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Immunogenicity Testing by Quantitative Mass Spectrometry: Overcoming the Interference of Excess Protein Therapeutics

presented by

Hendrik Neubert
Pfizer, Inc.

June 27, 2007

TSRI Faculty Club, Tennis Court Room


Background:

Abstract:

     The administration of biological therapeutics can lead to the generation of antibodies resulting in an increased clearance, sustenance or neutralization of the therapeutic. Modifications of the native protein such as pegylation increase the circulating half-life and may alter the immunogenic potential and, depending on the mechanism of action, peak drug concentrations can be in the micrograms per milliliter range in serum. Due to the interference of excess drug in the assay, conventional immunogenicity assays do not give reliable data. Therefore, in the presence of excess drug it is currently difficult to assess if lack of efficacy is due to the presence of neutralizing antibodies.


     In order to improve the assay tolerance to excess drug in the sample we have started exploring the suitability of alternative sample preparation and detection strategies. To this end, we have developed and validated a magnetic bead based immunoprecipitation method followed by quantitative LC/MS to determine anti-protein therapeutic antibodies in human and cynomolgus serum in the presence of high concentrations of drug. The method uses low microliter volumes of serum that is spiked with a high concentration of therapeutic protein to ensure saturation of all available specific antibody binding sites. The approach employs automated 96 well plate magnetic bead based Protein G affinity enrichment of IgG antibodies and their bound antigens. Isolated antibody-antigen complexes are eluted from the magnetic beads and stable isotope labeled peptide standards mimicking sequences of the targeted therapeutic protein are added. Following chemical proteolysis, multiple peptides of the target proteins are quantified by LC/MS inferring the presence of anti-drug IgG.


     This method currently supports clinical programs addressing the safety and tolerability of growth hormone analogues. It will add to the toolkit for the assessment of immunogenicity responses and is anticipated to be transferable to other protein therapeutics.

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