in which NA is Avogadro's constant (mol-1), λo is the Laser wavelength (cm), no is the solvent refractive index, and dn/dc is the the differential refractive index increment (cm3 g-1).
For particles that are smaller than the incident light (laser) used to measure the particle size, Equation 1 can be reduced
to the linear form shown in the following:13
MAb1 in a liquid formulation consisting of 24 mg/mL of IgG1 in a buffer, 150 mM NaCl, and PS-80, pH 6.0 in glass vials was
inspected visually at 25 °C and was determined to be opalescent (Figure 1). The MAb1 formulation was equivalent to a Reference
III standard [18–30 nephelometric turbidity units (NTU)] according to the EP (5.0, 2.2.1, Clarity and degree of opalescence of liquids). Following filtration of MAb1 through a 0.22-μm sterilizing filter,
the opalescence remained unchanged, indicating it was not caused by insoluble aggregates (data not shown).
The particle size of MAb1 was evaluated beginning at a concentration of 20 mg/mL of IgG1 in a buffer, 150 mM NaCl, and PS-80,
pH 6.0 in glass vials and was diluted in the same formulation to approximately 1 mg/mL (Figure 2). It was determined that
MAb1 had a larger apparent hydrodynamic radius at higher concentrations, and that it decreased as the formulation was diluted.
SEC–HPLC was then performed on MAb1 at a concentration of 1 mg/mL. It was determined that the sample was mostly monomeric
(>99%), with a small amount of dimer present (~1%) (Figure 3). In independent experiments, it was demonstrated that IgG monomers
have a retention time of approximately 16 to 19 min on a TSKgel 3000SWXL column (data not shown).