title: Absolute quantification of the total and antidrug antibody-bound concentrations of recombinant human α-glucosidase in human plasma using protein G extraction and LC-MS/MS.
abstract: The administration of protein-based pharmaceuticals can cause the in vivo formation of antidrug antibodies (ADAs), which may reduce the efficacy of the therapy by binding to the protein drug. An accurate determination of the total and ADA-bound concentrations of the drug gives information on the extent of this immune response and its consequences and may help develop improved therapeutic regimens. We present an absolute quantitative method to differentiate between total, free, and ADA-bound drug for recombinant human alpha acid glucosidase (rhGAA) in plasma from patients suffering from Pompe's disease. LC-MS/MS quantification of a signature peptide after trypsin digestion of plasma samples before and after an extraction of the total IgG content of plasma with protein G coated beads was used to determine the total and the ADA-bound fractions of rhGAA in samples from Pompe patients after enzyme infusion. The methods for total and ADA-bound rhGAA allow quantitation of the drug in the range of 0.5 to 500 μg/mL using 20 μL of plasma and met the regular bioanalytical validation requirements, both in the absence and presence of high levels of anti-rhGAA antibodies. This demonstrates that the ADA-bound rhGAA fraction can be accurately and precisely determined and is not influenced by sample dilution, repeated freezing and thawing, or extended benchtop or frozen storage. In samples from a patient with a reduced response to therapy due to ADAs, high ADA-bound concentrations of rhGAA were found, while in the samples from a patient lacking ADAs, no significant ADA-bound concentrations were found. Since protein G captures the complete IgG content of plasma, including all antidrug antibodies, the described extraction approach is universally applicable for the quantification of ADA-bound concentrations of all non-IgG-based biopharmaceuticals.
authors: †Bioanalytical Laboratory, PRA Health Sciences, Early Development Services, Westerbrink 3, 9405 BJ Assen, The Netherlands.|‡Analytical Biochemistry, Department of Pharmacy, University of Groningen, A. Deusinglaan 1, 9700 AV Groningen, The Netherlands.
authors: Kees J
authors: KJ
authors: Bronsema
authors: ‡Analytical Biochemistry, Department of Pharmacy, University of Groningen, A. Deusinglaan 1, 9700 AV Groningen, The Netherlands.
authors: Rainer
authors: R
authors: Bischoff
authors: §Center for Lysosomal and Metabolic Diseases, Erasmus University Medical Center, Dr. Molewaterplein 60, 3015 GJ Rotterdam, The Netherlands.|∥Molecular Stem Cell Biology, Department of Clinical Genetics, Erasmus MC University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands.|⊥Department of Pediatrics, Rotterdam Erasmus MC University Medical Center, Dr. Molewaterplein 60, 3015 GJ, Rotterdam, The Netherlands.
authors: W W M Pim
authors: WW
authors: Pijnappel
authors: §Center for Lysosomal and Metabolic Diseases, Erasmus University Medical Center, Dr. Molewaterplein 60, 3015 GJ Rotterdam, The Netherlands.|⊥Department of Pediatrics, Rotterdam Erasmus MC University Medical Center, Dr. Molewaterplein 60, 3015 GJ, Rotterdam, The Netherlands.
authors: Ans T
authors: AT
authors: van der Ploeg
authors: †Bioanalytical Laboratory, PRA Health Sciences, Early Development Services, Westerbrink 3, 9405 BJ Assen, The Netherlands.|‡Analytical Biochemistry, Department of Pharmacy, University of Groningen, A. Deusinglaan 1, 9700 AV Groningen, The Netherlands.
authors: Nico C
authors: NC
authors: van de Merbel
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D002853:Chromatography, Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006009:Glycogen Storage Disease Type II / Q000201:enzymology; D006801:Humans; D011994:Recombinant Proteins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry; D000520:alpha-Glucosidases / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; C509951:GAA protein, human; D000520:alpha-Glucosidases; D019204:GTP-Binding Proteins