Clarence Lew

Rapid Magnetic Bead Based Sample Preparation for Automated and High Throughput N-Glycan Analysis of Therapeutic Antibodies

Full automation to enable high throughput N-glycosylation profiling and sequencing with good reproducibility is vital to fulfill the contemporary needs of the biopharmaceutical industry and requirements of national regulatory agencies. The most prevalently used glycoanalytical methods of capillary electrophoresis and hydrophilic interaction liquid chromatography, while very efficient, both necessitate extensive sample preparation and cleanup, including glycoprotein capture, N-glycan release, fluorescent derivatization, purification, and preconcentration steps during the process. Currently used protocols to fulfill these tasks require multiple centrifugation and vacuum-centrifugation steps, making liquid handling robot mediated automated sample preparation difficult and expensive. In this paper we report on a rapid magnetic bead based sample preparation approach that enables full automation including all the process phases just in a couple of hours without requiring any centrifugation and/or vacuum centrifugation steps. This novel protocol has been compared to conventional glycan sample preparation strategies using standard glycoproteins (IgG, fetuin, and RNase B) and featured rapid processing time, high release and labeling efficiency, good reproducibility, and the potential of easy automation.

Semiconductor laser-induced fluorescence detection in capillary electrophoresis using a cyanine dye

Cy5, an activated carboxyl cyanine fluorophore, was characterized by capillary electrophoresis (CE) using a semiconductor laser at 652 nm to induce fluorescence. Hydrolysis of the activated Cy5 in the presence of ammonia results in the formation of a mono- and diamide and a dicarboxylic acid. A Cy5-labeled oligonucleotide M13 primer for DNA sequencing (M13mp18 template) was synthesized with a purity of better than 95%. The labeled primer was analyzed by liquid chromatography, using UV-visible detection, and by CE, monitored by laser-induced fluorescence (LIF) detection. Analysis of the Cy5-labeled oligonucleotide primer by CE-LIF in a 9% polyacrylamide gel-filled capillary indicated the purity of the major Cy5-oligonucleotide primer was greater than 90%. The detection sensitivity for Cy5-based CE-LIF detection system with a 2.5-mW red semiconductor laser is about 10(-10) M.

Rapid level-3 characterization of therapeutic antibodies by capillary electrophoresis electrospray ionization mass spectrometry

With the increase of the number of approved protein therapeutics in the market, comprehensive and reproducible characterization of these new generation drugs is crucial for the biopharmaceutical industry and regulatory agencies. One of the largest groups of biotherapeutics is monoclonal antibodies (mABs) possessing various posttranslational modifications and potential degradation hotspots during the manufacturing process that may affect efficacy and immunogenicity. The exceptionally high separation power of capillary electrophoresis (CE) in conjunction with mass spectrometry fulfills Level-3 characterization requirements necessary to reveal such modifications and degradations. In this paper, a comprehensive characterization example will be given for a representative mAB Trastuzumab (Herceptin), illustrating the benefits of the integration of CE and electrospray ionization in a unified bioanalytical process coupled with high-resolution mass spectrometry. Peptides separated in a wide size range (3-65 amino acids) were identified with 100% sequence coverage and quantified, including degradative hotspots such as glutamic acid cyclization, methionine oxidation, aspargine deamidation and C-terminal lysine heterogeneity using only 100 fmol of a single protease digest sample. The low flow rate of the system (>20 nL/min) ensured maximized ionization efficiency and dramatically reduced ion suppression.

Fully Automated Sample Preparation for Ultrafast N-Glycosylation Analysis of Antibody Therapeutics

There is a growing demand in the biopharmaceutical industry for high-throughput, large-scale N-glycosylation profiling of therapeutic antibodies in all phases of product development, but especially during clone selection when hundreds of samples should be analyzed in a short period of time to assure their glycosylation-based biological activity. Our group has recently developed a magnetic bead–based protocol for N-glycosylation analysis of glycoproteins to alleviate the hard-to-automate centrifugation and vacuum–centrifugation steps of the currently used protocols. Glycan release, fluorophore labeling, and cleanup were all optimized, resulting in a <4 h magnetic bead–based process with excellent yield and good repeatability. This article demonstrates the next level of this work by automating all steps of the optimized magnetic bead–based protocol from endoglycosidase digestion, through fluorophore labeling and cleanup with high-throughput sample processing in 96-well plate format, using an automated laboratory workstation. Capillary electrophoresis analysis of the fluorophore-labeled glycans was also optimized for rapid (<3 min) separation to accommodate the high-throughput processing of the automated sample preparation workflow. Ultrafast N-glycosylation analyses of several commercially relevant antibody therapeutics are also shown and compared to their biosimilar counterparts, addressing the biological significance of the differences.

Multi-Site N-glycan mapping study 1: Capillary electrophoresis – laser induced fluorescence

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.