Ferritin as a Natural Protein Scaffold: Building a Multivalent Ferritin-Fab Conjugate

The design of molecules with a high degree of valency can be useful for receptor clustering, T-cell recruiting, agonist activation, and half-life extension. Although this is traditionally accomplished using polymer-based molecular scaffolds, biocompatibility and the fate of polymer by-products are often of concern. Multivalent, self-assembling scaffolds such as ferritin, a ubiquitous protein found in most human cell types in addition to invertebrates, higher plants, fungi and bacteria, offer an attractive "natural" alternative to polymer-based scaffolds. In mammals, ferritins are composed of 24 subunits that form an icosahedron with an external hydrodynamic radius of 6 nm, and an overall molar mass of approximately 474 kDa, depending on the biological tissue from which it is derived. The utility of molecular cages, such as ferritin and its derivatives for applications in drug delivery, is well-known. Here, we describe the design, production, and characterization of a multimeric ferritin-antibody fragment conjugate. Following optimization of the conjugation strategy using LC-MS, in-depth characterization was performed using SEC-MALS-QELS. The combined results of this study confirmed that Ferritin-Fab conjugates were successfully generated.