Increase in Ionic Strength Correlates to an Increase in Opalescence
To determine if the ionic strength contributed to opalescence, NaCl (one of the excipients in the formulation) was tested
in a range of 0 to 200 mM in a formulation consisting of 24 mg/mL IgG1 and PS-80, pH 6.0. A visual observation of the vials
indicated that as the molarity of NaCl increased, the opalescence also increased (Figure 4). In the absence of NaCl, no opalescence
was observed and correlated to at or below a Reference I standard (3–6 NTU) according to EP 5.0, 2.2.1. These results indicated that an increase in the ionic strength played a major role in contributing to the opalescent
appearance of MAb1 (Figure 4).
Effect of Excipients on the Opalescence of MAb1
To determine if other components of the formulation influenced the opalescence of the MAb1 formulation, PS-80 was tested.
Four formulations consisting of 24 mg/mL IgG1 with and without PS-80 and NaCl were generated in glass vials and are depicted
in Figure 5. Opalescence was strongest in the formulation without PS-80 in the presence of 150 mM NaCl. This sample was more
opalescent than the Reference III standard according to EP 5.0, 2.2.1. When PS-80 was added to this formulation, the opalescence was reduced (Figure 5). PS-80 had no effect on formulations
that did not contain NaCl (Figure 5). The results indicated that PS-80 plays an important role in mediating the opalescence
of MAb1 formulations containing NaCl.
Opalescence and Impact of Different Salts
It was next evaluated whether salts other than NaCl could affect the opalescence of MAb1. Different salts were selected based
on their position in the Hofmeister series.14,15 Salts that were evaluated included KCl, MgCl2, KSCN, Na3PO4, CsCl, and Na2SO4. They were selected because they yield ions that are strong chaotropes, such as SCN¯, or strong kosmotropes, such as PO43-. Chaotropes have destabilizing effects and promote salting-in of proteins in solution, whereas kosmotropes have stabilizing
and salting-out effects on proteins.14,15 Each of these salts were added to MAb1 at a concentration of 150 mM in the absence of PS-80. The IgG1 concentration was
maintained at 24 mg/mL. The OD for each sample was measured and the results are depicted in Figure 6. It was determined that
addition of these salts resulted in a similar level of opalescence as was observed with 150 mM NaCl (Figure 6). Ionic strength
rather than the specific ion is therefore the major contributor to the opalescence of MAb1 (Figure 6).