunable, non-toxic, and biodegradable drug delivery system for the sustained release of proteins such as rhGALNS. Another potential benefit of a hydrogel delivery device is the potential rhGALNS stabilization by confinement. We have previously observed rhGALNS activity loss in buffers at physiological pH, and, in vivo, rhGALNS will remain within the hydrogel in interstitial fluid for the duration of the release window. Understanding the stability of rhGALNS within the hydrogel mesh and during transport to the lysosome will be critical to the efficacy of any sustained release device for rhGALNS. In this study, we investigate the stability of rhGALNS in buffers relevant to our PEG hydrogel delivery system, both in buffer alone and after release from the hydrogel.

Materials and

Methods: rhGALNS was expressed in CHO cells and purified from the spent media. Specific activity was determined from concentration by the Bradford protein assay on Nanodrop and enzymatic activity by fluorescent substrate 4-MU-β-D-galactopyranoside-6-sulfate. rhGALNS was encapsulated in PEG hydrogels by Michael-Type addition between 8-arm PEG-Acrylate (20 kDa) and PEG-dithiol (3.4 kDa) in 100mM HEPES, pH 7.4. The specific activity of rhGALNS was monitored upon incubation in Acetate at pH 5.5, PBS at pH 7.4, and HEPES at pH 7.4. These buffer conditions broadly reflect the pH of the lysosome, the pH of the interstitial fluid, and our gelation buffer, respectively. When noted, samples were buffer-exchanged into Acetate pH 5.5 prior to activity measurements via concentration and dilution on Amicon Ultra centrifugal filters. The hydrodynamic radius of Alexa-647-labeled rhGALNS was also determined by Fluorescence Correlation Spectroscopy. The impact of confinement was compared to macromolecular crowding by both unmodified PEG and negatively-charged kappa-carrageenan.

Results, Conclusions, and Discussions: rhGALNS was found susceptible to activity loss both by inhibition and degradation. PBS inhibited rhGALNS, but activity was recovered after buffer exchange. This effect was not linked to pH and may have been caused by PBS components binding to the rhGALNS active site. Irreversible activity degradation was observed in our gelation buffer, 100 mM HEPS, pH 7.4 as well as PBS. The activity degradation in PBS did not correlate with a change in protein radius as measured by FCS. This activity degradation poses a challenge for the sustained release of rhGALNS from an in-situ drug reservoir stored under physiological conditions and pH, such as in subcutaneous tissue interstitial fluid. However, rhGALNS encapsulated in a PEG hydrogel and released into PBS retained its specific activity for up to seven days. Although incubation with PEG-Ac alone resulted in significant activity loss, the gelation procedure itself, where PEG-Ac is consumed by hydrogel crosslinking, did not impact rhGALNS activity. We compared the impact of confinement within the gel to macromolecular crowding with both PEG and carrageenan. PEG did not impact activity and carrageenan significantly reduced activity. This study confirms that a sustained release device for rhGALNS is feasible despite the limited stability of rhGALNS.

Acknowledgements (Optional): This work was funded by the Research Innovation Fund of Saint Louis University.