A high-throughput method for quantification of glycoprotein sialylation.

Sialic acid can improve qualities of therapeutic glycoproteins such as circulatory half-life, biological activity, and solubility. In production of therapeutic glycoproteins, a high-throughput method is required for process monitoring and optimization to ensure consistent and optimal sialic acid content. Current methods for quantifying sialic acid, however, require chromatographic separation that is time-consuming and cannot rapidly analyze many samples in parallel. Here we present a novel high-throughput method for quantifying glycoprotein sialylation. Using chemical reduction, enzymatic release of sialic acid, and chemical derivatization of the sialic acid, the method can accurately, rapidly (15 min), and specifically analyze many samples in parallel. It requires only 45 µl of sample and has a quantitation limit of 2 µM sialic acid. It has also been validated for monitoring sialylation of recombinant interferon gamma (IFN-γ) produced in Chinese hamster ovary (CHO) cell culture. This method is useful for various applications in upstream and downstream bioprocesses.

Eggshell matrix protein mimics: designer peptides to induce the nucleation of calcite crystal aggregates in solution.

Ansocalcin is a novel goose eggshell matrix protein with 132 amino acid residues, which induces the formation of polycrystalline calcite aggregates in in vitro crystallization experiments. The central region of ansocalcin is characterized by the presence of multiplets of charged amino acids. To investigate the specific role of charged amino acid multiplets in the crystal nucleation, three short peptides REWD-16, REWDP-17 (containing charged doublets), and RADA-16 (alternating charged residues) were synthesized and characterized. The aggregation of these peptides in solution was investigated using circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering experiments. The peptides REWD-16 and REWDP-17 induced the polycrystalline calcite crystal aggregates, whereas RADA-16 did not induce significant changes in calcite crystal morphology or aggregate formation in in vitro crystallization experiments. The lattice and morphology of the calcite crystals were characterized using X-ray diffraction and scanning electron microscope. The results discussed in this paper reveal the importance of multiplets of charged amino acid residues toward the nucleation of polycrystalline calcite crystal aggregates in solution.