Opalescence is a phenomenon that has been observed in several commercially available monoclonal antibodies, both in liquid
and reconstituted lyophilized formulations. In this article, we demonstrated that an increase in the ionic strength of a monoclonal
antibody formulation (MAb1) led to opalescence and higher viscosity. When the ionic strength was reduced, no opalescence in
the MAb1 formulation was observed. The removal of polysorbate-80 (PS-80) from the formulation resulted in an increase in opalescence
in NaCl-containing formulations, whereas it had no effect on formulations lacking NaCl. Differential scanning calorimetry
with MAb1 formulations containing increasing amounts of NaCl indicated that formulations with higher ionic strength present
a lower apparent melting temperature. Opalescent MAb1 formulations placed on stability remained unchanged after four weeks
at 4 °C, whereas at 45 °C, an increase in dimers was observed. Using multi-angle light scattering, the MAb1 formulation was
found to have a negative second virial coefficient.
There are more than 23 therapeutic monoclonal antibodies that are commercially available for treating a wide variety of diseases.1 Of these products, several have an opalescent appearance either as liquids or following reconstitution of a lyophilized
formulation.2 Opalescence is defined as exhibiting a play of colors like that of the opal, or having a milky iridescence.3
(Merck & Co.)
Factors influencing the opalescence of an IgG1 monoclonal antibody formulation include the concentration of the antibody as
well as formulation components such as buffers, ionic strength, excipients, and pH. It was demonstrated that an increase in
the concentration of the antibody and a decrease in temperature (5 °C) of an IgG1 formulation resulted in opalescence.4
It is well known that ionic strength can affect the behavior of proteins in solution. In most cases, at low and high salt
concentrations either salting-in or salting-out occurs, respectively.5 Salting-in is observed when electrostatic interactions between the salt ions and charged residues of the protein are favorable.5 Salting-out occurs when the salt ions are excluded from the protein, which is mainly caused by unfavorable interactions
between the salts and hydrophobic regions of the protein.5 There are examples, however, in which proteins are soluble at high salt concentrations.5–7
The ionic strength can also mediate protein–protein interactions. One example is with the protein b-lactoglobulin, which is
predominantly monomeric at pH 3 in the absence of salt, but is dimeric in the presence of salt.8 Additionally, the type of salt may also affect the stability of the protein. Bovine serum albumin is stabilized against
thermal unfolding with NaSCN and NaClO4, both kosmotropic salts, yet destabilized by chaotropic salts at high ionic strength.9,10 In other examples, aggregation was decreased in the presence of NaCl for both recombinant factor VIII SQ and recombinant
keratinocyte growth factor, whereas aggregation increased in the presence of NaCl with recombinant human granulocyte colony
In the following article, we sought to determine whether ionic strength and excipients mediated the opalescence of an IgG1
formulation (MAb1). It was demonstrated that MAb1 formulations become opalescent as the ionic strength of the formulation
is increased. Conversely, MAb1 formulations without salt lack opalescence. The second virial coefficient of MAb1 was negative.
Stability studies indicated that opalescent MAb1 formulations have increased amount of irreversible dimers at elevated temperatures.
MATERIALS AND METHODS
Materials and Reagents
MAb1 is a fully human IgG1 monoclonal antibody. MAb1 was purified from Chinese hamster ovary (CHO) cells by Bioprocess Research
and Development, Merck Research Laboratories (Whitehouse Station, NJ). The MAb1 formulation contains 24 mg/mL IgG1 in a formulation
containing a buffer, NaCl, and polysorbate-80 (PS-80), pH 6. Polysorbate-80 was from Croda Incorporated (Mill Hall, PA). NaCl,
KCl, MgCl2, KSCN, Na3PO4, CsCl, Na2SO4, hexamethylenetetramine, and hydrazine sulfate were from Sigma (St. Louis, MO). The filter used was a 0.22 μm Millex GV from
Millipore (Billerica, MA).