Characterization and Control of Dynamic Rearrangement in a Self-Assembled Antibody Carrier

Abstract

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.

Development of Self-Assembled Protein Nanocage Spatially Functionalized with HA Stalk as a Broadly Cross-Reactive Influenza Vaccine Platform

“…A Malvern OmniSEC integrated system (Malvern Panalytical) with a SRT SEC-300 analytical SEC column (Sepax) was used to assess structures of HrHA-tGCN4 and OVA-tGCN4 as previously described . A bovine serum albumin standard was used to perform calibration, and the chromatogram profiles were assessed by a refractive index, right angle light scattering, and viscometer.…”

“…A Malvern OmniSEC integrated system (Malvern Panalytical) with a SRT SEC-300 analytical SEC column (Sepax) was used to assess structures of HrHA-tGCN4 and OVA-tGCN4 as previously described. 87 A bovine serum albumin standard was used to perform calibration, and the chromatogram profiles were assessed by a refractive index, right angle light scattering, and viscometer. A refractive index increment value (d n /d c ) of 0.185 was provisioned into the software to calculate the molecular weight of the peaks, and each peak area was quantified at 254 nm wavelength to determine percent fractions of sample.…”

Supramolecular “Click Chemistry” for Targeting in the Body

“…Engineered coiled-coil peptides, characterized by the arrangement of α-helical peptides into a superhelix bundle, afford recognition properties with utility in the design of functional materials and systems. , Their biological relevance as a common structural motif found in nature has inspired significant study into both mechanisms of formation and strategies for sequence manipulation to realize coiled-coil motifs comprising a different number ( n = 2–6) of both homo- or hetero­[ n ]­meric α-helical peptides. − Various naturally derived and de novo designs have thus been demonstrated for coiled-coil recognition, with synthetic heterodimeric variants having K eq values up to 10 14 M –1 . − Such interactions are thus comparable to (or higher than) high-affinity host–guest or oligonucleotide motifs. The predictable nature of these associations has been used to recreate the complex higher-ordered structures of natural proteins with synthetic variants, for instance, in the preparation of discrete cage-like assemblies. , In addition, coiled-coil motifs have been incorporated as a modular associating unit in noncovalent preparation of modular drug carriers. − Accordingly, these interactions offer another class of synthetic noncovalent interactions with promise for in situ recognition in the body.…”

Multi-Detection Size Exclusion Chromatography as an Advanced Tool for Monitoring Enzyme–Antibody Conjugation Reaction and Quality Control of a Final Product

“…It provides a comprehensive insight into the molecular characterization data of the protein sample, including absolute molecular weight (Mw), molecular weight distribution or polydispersity, molecular size, intrinsic viscosity (IV), and recovery. Moreover, it is possible to obtain additional information on the macromolecular structure, conformation, aggregation, branching, and copolymer/conjugate composition [7][8][9][10].…”