Comparability and monitoring immunogenic N-linked oligosaccharides from recombinant monoclonal antibodies from two different cell lines using HPLC with fluorescence detection and mass spectrometry.

One of the most important structural features of recombinant monoclonal antibodies produced in mammalian cells is the N-linked oligosaccharide profile. These profiles impact recombinant therapeutics in a multitude of ways affecting distribution, efficacy, and immunogenicity. High mannose, alpha-gal and other oligosaccharide species are highly immunogenic and in most cases should be minimized during manufacturing. A recombinant monoclonal antibody, h5G1.1, was produced in NS0 and CHO cell lines and tested to identify changes in the N-linked oligosaccharide profiles caused from a change in cell line. Traditional peak analysis using HPLC with fluorescence detection was augmented by mass spectrometric analysis. Nano LC-MS following tryptic digestion corroborated HPLC findings of the presence of several alpha-gal oligosaccharide species in the recombinant IgG (rIgG) from NS0 cell line. Both cell lines possessed rIgGs with complex and small amounts of high mannose glycans.

Mass spectrometry and HPLC with fluorescent detection-based orthogonal approaches to characterize N-linked oligosaccharides of recombinant monoclonal antibodies.

A number of HPLC and mass spectrometric techniques are used to characterize post-translational modification in recombinant monoclonal antibodies (MAbs) using the intact glycoprotein and free glycans. LC separation utilizing fluorescent detection technique allows tentative structural assignment of MAb oligosaccharides. Intact molecular weight analysis via electrospray allows for an accurate mass determination and observation of the native glycoform mass envelope. N-linked oligosaccharides are then analyzed by MALDI-ToF. Their structures are further confirmed by analyzing the fragmentation patterns formed by MS/MS. All these techniques provide useful information when performed in isolation. However, the combined information allows for definitive and robust characterization of the N-linked glycans from recombinant MAbs.