Branching Analysis of Multivalent Conjugates Using Size Exclusion Chromatography–Multiangle Light Scattering

Abstract

Multivalent conjugates (MVCs) (conjugation of multiple proteins to a linear polymer chain) are powerful for improving the bioactivity and pharmacokinetics of a bioactive molecule. Since this effect is highly dependent upon the valency of the conjugated proteins, it is imperative to have a technique for analysis of the conjugation ratio. Studies of MVCs have used size exclusion chromatography-multiangle light scattering (SEC-MALS), which allows for the separate and individual analysis of the protein and biopolymer components based on their specific refractive index increment and UV extinction coefficient constants to determine the number of proteins bound per biopolymer molecule. In this work, we have applied traditional branching analysis to the SEC-MALS data, with the primary assumption that the polymer backbone can be used as the linear counterpart. We demonstrated good agreement between the branching values and the valency determined by traditional analysis, demonstrating that branching analysis can be used as an alternative technique to approximate the valency of MVCs. The branching analysis method also provides a more complete picture of the distribution of the measured values, provides important branching information about the molecules, and lowers the cost and complexity of the characterization. However, since MVC molecules are both conjugate molecules and branched molecules, the most powerful approach to their characterization would be to use both traditional multivalent conjugate analysis and branching analysis in conjunction.

Multivalent conjugates of basic fibroblast growth factor enhance in vitro proliferation and migration of endothelial cells

“…In previous studies, we have analyzed multivalent protein conjugates as branched polymers by modeling the conjugated proteins as branches emanating from the biopolymer backbone. 25,39 Based on branching analysis, 39 the number of branch units per HyA molecule was 7.5 and 14.9 for the 10:1 and 30:1 conjugates, respectively. Since MVCs behave as branched molecules, at a given molar mass, as the protein-to-polymer ratio or conjugation ratio of the growth factor increases, the molecular size (RMS radius) should decrease.…”

“…The radius of gyration (Rg) values were calculated by obtaining a linear regression in the form of RMS radius (R) = kM b , where M was the molar mass, from the conformation plots for two online runs of the unmodified 200 kDa hyaluronic acid. 39 The resulting equation was used to calculate the predicted linear HyA Rg value at the M value equivalent to M w for each of the conjugates.…”

Probing Polyester Branching by Hybrid Trapped Ion-Mobility Spectrometry–Tandem Mass Spectrometry

“…SEC is the most used technique to determine the molecular weight distribution ( M w ) , of polymers, but can also provide insights in the overall DB. Branched molecules exhibit reduced hydrodynamic volumes ( V H ) relative to their linear counterparts of identical molar mass and elute later in SEC .…”