Structural refinement of protein A mimetic peptide

✍ Scribed by Francesca Dinon; Matteo Salvalaglio; Andrea Gallotta; Luca Beneduce; Paolo Pengo; Carlo Cavallotti; Giorgio Fassina

• Publisher: John Wiley and Sons
• Year: 2011
• Tongue: English
• Weight: 629 KB
• Volume: 24
• Category: Article
• ISSN: 0952-3499
• DOI: 10.1002/jmr.1157

✦ Synopsis

A novel dendrimeric peptide ligand dubbed D‐PAM‐Φ was designed to achieve a high capacity for human IgG through the decoration of the D‐PAM scaffold. The design criteria based on the introduction of small hydrophobic groups on the D‐PAM structure were supported by the recently published solid‐state structure of D‐PAM complexed to the Fc fragment of a recombinant human IgG1 and by molecular dynamic simulations that provided information on the mode of binding of phenylacetyl‐D‐PAM (D‐PAM‐Φ). D‐PAM‐Φ was immobilised on an activated solid support and compared with the parent D‐PAM affinity matrix. The newly obtained affinity sorbent was evaluated for its capacity to selectively capture polyclonal human IgG; the binding capacity was approximately 10 mg/ml, an almost 10‐fold enhancement with respect to the D‐PAM–functionalised matrices without the specificity of binding being reduced. The new ligand was also effective in the capturing of recombinant humanised IgG1 from a clarified cell culture supernatant. Under a typical laboratory‐scale affinity chromatography assembly and preliminarily optimised binding conditions, the affinity purification of humanised IgG1 from culture supernatants rendered the desired product, with purity higher than 90%. The results suggest that the application of the computational approach on the structure of the D‐PAM–Fc complex may be very valuable in the development of novel lead molecules for the downstream processing of human or humanised antibodies used in therapy. Copyright © 2011 John Wiley & Sons, Ltd.