Shedding Light into the Subcutis: A Mass Spectrometry Based Immunocapture Assay Enabling Full Characterization of Therapeutic Antibodies after Injection in Vivo.

An understanding of what happens to therapeutic antibodies in vivo after subcutaneous injection is of high interest. Therefore, we applied the open flow microperfusion technique to extract interstitial fluid from the subcutaneous tissue. In order to analyze those biological samples, a specific and sensitive workflow was required. In this study, we present a complete workflow that enables full characterization of therapeutic antibodies after subcutaneous injection. Compared to classical pharmacokinetic approaches where only a limited number of peptides are detected, our workflow provides full sequence coverage and even enables the identification of potential quality attributes. The efficiency to purify therapeutic antibodies from biological matrixes of two different antibody capture molecules and two types of magnetic beads was compared. Furthermore, several desalting protocols were tested in the development of a miniaturized peptide map procedure. The best results were achieved using a commercial anti-human capture mAb fragment in combination with streptavidin coated magnetic beads, providing capture efficiencies of 90-100%. The optimized peptide map protocol that requires <1 µg of mAb includes two desalting steps and showed sequence coverages of 95-100%. The final method was successfully used for analysis of interstitial fluid and serum samples after a subcutaneous injection of a therapeutic antibody into a domestic pig.

N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain.

Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions.Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau.Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.