Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands

• Published in: The Journal of biological chemistry Vol. 299; no. 7; p. 104910
• Main Authors: Suderman, Richard J., Gibson, Shane D., Strecker, Mary, Bonner, Amanda M., Chao, David M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-07-2023; American Society for Biochemistry and Molecular Biology
• Subjects: affinity capture ligand; affinity chromatography; Antibodies - chemistry; Antibodies - immunology; Antibodies - metabolism; Antibody Affinity; antibody mimetic; biotechnology; chromatography; Chromatography, Affinity - methods; Hot Temperature; Ligands; Methods and Resources; Molecular Mimicry; Protein Binding; protein engineering; Protein Engineering - methods; protein purification; Protein Stability - drug effects; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sodium Hydroxide - pharmacology; Trypsin - metabolism; affinity chromatography; biotechnology; protein engineering; RBD; affinity capture ligand; chromatography; ECD; antibody mimetic; protein purification; nanoCLAMPs
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2023.104910

Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents. This work describes a protein engineering campaign to develop a more robust nanoCLAMP scaffold compatible with harsh bioprocessing conditions. The campaign generated an improved scaffold with dramatically improved resistance to heat, proteases, and NaOH. To isolate additional nanoCLAMPs based on this scaffold, we constructed a randomized library of 1 × 1010 clones and isolated binders to several targets. We then performed an in-depth characterization of nanoCLAMPs recognizing yeast SUMO, a fusion partner used for the purification of recombinant proteins. These second-generation nanoCLAMPs typically had a Kd of <80 nM, a Tm of >70 °C, and a t1/2 in 0.1 mg/ml trypsin of >20 h. Affinity chromatography resins bearing these next-generation nanoCLAMPs enabled single-step purifications of SUMO fusions. Bound target proteins could be eluted at neutral or acidic pH. These affinity resins maintained binding capacity and selectivity over 20 purification cycles, each including 10 min of cleaning-in-place with 0.1 M NaOH, and remained functional after exposure to 100% DMF and autoclaving. The improved nanoCLAMP scaffold will enable the development of robust, high-performance affinity chromatography resins against a wide range of protein targets.