Abstract
The monoclonal anti-CD20 IgG1 antibody rituximab is used as a first-line treatment for B cell lymphoma. Like all therapeutic antibodies, it is a complex protein for which both safety and efficacy heavily depend on the integrity of its three-dimensional structure. Aptamers, short oligonucleotides with a distinct fold, can be used to detect minor modifications or structural variations of a molecule or protein. To detect antibody molecules in a fold state occurring prior to protein precipitation, we generated DNA aptamers that were selected for extensively heat-treated rituximab. Using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX), we obtained six DNA aptamer sequences (40-mers) specific for 80°C heat-treated rituximab. In silico fold prediction and circular dichroism analysis revealed a G-quadruplex structure for one aptamer, while all others exhibited a B-DNA helix. Binding affinities ranging from 8.8–86.7 nM were determined by an enzyme-linked apta-sorbent assay (ELASA). Aptamers additionally detected structural changes in rituximab treated for 5 min at 70°C, although with lower binding activity. Notably, none of the aptamers recognized rituximab in its native state nor did they detect the antibody after it was exposed to lower temperatures or different physical stressors. Aptamers also reacted with the therapeutic antibody adalimumab incubated at 80°C suggesting similar aptamer binding motifs located on extensively heat-treated IgG1 antibodies. Within this work, we obtained the first aptamer panel, which is specific for an antibody fold state specifically present prior to protein aggregation. This study demonstrates the potential of aptamer selection for specific stress-based protein variants, which has potential impact for quality control of biopharmaceuticals.
Highlights
Biopharmaceuticals are therapeutic agents which are produced in living cells or organisms and require tightly controlled production processes and product characterization [1,2,3]
To obtain DNA aptamers against the heat-treated monoclonal IgG1 antibody rituximab, a DNA-library consisting of 1015 different single-strand oligonucleotides with a random part of 40 bp was used as template for initial selection
Six aptamer selection cycles and one counter cycle were performed to enrich DNA sequences with specificity to rituximab treated at 80 ̊C during the aptamer elution process
Summary
Biopharmaceuticals are therapeutic agents which are produced in living cells or organisms and require tightly controlled production processes and product characterization [1,2,3]. Complex molecules like monoclonal antibodies (mAbs), produced in eukaryotic cell lines, depend on an orchestrated subunit assembly and distinct post-translational modifications [4]. The function and efficacy of therapeutic mAbs depends on their correct three-. Authors MK, SW, and CGH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section
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