The Effects of Linker Length and Flexibility on Fc-Fusion Proteins

Abstract

Aggregation, yield, and overall stability of molecules are of enormous importance for development of biopharmaceuticals. Several construct and cell-based optimization approaches are being undertaken to generate molecules with increased stability and yield. Different element of the DNA construct can affect expression and aggregation levels of a protein, including the type of linker used to connect the extracellular domain to the Fc of a fusion molecule. The choice of linker is protein specific depending on the function of the molecule and may require distance between the domains for correct folding. The aim is to examine the effects of linker rigidity and length on folding, yield, and aggregation of ActRIIB-Fc homodimer. In particular, we selected hIgA1 hinge and (AP)x11 from literature as the two linkers to test, due to differences in structure, rigidity, and composition. In this study, ActRIIB-Fc was cloned with hIgA1 hinge and (AP)x11, two linkers with rigidity added to its structure due to proline residues, along with two flexible control linkers composed of GGG and (G4S)4. All four constructs were transiently expressed in ExpiCHO cells and purified using protein A affinity resin followed by Preparative SEC. The quality of each product was analyzed for changes in titer, aggregation and other contaminants in culture by western blots and size exclusion chromatography (SEC). They were further characterized for binding and inhibition of its interacting partners. Proteins were also assessed for any changes in thermal stability and folding due to the rigidity and length of the linkers. We observed an improvement in overall affinity of the product to a proprietary anti-ActRIIB antibody affinity column. Evaluation of the cultures revealed no change in aggregation between the rigid and flexible linkers but there was an increase in low molecular weight species, which translated to a lower percentage of correct size molecule, seen with rigid linkers. There was no change in binding and inhibition of the ligands, and we noted no difference in thermal stability of these constructs. Based on these results, we observed some improvements to ActRIIB-Fc homodimers by replacing the linkers with a more rigid one, but even these linkers can be further optimized to prevent sequence-related clipping and increasing folding rate.