Impact of Ionic Strength on Viscosity
To further explore the effect of ionic strength on the MAb1 formulation, the viscosity of formulations with increasing amounts
of NaCl and concentration were evaluated. It was determined that the viscosity increased in a concentration-dependent manner
(Figure 7). Additionally, as the ionic strength was increased with NaCl, the viscosity also increased (Figure 7). MAb1 at
100 mg/mL in 150 mM NaCl had a viscosity of ~20 cP (Figure 7). The most significant change in viscosity (~90 cP) occurred
in the 100 mg/mL sample containing 500 mM NaCl (Figure 7).
Differential Scanning Calorimetry of MAb1 with Increasing Amounts of NaCl
Differential scanning calorimetry (DSC) was performed on MAb1 at a concentration of 1 mg/mL IgG1, PS-80, pH 6.0 with increasing
amounts of NaCl. It was determined that the first transition (Tm1 = 69.7 °C) shifted to a lower temperature (67.1 °C) as the
amount of NaCl was increased to 250 mM. The second transition (Tm2 = 88.1 °C) also decreased (85.5 °C) as the NaCl concentration
was increased to 250 mM (Figure 8).
It was also investigated whether the ionic strength had an effect on the stability of MAb1 formulations. MAb1 formulations
at 24 mg/mL IgG1 containing increasing amounts of NaCl, a buffer, and PS-80 were placed on stability for four weeks at 4 °C
and 45 °C. Samples were analyzed using SEC–HPLC at different time points. In formulations stored for four weeks at 4 °C, there
were no changes detected (data not shown). After four weeks at 45 °C in the absence of NaCl, approximately 2.5% dimers were
present (Figure 9). However, in samples containing 250 mM NaCl that were incubated for four weeks at 45 °C, the amount of
dimers increased to approximately 5% (Figure 9). A systematic trend of increasing dimers was observed with an increase in
NaCl concentration (Figure 9).
Osmotic Second Virial Coefficient of the MAb1 Formulation
The osmotic second virial coefficient is commonly indicated by the term A2 or B22. When the second virial coefficient is measured using light scattering, it reflects protein–protein interactions as well
as contributions from protein–cosolute interactions and protein nonideality.16,17 A positive B22 value correlates to net repulsive forces between solute molecules, and negative B22 values indicate net attractive forces between solute molecules.16,18,19 Several methods can be used to measure the B22 value of a formulation, one of which is the use of multi-angle light scattering with dilutions of the formulation. This method
was used to obtain second virial coefficient values for MAb1.