Identification of model peptides as affinity ligands for the purification of humanized monoclonal antibodies by means of phage display.
A proof-of-principle study was initiated to determine whether phage-display technology could be used to identify peptides as leads in the customization of ligands for affinity chromatography and to identify a peptide or peptidomimetic for use as a Protein A alternative in the affinity purification of monoclonal antibodies. The constant region of humanized anti-Tac (HAT), prepared by pepsin digestion and receptor-affinity chromatography, was used as the target for phage display in this study. As such, 20 phage-derived peptide sequences were identified from four rounds of biopanning with two linear phage-display libraries (7-mer, containing 100 copies of 2 x 10(9) sequences and 12-mer, containing 70 copies of 1.4 x 10(9) sequences). Five peptides were synthesized for use as affinity ligands, based on sequence homology to Protein A, sequence redundancy, and amino acid motifs. The best HAT binding immobilized peptide was EPIHRSTLTALL. The best-fit analysis of this peptide sequence with Protein A yielded an alignment well within the Fc binding domain of Protein A. These results suggest that phage display can serve as a tool in the identification of peptides as model ligands for affinity chromatography.
Ehrlich
G K
GK
Pharmaceutical and Analytical R&D, Hoffmann La Roche, Nutley, NJ 07110, USA. george.erlich@roche.com
Bailon
P
P
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000302:isolation & purification; D008024:Ligands; D008969:Molecular Sequence Data; D010434:Pepsin A; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D008024:Ligands; D010446:Peptide Fragments; D019151:Peptide Library; D010455:Peptides; D013205:Staphylococcal Protein A; D010434:Pepsin A