Biophysical and Functional Characterization of Recombinant Human Lubricin Proteins: rhPRG4 vs ECF843

Abstract

Purpose : Proteoglycan 4 (PRG4, or lubricin) is a mucin-like glycoprotein found on the ocular surface and in tears. Recombinant human PRG4 (rhPRG4, Lubris LLC) was shown to be clinically effective at improving signs and symptoms in patients with dry eye disease (DED). Conversely, the recombinant human lubricin ECF843 (Novartis) showed no signs of clinical efficacy for DED. Here, we examined the biophysical and functional properties of rhPRG4 and ECF843.

Methods : Size (hydrodynamic radius, R_h) and charge were measured using dynamic light scattering (DLS) and zeta potential, respectively. Molecular weight was characterized by size exclusion chromatography with inline multiangle light scattering (MALLS). Sedimentation parameters were measured by analytical ultracentrifugation (AUC), and chemical structure was analyzed by Raman spectroscopy. Inhibition of MMP9 activity was assessed by commercial assay. Boundary lubricating ability was assessed at a latex glass interface and anti-adhesion properties by a cell-based assay. Kinetics of binding to Col1 were assessed by quartz crystal microbalance (QCM-D). All results are n=3-6.

Results : ECF843 had a smaller z-avg R_h (58.9±3.3nm vs 157.9±2.3nm, p<0.001) and was more positively charged (-11.6±1.1 mV vs -18.4±2.2 mV, p<0.05) than rhPRG4. MALLS analysis showed rhPRG4 as a 350±43kDa monomer and 713±36kDa dimer, while ECF843 was a 369±15kDa monomer and smaller 280±54kDa monomer. AUC showed rhPRG4 as a ~4.25S primary peak, while ECF843 had an additional major peak at 6S, showing it was more compact. Raman showed a significant shift in the 550/987 peak area ratio for ECF843 vs rhPRG4 (1.18±0.3 vs 0.36±0.07, p<0.05), indicating an altered ratio of disulfide bonds to carbon backbone. ECF843 did not inhibit MMP9 activity as well as rhPRG4 at 50μg/mL (73.9±1.4% vs 24.1±1%, p<0.001) and 150μg/mL (29.2±2.8% vs 8.4±0.8%, p<0.01). ECF843 was less anti-adhesive, as more cells adhered to ECF843-coated surfaces vs rhPRG4 (12.5-50μg/mL, p<0.01 – <0.001), and showed diminished lubricating ability with a significantly higher friction coefficient vs rhPRG4 (0.089±0.016 vs 0.048±0.02, p<0.05). ECF843 had a binding rate constant to Col1 ~5X slower than rhPRG4.

Conclusions : ECF843 has altered protein size, charge and structure from rhPRG4, along with diminished function. It is likely these differences played a role in the disparate clinical outcomes observed between the two molecules.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.