A versatile design platform for glycoengineering therapeutic antibodies.

Manipulation of glycosylation patterns, i.e., glycoengineering, is incorporated in the therapeutic antibody development workflow to ensure clinical safety, and this approach has also been used to modulate the biological activities, functions, or pharmacological properties of antibody drugs. Whereas most existing glycoengineering strategies focus on the canonical glycans found in the constant domain of immunoglobulin G (IgG) antibodies, we report a new strategy to leverage the untapped potential of atypical glycosylation patterns in the variable domains, which naturally occur in 15% to 25% of IgG antibodies. Glycosylation sites were added to the antigen-binding regions of two functionally divergent, interleukin-2-binding monoclonal antibodies. We used computational tools to rationally install various N-glycosylation consensus sequences into the antibody variable domains, creating "glycovariants" of these molecules. Strikingly, almost all the glycovariants were successfully glycosylated at their newly installed N-glycan sites, without reduction of the antibody's native function. Importantly, certain glycovariants exhibited modified activities compared to the parent antibody, showing the potential of our glycoengineering strategy to modulate biological function of antibodies involved in multi-component receptor systems. Finally, when coupled with a high-flux sialic acid precursor, a glycovariant with two installed glycosylation sites demonstrated superior in vivo half-life. Collectively, these findings validate a versatile glycoengineering strategy that introduces atypical glycosylation into therapeutic antibodies in order to improve their efficacy and, in certain instances, modulate their activity early in the drug development process.

A study of phospholipids by ion mobility TOFMS.

Combining matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with ion mobility (IM) results in the fast sorting of biomolecules in complex mixtures along trend lines. In this two-dimensional (2D) analysis of biological families, lipids, peptides, and nucleotides are separated from each other by differences in their ion mobility drift times in a timescale of hundreds of microseconds. Molecular ions of similar chemical type fall along trend lines when plotted in 2D plots of ion mobility drift time as a function of m/z. In this study, MALDI-IM MS is used to analyze species from all of the major phospholipid classes. Complex samples, including tissue extracts and sections, were probed to demonstrate the effects that radyl chain length, degree of unsaturation, and class/head group have upon an ion's cross section in the gas phase. We illustrate how these changes can be used to identify individual lipid species in complex mixtures, as well as the effects of cationization on ion cross section and ionization efficiency.

MALDI-ion mobility-TOFMS imaging of lipids in rat brain tissue.

While maintaining anatomical integrity, matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) has allowed researchers to directly probe tissue, map the distribution of analytes and elucidate molecular structure with minimal preparation. MALDI-ion mobility (IM)-orthogonal time-of-flight mass spectrometry (oTOFMS) provides an advantage by initially separating different classes of biomolecules such as lipids, peptides, and nucleotides by their IM drift times prior to mass analysis. In the present work the distribution of phosphatidlycholine and cerebroside species was mapped from 16 microm thick coronal rat brain sections using MALDI-IM-oTOFMS. Furthermore, the use of gold nanoparticles as a matrix enables detection of cerebrosides, which although highly concentrated in brain tissue, are not easily observed as positive ions because of intense signals from lipids such as phosphatidlycholines and sphingomyelins.