Structural refinement of protein A mimetic peptide.
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.
Xeptagen SpA, I-30175 Marghera, Venice, Italy.
Francesca
F
Dinon
Matteo
M
Salvalaglio
Andrea
A
Gallotta
Luca
L
Beneduce
Paolo
P
Pengo
Carlo
C
Cavallotti
Giorgio
G
Fassina
D040761:Biomimetic Materials / Q000737:chemistry*; D050091:Dendrimers / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification*; D008024:Ligands; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D011487:Protein Conformation; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000276:immunology
D050091:Dendrimers; D007074:Immunoglobulin G; D008024:Ligands; D010455:Peptides; D013205:Staphylococcal Protein A