Development of immunoprecipitation-two-dimensional liquid chromatography-mass spectrometry methodology as biomarker read-out to quantify phosphorylated tau in cerebrospinal fluid from Alzheimer disease patients

In Alzheimer's disease (AD) brain, one of the histopathological hallmarks is the neurofibrillary tangles consisting of aggregated and hyperphosphorylated tau. Currently many tau binding antibodies are under development to target the extracellular species responsible for the spreading of the disease in the brain. As such, an in-house developed antibody JNJ-63733657 with picomolar affinity towards tau phosphorylated at both T212 and T217 (further named p217+tau) was recently tested in phase I clinical trial NCT03375697. Following multiple dose administration in healthy subjects and subjects with AD, there were dose dependant reductions in free p217+tau fragments in cerebrospinal fluid (CSF) following antibody administration, as measured with a novel single molecule ELISA assay (Simoa PT3 x PT82 assay), demonstrating epitope engagement of the therapeutic antibody [Galpern, Haeverans, Janssens, Triana-Baltzer, Kolb, Li, Nandy, Mercken, Van Kolen, Sun, Van Nueten, 2020]. Total p217+tau levels also were reduced in CSF as measured with the Simoa PT3 x PT82 assay. In this study we developed an orthogonal immunoprecipitation - liquid chromatography - triple quadrupole mass spectrometry (IP-LC-TQMS) assay to verify the observed reductions in total p217+ tau levels. In this assay, an excess of JNJ-63733657 is added to the clinical CSF to ensure all p217+tau is bound by the antibody instead of having a pool of bound and unbound antigen and to immunoprecipitate all p217+tau, which is followed by on-bead digestion with trypsin to release surrogate peptides. Tryptic peptides with missed cleavages were monitored when phosphorylation occurred close to the cleavage site as this induced miscleavages. Compared with acidified mobile phases typically used for peptide analysis, reversed phase LC with mobile phase at basic pH resulted in sharper peaks and improved selectivity and sensitivity for the target peptides. With this setup a diphospho-tau tryptic peptide SRTPSLPTPPTREPK*2 could be measured with pT217 accounting for at least one of the phospho-sites. This is the first time that the presence of a diphopsho-tau peptide is reported to be present in human CSF. A two-dimensional LC-TQMS method was developed to remove matrix interferences. Selective trapping of diphospho-peptides via a metal oxide chromatography mechanism was achieved in a first dimension with a conventional reversed phase stationary phase and acidified mobile phase. Subsequent elution at basic pH enabled detec