title: Preparation and extensive characterization of hyaluronan with narrow molecular weight distribution.
abstract: The physicochemical properties and biological functions of hyaluronan (HA) are closely related to its molecular weight (MW) and molecular weight distribution (MWD). Therefore, it is crucially important to provide a reliable characterization of these parameters for proper use of HA and its degradation products in both chemical and clinical fields. In this study, we present a novel method for the preparation of HA fragments of defined size with narrow molecular weight distribution. The HA fractionation was performed using an anion-exchange chromatography and is applicable either after enzymatic or chemical hydrolysis of polymeric HA. Isolated fractions with a molecular weight ranging from 3000-420,000gmol-1were analyzed by size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS). Hundred-milligram scale HA fragments were obtained from 5g hyaluronan starting material. Independently on weight-average molecular weight (Mw), the polydispersity index (PDI) of the HA fractions was less than 1.23. The fractionation methodology can be easily up-scaled and is applicable on any negatively charged polymers. We have also found that PDI is insufficient to characterize almost monodisperse fractions and for proper material characterization we proposed a new characteristic termed "distribution angle", calculated from the slope of the cumulative molecular weight distribution curve. Compared to PDI, the distribution angle reflects more efficiently changes in size distribution and thus is highly recommended to be used along with Mwdetermination of any polymer.
authors.affiliation: Contipro a.s., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czechia.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Čožíková
authors.affiliation: Contipro a.s., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czechia.
authors.forename: Tereza
authors.initials: T
authors.lastname: Šílová
authors.affiliation: Contipro a.s., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czechia.
authors.forename: Veronika
authors.initials: V
authors.lastname: Moravcová
authors.affiliation: Contipro a.s., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czechia.
authors.forename: Daniela
authors.initials: D
authors.lastname: Šmejkalová
authors.affiliation: Contipro a.s., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czechia.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Pepeliaev
authors.affiliation: Contipro a.s., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czechia.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Velebný
authors.affiliation: Contipro a.s., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czechia. Electronic address: martina.hermannova@contipro.com.
authors.forename: Martina
authors.initials: M
authors.lastname: Hermannová
mesh_terms: D002850:Chromatography, Gel; D006820:Hyaluronic Acid / Q000737:chemistry*; D006868:Hydrolysis; D008970:Molecular Weight; D011108:Polymers
chemical_list: D011108:Polymers; D006820:Hyaluronic Acid
keywords: Anion-exchange chromatography; Hyaluronan fragmentation; Molecular weight; Polydispersity; Size exclusion chromatography