Capturing and amplifying impurities from purified recombinant monoclonal antibodies via peptide library beads: a proteomic study.
Capture and amplification of low-level contaminants in purified preparations of recombinant DNA products is described here in the case of mAb meant for human consumption. Such a process is based on treatment with a vastly heterogeneous ligand library composed of hexapeptides bound to a polyhydroxymethacrylate resin. Upon this treatment, a protein solution is recovered with "normalized" relative concentration ratios, in which high-abundance proteins are strongly reduced and rare proteins are highly concentrated. Upon 2-D map analysis, the relatively few spots present in control monoclonals were seen to increase in number, reaching >100 visible polypeptide chains in the pI/M(r) plane. Most of these newly emerged spots were subjected to MS analysis and were found to be composed mainly of three classes of proteins: those derived from proteins present in the culture broth (notably albumin and transferrin), fragments of the desired final product, covering M(r) ranges from as low as 5 up to 45 kDa and some aggregates of light and heavy chains of Igs (mostly dimers and trimers). This ligand library thus appears to be a formidable tool for exploring and bringing to the limelight the "hidden proteome".
Antonioli
Paolo
P
Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Via Mancinelli 7, Milano, Italy.
Fortis
Fréderic
F
Guerrier
Luc
L
Rinalducci
Sara
S
Zolla
Lello
L
Righetti
Pier Giorgio
PG
Boschetti
Egisto
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019151:Peptide Library; D020543:Proteome / Q000032:analysis*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D019151:Peptide Library; D020543:Proteome; D011994:Recombinant Proteins