(729e) Affinity Ligands Derived From Hyperthermophilic Protein Scaffolds for Chromatographic Separations

Affinity chromatography has great potential to reduce the cost and complexity of protein purification steps in the production of biopharmaceuticals. However, availability of robust affinity ligands for use in industrial chromatography remains a challenge. Ideally, an affinity ligand must have all of the following properties: high binding affinity and specificity for the target protein to be purified, mild elution conditions, ability to withstand repeated sterilization cycles using procedures such as “clean in place” (CIP) that involves a wash step with sodium hydroxide (NaOH) solution) and low cost. Conventional protein ligands for chromatography such as Protein A or antibodies have one or more of the following limitations: high cost, low stability, harsh elution conditions. Small ligands such as peptides can offer milder elution conditions and higher stability relative to protein ligands, in addition to lower cost. However small ligands typically have low affinities for their targets and lack the selectivity required for many applications. Here we show that the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus can be engineered to generate highly stable affinity ligands for a wide spectrum for targets.

To validate the versatility of the Sso7d scaffold, we generated a library of 10⁸ Sso7d mutants by randomizing 10 surface exposed amino acid residues on Sso7d, using yeast surface display and subsequently isolated binding proteins for a diverse set of model targets; our chosen targets included a small organic molecule (fluorescein), a 12 amino acid peptide fragment from the C‑terminus of beta-catenin, the model proteins hen egg lysozyme and streptavidin, and immunoglobulins from chicken and mouse (1). Mutant proteins based on Sso7d can be expressed at high yields in the E. coli cytoplasm. Further, these mutants have high thermal stability, resistant to chemical denaturation by Guanidine Hydrochloride and retain their secondary structure after extended incubation in extreme pH conditions. Taken together, our results show that Sso7d-derived ligands have properties that make them likely to be well-suited for chromatographic separations.

To further explore the use of Sso7d-based ligands in the context of protein purification, we isolated Sso7d-based proteins that bind the Fc portion of human IgG (hFc). These hFc binders could be used to isolate human IgG from complex media. We systematically characterized the binding of the Sso7d-based ligands to various IgG isotypes as well the effect of glycosylation on binding. Further, using a combination of histidine scanning mutagenesis and directed evolution, we isolated Sso7d variants that can elute human IgG under milder conditions. We expect that Sso7d-based hFc binders identified in this study can be used for purification of humanized antibodies as well as human IgG from plasma for use in intravenous immunoglobulin therapy.

(1) Gera, N. et al., Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold, J. Mol. Biol. (2011) http://dx.doi.org/10.1016/j.jmb.2011.04.020