Opalescent appearance of an IgG1 antibody at high concentrations and its relationship to noncovalent association.
OBJECTIVE
Therapeutic antibodies are often formulated at a high concentration where they may have an opalescent appearance. The aim of this study is to understand the origin of this opalescence, especially its relationship to noncovalent association and physical stability.


METHODS
The turbidity and the association state of an IgG1 antibody were investigated as a function of concentration and temperature using static and dynamic light scattering, nephelometric turbidity, and analytical ultracentrifugation. RESULTS. The antibody had increasingly opalescent appearance in the concentration range 5-50 mg/ml. The opalescence was greater at refrigerated temperature but was readily reversible upon warming to room temperature. Turbidity measured at 25 degrees C was linear with concentration, as expected for Rayleigh scatter in the absence of association. In the concentration range 1-50 mg/ml, the weight average molecular weights were close to that expected for a monomer. Zimm plot analysis of the data yielded a negative second virial coefficient, indicative of attractive solute-solute interactions. The hydrodynamic diameter was independent of concentration and remained unchanged as a function of aging at room temperature.


CONCLUSIONS
The results indicate that opalescent appearance is not due to self-association but is a simple consequence of Rayleigh scatter. Opalescent appearance did not result in physical instability.
Biopharmaceutical Research and Development, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. sukumar@lilly.com
Muppalla
M
Sukumar
Brandon L
BL
Doyle
Jessica L
JL
Combs
Allen H
AH
Pekar
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004355:Drug Stability; D007074:Immunoglobulin G / Q000737:chemistry*; D008027:Light; D009391:Nephelometry and Turbidimetry; D012034:Refrigeration; D012542:Scattering, Radiation; D013696:Temperature; D014461:Ultracentrifugation
D007074:Immunoglobulin G