A Unique SEC Method Overcomes the Multiple-Monomeric-Peak Profile of the Broadly Neutralizing HIV-1 Antibody 10E8

10E8 is a potent broadly neutralizing antibody (bNAb), which targets the membrane-proximal external region (MPER) of the HIV virus. During early analytical development of this bNAb directed towards clinical evaluation, 10E8 exhibited a multiple-monomeric-peak profile caused by secondary interactions on traditional size exclusion chromatography (SEC), thereby rendering SEC unfit for purpose of assessing aggregation, a target critical quality attribute. To overcome this challenge, an innovative and robust SEC method was successfully developed in which the mobile phase was tested for excipients capable of reducing the secondary interactions responsible for the multi-peak profile, and an optimal mobile phase comprising of 2X PBS, 100 mM arginine, and pH 10.55 was established. Application of this optimized mobile phase was shown to allow quantification of the intrinsic level of aggregation of 10E8 without alteration to the SEC matrix itself. Furthermore, the newly developed method was linear, specific, accurate, and precise over an established range. Overall, an SEC method involving optimization of the mobile phase has been successfully developed which allowed assessment of antibody aggregation throughout process development, manufacturing release, and stability testing.