Toward understanding molecular heterogeneity of polysorbates by application of liquid chromatography-mass spectrometry with computer-aided data analysis.

Polysorbates (PS) are widely used as oil-in-water emulsifiers, stabilizers, wetting agents, solubilizers, and dispersants in the agricultural, food, personal care, and pharmaceutical industries due to their cost effectiveness, biocompatibility, formulation flexibility, low toxicity, and good stabilizing and protecting properties. The polysorbates are often pictured as polyoxyethylated sorbitan monoesters of saturated and/or unsaturated fatty acids. In reality, polysorbates are complex mixtures of multiple components, as follows from the reactions involved in their production. In this work, we report a novel application of liquid chromatography-mass spectrometry (LC-MS) for the characterization of polysorbates. This method takes advantage of accurate mass measurements and information on the identity of a fatty acid from "in-source" generated characteristic dioxolanylium ions. The method allowed us to perform quick profiling of fatty acids in PS 20 and 80 which, combined with a computer-aided peak assignment algorithm, facilitated detailed characterization of their constituents. As a major finding, we determined that different samples of PS 20 varied from 0% to 15% in relative amounts of unsaturated oleic acid. Although the consequences of this difference were not fully evaluated in this work, one might expect that PS 20 with larger amounts of oleic acid will be more prone to autoxidation, thus potentially having greater impact on the oxidative degradation of the biotherapeutics it formulates.

On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format.

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography-mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.

Characterization of oligosaccharides in recombinant tissue plasminogen activator produced in Chinese hamster ovary cells: two decades of analytical technology development.

Recombinant tissue plasminogen activator (rt-PA) is a well-characterized glycoprotein with a great deal of published information on its structure, post-translational modifications, and O- and N-glycosylation. Most of the characterization was accomplished in the late 1980s. During the past 2 decades, however, mass spectrometry has made a quantum leap forward offering new capabilities in soft electrospray ionization, speed, resolution, and accuracy of mass measurements. From this point of view, it is worthwhile to revisit the characterization of familiar proteins, such as rt-PA, using the new capabilities of modern analytical technology. In this work, we applied LC-MS with state-of-the-art instrumentation to the characterization of glycoforms of rt-PA. This method takes advantage of accurate mass measurements along with a fast "in-source" voltage switching for the detection of characteristic oxonium ions of saccharides. This method confirmed previously identified glycan structures based on existing knowledge of rt-PA glycans. In addition, we identified two novel glycan structures in rt-PA. A low level of Asn142 N-glycosylation was detected at an atypical Asn-Xaa-Cys consensus motif. It was found to be modified predominantly by biantennary hybrid structures. This N-glycosylation site was confirmed using a recently developed electron-transfer dissociation (ETD) technique. Also using this method, we detected low levels of elongation of fucose-O-Thr61 to di-, tri-, and tetrasaccharides, not previously observed in rt-PA. The results demonstrate that use of state-of-the-art analytical methods can reveal low-level, previously undetected modifications of well-characterized biopharmaceuticals.