The Fluorescent Enzyme Cascade Detects Low Abundance Protein Modifications Suitable for the Assembly of Functionally Annotated Modificatome Databases.

Pathophysiological functions of proteins critically depend on both their chemical composition, including post-translational modifications, and their three-dimensional structure, commonly referred to as structure-activity relationship. Current analytical methods, like capillary electrophoresis or mass spectrometry, suffer from limitations, such as the detection of unexpected modifications at low abundance and their insensitivity to conformational changes. Building on previous enzyme-based analytical methods, we here introduce a fluorescence-based enzyme cascade (fEC), which can detect diverse chemical and conformational variations in protein samples and assemble them into digital databases. Together with complementary analytical methods an automated fEC analysis established unique modification-function relationships, which can be expanded to a proteome-wide scale, i. e. a functionally annotated modificatome. The fEC offers diverse applications, including hypersensitive biomarker detection in complex samples.

Analytical Cascades of Enzymes for Sensitive Detection of Structural Variations in Protein Samples.

Protein function critically depends on structure. However, current analytical tools to monitor consistent higher-order structure with high sensitivity, as for instance required in the development of biopharmaceuticals, are limited. To complement existing assays, we present the analytical cascade of enzymes (ACE), a method based on enzymatic modifications of target proteins, which serve to exponentially amplify structural differences between them. The method enables conformational and chemical fingerprinting of closely related proteins, allowing for the sensitive detection of heterogeneities in protein preparations with high precision. Using this method, we detect protein variants differing in conformation only, as well as structural changes induced by diverse covalent modifications. Additionally, we employ this method to identify the nature of structural variants. Moreover, the ACE method should help to address the limited reproducibility in fundamental research, which partly relates to sample heterogeneities.

Aptamers as quality control tool for production, storage and biosimilarity of the anti-CD20 biopharmaceutical rituximab.

Detailed analysis of biopharmaceuticals is crucial for safety, efficacy and stability. Aptamers, which are folded, single-stranded oligonucleotides, can be used as surrogate antibodies to detect subtle conformational changes. We aimed to generate and assess DNA aptamers against the therapeutic anti-CD20 antibody rituximab. Six rituximab-specific aptamers with Kd = 354-887 nM were obtained using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX) technology. Aptamer folds were analysed by online prediction tools and circular dichroism spectroscopy suggesting quadruplex structures for two aptamers while others present B-DNA helices. Aptamer binding and robustness with respect to minor differences in buffer composition or aptamer folding were verified in the enzyme-linked apta-sorbent assay. Five aptamers showed exclusive specificity to the Fab-fragment of rituximab while one aptamer revealed a broader recognition pattern to other monoclonal antibodies. Structural differences upon incubation at 40 °C for 72 h or UV exposure of rituximab were uncovered by four aptamers. High similarity between rituximab originator and biosimilar lots was demonstrated. The most sensitive aptamer (RA2) detected signal changes for all lots of a copy product suggesting conformational differences. For the first time, a panel of rituximab-specific aptamers was generated allowing the assessment of conformational coherence during production, storage, and biosimilarity of different products.