Determination of pituitary and recombinant human growth hormone molecular weights by modern high-performance liquid...

The determination of molecular weight for pituitary and recombinant human growth hormone (p-hGH/Crescormon and r-hGH/Protropin) has been performed. This has involved on-line coupling of size-exclusion chromatography (SEC) and gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A 5-microns, 300 A, Delta-bond octyl column was used. Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for p-hGH and r-hGH. Known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data has been obtained for both proteins under conventional RP-HPLC gradient elution conditions. SEC data of both hGHs were found to be concentration, mobile phase, and column dependent for the particular analyses. Both medium- and high-resolution SEC-LALLS studies were performed, and all of these determinations further confirmed our RP-HPLC results. On-line LALLs provides certain advantages in identifying aggregates that may be present, even in medium-resolution SEC, where incomplete resolution occurs. The on-line coupling of modern RP-HPLC for biopolymers with LALLS detection represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus higher-order aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.