Second virial coefficient determination of a therapeutic peptide by self‐interaction chromatography

Abstract

Self-interaction of macromolecules has been shown to play an important role in a number of physical processes, including crystallization, solubility, viscosity, and aggregation. Peptide self-interaction is not as well studied as for larger proteins, but should play an equally important role. The osmotic second virial coefficient, B, can be used to quantify peptide and protein self-interaction. B values are typically measured using static light scattering (SLS). Peptides, however, do not scatter enough light to allow such measurements. This study describes the first use of self-interaction chromatography (SIC) for the measurement of peptide B values because SIC does not have the molecular size limitations of SLS. In the present work, SIC was used to measure B for enfuvirtide, a 36-amino acid therapeutic peptide, as a function of salt concentration, salt type, and pH. B was found to correlate strongly with solubility and apparent molecular weight. In general, the solubility of enfuvirtide increases with pH from 6 to 10 and decreases as the salt concentration increases from 0 to 0.5M for three different salts. The effect of peptide concentration on B was also investigated and shown to have a significant effect, but only at high concentrations (>80 mg/mL).

Colloidal Behavior of Proteins: Effects of the Second Virial Coefficient on Solubility, Crystallization and Aggregation of Proteins in Aqueous Solution

“…In a recent study of the peptide, enfuvirtide, the B value was measured by SIC over a wide range of solution concentrations (1-120 mg/ml) [57]. Only at the highest concentration was B statistically significantly different than the other measured B values (Fig.…”

An Application of Ultraviolet Spectroscopy to Study Interactions in Proteins Solutions at High Concentrations

“…The magnitude of Abs, however, clearly increases at higher BSA and lysozyme concentrations (>100 mg/mL), suggesting that, at these concentrations, PPIs may result in nonideal solution behavior of these two proteins. Furthermore, self-interaction chromatographic studies of a peptide 45 and light scattering measurements using model proteins 32,33 suggest attractive interactions to be predominant at higher (>100 mg/mL) concentrations. Similarly, the increase in magnitude of Abs values observed at high (>100 mg/mL) BSA and lysozyme concentrations may therefore represent increase in the extent of attractive interactions between protein molecules.…”

Low-volume solubility assessment during high-concentration protein formulation development

“…stated that concentration dependence of reduced osmotic pressure and therefore B 22 varies little up to 50 mg/ml at a maximum. But HCF development focuses on protein concentrations up to 200 mg/ml or higher, and non‐ideality at high protein concentrations has been observed several times . The influence of short ranging and multiple particle interactions strongly differs between single proteins but cannot generally be neglected at certain concentrations.…”