Static light scattering and small-angle neutron scattering study on aggregated recombinant gelatin in aqueous solution

Recombinant gelatins are currently evaluated as new excipients for pharmaceutical formulations. They can differ from nonrecombinant gelatins because of intentional alteration of the amino acid sequence and specific properties of the expression systems used. This may affect their solution behavior. In the present work, aqueous solutions of a histidine-containing recombinant gelatin (RG-15-His) were analyzed. Dynamic light scattering (DLS) and loss of absorbance at 200 nm upon centrifugation indicated the formation of aggregates within 1 day upon sample preparation. Static light scattering (SLS) and small-angle neutron scattering (SANS) experiments showed that the aggregate's size was ≥300 nm, and that aggregates are composed of thin, rigid rods of 37 ± 5 nm in length. The observed aggregation was not detectable by circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and cryo transmission electron microscopy (cryo-TEM). SANS experiments, which are not frequently used in the pharmaceutical field, provided additional morphological information about the recombinant gelatin in solution. The results show that combining SLS and SANS is a broadly applicable, complementary approach for detecting aggregation of proteins and other biomolecules and for obtaining structural information about the aggregates.