title: Bioanalysis of therapeutic peptides: differentiating between total and anti-drug antibody bound drug using liquid chromatography-tandem mass spectrometry quantitation.
abstract: An acylated peptide with MW ~4.5 kDa was measured in samples from pharmacokinetic, toxicology and clinical studies using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lower limits of quantitation of 2 ng/mL and 50 pg/mL were achieved for animal and human plasma, respectively. Repeated drug administration may lead to anti-drug antibodies (ADA) which can inactivate the drug by formation of drug-ADA complexes. Hence, the LC-MS/MS assay incorporated cleavage of potential drug-ADA complexes to quantify the total plasma concentration. To obtain information on active drug levels, an assay that measures the free concentration or alternatively the ADA-unbound concentration would be needed. Ultrafiltration experiments through 100 kD cutoff membranes to remove Ig-bound peptide were not successful due to nonspecific binding. Extraction of Ig-bound drug using Protein A or G (bacterial cell wall proteins with high affinity to the Fc region of IgG) was suitable to distinguish between ADA-bound drug and [free+protein bound (not ADA-bound)] drug and correlated with findings from ELISA ADA measurement.
authors.affiliation: F. Hoffmann-La Roche Ltd., Non-Clinical Drug Safety, DMPK and Bioanalytical R&D, Basel, Switzerland. Electronic address: katja.heinig@roche.com.
authors.forename: Katja
authors.initials: K
authors.lastname: Heinig
authors.forename: Thomas
authors.initials: T
authors.lastname: Wirz
authors.forename: Eginhard
authors.initials: E
authors.lastname: Schick
authors.forename: Alberto
authors.initials: A
authors.lastname: Guenzi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D057230:Limit of Detection; D008252:Macaca fascicularis; D010455:Peptides / Q000097:blood / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D013205:Staphylococcal Protein A; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014462:Ultrafiltration
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D010455:Peptides; D004364:Pharmaceutical Preparations; D013205:Staphylococcal Protein A
keywords: Anti-drug antibody; Free vs. total; Liquid-chromatography–tandem mass spectrometry; Peptide; Protein A/G; Ultrafiltration