TY - JOUR

T1 - Complex coacervates of hyaluronic acid and lysozyme

T2 - Effect on protein structure and physical stability

AU - Water, Jorrit J.

AU - Schack, Malthe M.

AU - Velazquez-Campoy, Adrian

AU - Maltesen, Morten J.

AU - van de Weert, Marco

AU - Jorgensen, Lene

PY - 2014/1/1

Y1 - 2014/1/1

N2 - Complex coacervates of hyaluronic acid and lysozyme, a model protein, were formed by ionic interaction using bulk mixing and were characterized in terms of binding stoichiometry and protein structure and stability. The complexes were formed at pH 7.2 at low ionic strength (6 mM) and the binding stoichiometry was determined using solution depletion and isothermal titration calorimetry. The binding stoichiometry of lysozyme to hyaluronic acid (870 kDa) determined by solution depletion was found to be 225.9 ± 6.6 mol, or 0.1 bound lysozyme molecules per hyaluronic acid monomer. This corresponded well with that obtained by isothermal titration calorimetry of 0.09 bound lysozyme molecules per hyaluronic acid monomer. The complexation did not alter the secondary structure of lysozyme measured by Fourier-transform infrared spectroscopy overlap analysis and had no significant impact on the Tm of lysozyme determined by differential scanning calorimetry. Furthermore, the protein stability of lysozyme was found to be improved upon complexation during a 12-weeks storage study at room temperature, as shown by a significant increase in recovered protein when complexed (94 ± 2% and 102 ± 5% depending on the polymer-protein weight to weight ratio) compared to 89 ± 2% recovery for uncomplexed protein. This study shows the potential of hyaluronic acid to be used in combination with complex coacervation to increase the physical stability of pharmaceutical protein formulations.

AB - Complex coacervates of hyaluronic acid and lysozyme, a model protein, were formed by ionic interaction using bulk mixing and were characterized in terms of binding stoichiometry and protein structure and stability. The complexes were formed at pH 7.2 at low ionic strength (6 mM) and the binding stoichiometry was determined using solution depletion and isothermal titration calorimetry. The binding stoichiometry of lysozyme to hyaluronic acid (870 kDa) determined by solution depletion was found to be 225.9 ± 6.6 mol, or 0.1 bound lysozyme molecules per hyaluronic acid monomer. This corresponded well with that obtained by isothermal titration calorimetry of 0.09 bound lysozyme molecules per hyaluronic acid monomer. The complexation did not alter the secondary structure of lysozyme measured by Fourier-transform infrared spectroscopy overlap analysis and had no significant impact on the Tm of lysozyme determined by differential scanning calorimetry. Furthermore, the protein stability of lysozyme was found to be improved upon complexation during a 12-weeks storage study at room temperature, as shown by a significant increase in recovered protein when complexed (94 ± 2% and 102 ± 5% depending on the polymer-protein weight to weight ratio) compared to 89 ± 2% recovery for uncomplexed protein. This study shows the potential of hyaluronic acid to be used in combination with complex coacervation to increase the physical stability of pharmaceutical protein formulations.

KW - Binding characteristics

KW - Bovine serum albumin

KW - Complex coacervation

KW - Differential scanning calorimetry

KW - Differential scanning fluorimetry

KW - Hyaluronan

KW - Hydrogel

KW - Isothermal titration calorimetry

KW - Sodium hyaluronate

KW - Stability

U2 - 10.1016/j.ejpb.2014.09.001

DO - 10.1016/j.ejpb.2014.09.001

M3 - Journal article

C2 - 25218319

AN - SCOPUS:84908368377

VL - 88

SP - 325

EP - 331

JO - European Journal of Pharmaceutics and Biopharmaceutics

JF - European Journal of Pharmaceutics and Biopharmaceutics

SN - 0939-6411

IS - 2

ER -