Minimization of recombinant human Flt3 ligand aggregation at the T-m Plateau: A matter of thermal reversibility

This study elucidates the importance of thermal reversibility as it pertain s to the minimization of recombinant human Flt3 ligand aggregation and its potential role for determining solution conditions that can achieve the gre atest long-term storage stability. Both thermal reversibility and T-m were evaluated as microcalorimetric parameters of stability within the range ext ending from pH 6 to 9, where the T-m was shown to plateau near 80 degrees C . Within this region, the reversibility was shown to decrease from 96.6% to 15.2% while the pH was increased from 6 to 9,respectively. Accelerated sta bility studies conducted at 50 degrees C exhibited rates of aggregation aug mented by pH that inversely correlated with the thermal reversibility data. Namely, high thermal reversibility at the T-m plateau correlated with slow er rates of aggregation. Enthalpic calorimetric to van't Hoff ratios (Delta H-1/Delta H-v) yielded results close to unity within the plateau region, s uggesting that the unfolding of rhFlt3 ligand was approximately two-state. Evidence that unfolding preceded the formation of the aggregate was provide d by far-UV CD data of a soluble islolate of the aggregated product exhibit ing a 28% loss of alpha-helix offset by a 31% gain in beta-sheet. This info rmation combined with the thermal reversibility data provided compelling ev idence that unfolding was a key event in the aggregation pathway at 50 OC. Minimization of aggregation was achieved at pH 6 and corroborated by eviden ce acquired from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion data. Correspondingly, the bioactivity was found to be o ptimal at pH 6. The findings link thermal reversibility to the propensity o f Flt3 ligand to aggregate once unfolded in the T-m plateau region and prov ide a basis for relating the reversibility of thermal denaturation to the p rediction of long-term storage stability in aqueous solution.