Optimization of Acidic Degradation of Hyaluronic Acid using Design of Experiments

Abstract: Hyaluronic acid (HA) is an unbranched polysaccharide consisting of the repeating disaccharide unit β(1→4)-GlcA-β(1→3)-GlcNAc and is a naturally occurring biopolymer in bacteria and vertebras. HA is predominantly found in the extracellular matrix (ECM) and the in vivo function of HA can vary depending on molecular weight (Mw) for instance high Mw HA is reported to be anti-angiogenic while low Mw HA induces angiogenesis. HA is a popular component for hydrogels such as dermal fillers. HA is commonly used in dermal fillers. However, other materials, such as other polymers, can be used as well. The project goal was to investigate different degradation processes for production of target Mw HA. Alkaline and acidic degradation processes in combination with increased temperatures seemed as the most promising methods. Degradation tests performed both in aqueous solution as well as heterogeneously in ethanol were evaluated. The acidic degradation in aqueous solution was proven to have the largest degradation constant. Both a robustness test as well as a Design of Experiments (DoE) was performed to investigate the influence different factors had on the degradation speed. The investigated factors were HA concentration, HCl concentration and temperature. Temperature and HCl concentrations proved to be the most influencing factors and a model was developed in the DoE software MODDE to describe how the factors influenced the degradation constant. The model was established as a good significant model with a Q2 value of 0.998 and relative standard deviation (RSD) value of 0.022 after a logarithmic transformation was performed as well as a simplification of the model by excluding some of the factor interactions. The acidic degradation method also proved to be a highly robust method which easily could be used to produce target Mw HA.