Improving the accuracy of hyaluronic acid molecular weight estimation by conventional size exclusion chromatography

There is an unreasonably high variation in the literature reports on molecular weight of hyaluronic acid (HA) estimated using conventional size exclusion chromatography (SEC). This variation is most likely due to errors in estimation. Working with commercially available HA molecular weight standards, this work examines the extent of error in molecular weight estimation due to two factors: use of non-HA based calibration and concentration of sample injected into the SEC column. We develop a multivariate regression correlation to correct for concentration effect. Our analysis showed that, SEC calibration based on non-HA standards like polyethylene oxide and pullulan led to approximately 2 and 10 times overestimation, respectively, when compared to HA-based calibration. Further, we found that injected sample concentration has an effect on molecular weight estimation. Even at 1 g/1 injected sample concentration, HA molecular weight standards of 0.7 and 1.64 MDa showed appreciable underestimation of 11-24%. The multivariate correlation developed was found to reduce error in estimations at 1 g/1 to < 4%. The correlation was also successfully applied to accurately estimate the molecular weight of HA produced by a recombinant Lactococcus lactis fermentation.