(T1430-01-01) Static Light Scattering Signatures Used to Identify and Minimize Aggregation During Formulation Development

Purpose: Right angle light scattering (RALS) was used to characterize the monomer to dimer transition rate of Immunoglobulin-G (IgG) as related to buffer conditions. ‘Time to dimer’ is defined as the time dependent, linear rate of increasing aggregate mass (increase in light scattering intensity) with respect to the initial unaggregated mass as the protein dimerizes (weighted average). Using this data, a formulation was developed which increased colloidal stability of IgG ~10-fold under thermal stress. In conclusion, ARGEN offers users a versatile platform to provide comprehensive stability landscapes for all classes of biopolymers (RNA, proteins, peptides, sugars and AAVs). under various thermal and solution conditions.

Methods: Eight samples of [IgG] = 1 mg/ml were prepared with increasing concentrations of sodium acetate to determine the minimum concentration of sodium acetate to minimize aggregation. In parallel, eight samples of [IgG] = 1 mg/ml were prepared with increasing concentrations of sucrose to determine the minimum concentration of sucrose required to minimize aggregation. All samples were analyzed at 55°C, below the T_m for IgG, which is necessary to generate linear and consistent aggregation rates.

Results: Figure 2 illustrates ‘time to dimer’ vs. [sodium acetate] and clearly defines the maximum time for monomer to dimer transition of IgG at [sodium acetate] = 25mM. At increasing [sodium acetate], ‘time to dimer’ begins to decrease as colloidal stability is reduced.  Figure 3 illustrates the effects of [sucrose] on ‘time to dimer’ where the rate is maximized at [sucrose] = 300mM, and further increases in [sucrose] has no effect on colloidal stability.  Figure 4 displays Arrhenius plots of the optimized and unoptimized IgG samples within a thermal range of 30°C to 77°C. The optimized sample (blue) demonstrates improved thermal stability at elevated temperatures and a significant increase in thermal stability at lower temperatures.

Conclusion: Right angle light scattering (RALS) was utilized for early detection and rate determination for the monomer to dimer transition of IgG. Formulation conditions which have a higher comparative ‘time to dimer’ rate indicate an increase in colloidal stability and permit users to modify solution conditions for optimized stability.  This data demonstrated the stability of IgG was optimized in [sodium acetate] = 25mM and [sucrose] = 300mM as evidenced by the increase ‘time to dimer’ and Arrhenius behavior.

Monitoring aggregation via static light scattering

Optimizing [sodium acetate] to increase colloidal stability

The effect of [sucrose] on 'time to dimer' rates