Thorough characterization
of protein assemblies is required for
the control of structure and robust performance in any given application,
especially for the safety and stability of protein therapeutics. Here,
we report the use of multiple, orthogonal characterization techniques
to enable control over the structure of a multivalent antibody carrier
for future use in drug delivery applications. The carrier, known as
Hex, contains six antibody binding domains that bind the Fc region
of antibodies. Using size exclusion chromatography, analytical ultracentrifugation,
and dynamic light scattering, we identified the stoichiometry of assembled
Hex–antibody complexes and observed changes in the stoichiometry
of nanocarriers when incubated at higher temperatures over time. The
characterization data informed the modification of Hex to achieve
tighter control over the protein assembly structure for future therapeutic
applications. This work demonstrates the importance of using orthogonal
characterization techniques and observing protein assembly in different
conditions over time to fully understand and control structure and
dynamics.
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