Figure 10
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A Zimm plot of MAb1 in the formulation containing a buffer and 150 mM NaCl is depicted in Figure 10. PS-80 was removed from
the formulation to prevent potential interference from micelles. It was determined that this formulation had a negative second
virial coefficient (–4.3 x 10-5 mol mL/g2). The molecular weight calculated for MAb1 was 1.46 x 105 g/mol (146 kDa), which is close to the molecular weight of MAb1, which is 148 kDa.
DISCUSSION
The purpose of this study was to investigate whether the ionic strength and excipients in the MAb1 formulation were associated
with the opalescence observed. We identified that the ionic strength of the MAb1 formulation played a major role in mediating
opalescence.
Effects of Ionic Strength
The opalescence of MAb1 was demonstrated to increase with ionic strength. In the MAb1 formulation containing NaCl, opalescence
was pronounced (Reference III opalescence standard) whereas in its absence, the solution was clear (Reference I opalescence
standard). It was also observed that the apparent hydrodynamic radius of the MAb1 formulation was larger at higher concentrations
(20 mg/mL) and lower at more dilute concentrations (1 mg/mL). The reason for the larger particle size at higher concentrations
of MAb1 is not known, and will require further investigation.
We explored whether salts other than NaCl had an impact on opalescence. Salts were selected based on their position in the
Hofmeister series, and included KCl, MgCl2, KSCN, Na3PO4, CsCl, and Na2SO4.14,15 Opalescence was observed in the presence of all salts tested, and no specific ion effects were observed.
As the concentration of NaCl was increased, the electrostatic shielding becomes saturated and it is possible that interactions
between the hydrophobic regions of MAb1 become dominant. An example of electrostatic shielding and enhancement of hydrophobic
effects in the presence of NaCl was demonstrated in studies using lysozyme.5,20
There are other examples of the effect of ionic strength on proteins. It was demonstrated that when recombinant factor VIII
SQ was placed in a solution containing 100 mM NaCl at pH 7, the solution turned opalescent within the first hour.7 However, after the first hour, the protein precipitated out of solution.7 At higher concentrations of NaCl, the precipitate could be dissolved, and activity was observed to recover.7
The interactions between MAb1 molecules described in these studies were not strong enough to precipitate the MAb. It is not
known, however, if the increase in opalescence correlates to molecular self-association of MAb1 molecules. Sukumar and colleagues
have attributed opalescence of an IgG1 formulation to Rayleigh scatter and indicated that opalescence is not caused by noncovalent
association.4
Effects of Excipients
PS-80 has been widely used in liquid parenteral formulations to reduce aggregation caused by surface interactions, to prevent
denaturation at the air–liquid interface, and to reduce agitation and temperature-induced aggregation.12,21 In this study, it was demonstrated that PS-80 was able to reduce the opalescence of the MAb1 formulation containing 150
mM NaCl. It is possible that PS-80 partially disrupted the hydrophobic–hydrophobic interactions that are dominant following
the initial shielding of electrostatic charges.
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