Relevant Parameters in Developing Protein Aggregation Methods

Introduction

A new high efficiency GFC column, Yarra, was recently introduced and is significantly more efficient than other GFC columns on the market. In addition to higher efficiency, Yarra columns demonstrate significantly higher inertness to ionic interactions versus other GFC columns; however, such chemical characteristics sometimes require changes to operating parameters. Performing method development for protein aggregation analysis using next-generation Yarra GFC columns will be discussed. 

Being an isocratic method, gel filtration chromatography is assumed to be a simple method with little or no method development involved. On closer inspection, however, subtle changes in mobile phase and other parameters can have significant results on separation performance and accuracy of determining the aggregation state of a protein. Protein gel filtration columns are typically made by bonding a highly polar “diol-like” ligand to a porous silica matrix of a specific pore size. This polar “diol” coat on the silica is intended to minimize surface interactions between the silica and proteins, resulting in separations based on the size of a protein in solution as proteins are differentially excluded from the pores of the silica particle. One typically uses different pore-sized columns that provide maximum resolution of a specific molecular weight range based on the protein being separated. Often, two different pore-sized columns overlap in a molecular weight range, resulting in different selectivities based on the column being used.