16
August/September 2011
The mAb-Glyco Chip Kit – a Workflow Solution for Rapid and Fully Automated Characterisation of N-linked Glycans from Monoclonal Antibodies
Lukas Trojer1(2) , Kirill Gromadski1(2) , Tom van de Goor1 and Stephan Buckenmaier1 *
*Corresponding author. (1) Agilent Technologies R&D and Marketing GmbH & Co. KG, Hewlett-Packard-Str. 8, 76337 Waldbronn, Germany (2) New adress: Sandoz GmbH, Biochemiestr. 10, 6250 Kundl, Austria
Monoclonal antibodies (mAbs) are an important class in the group of new biological entities with about 30 antibody drugs licensed for treatment of various diseases [1]. These glycoproteins bear complex oligosaccharide moieties within their structure, whose presence, absence, and profile can have significant impact on therapeutic efficacy, pharmacokinetics, and immunogenicity. Glycosylation is influenced by many factors such as the cell line in which the mAb is produced as well as specific production parameters including pH or temperature. Thus, characterisation of glycan profiles is of vital importance throughout the various phases of drug development.
The challenge for the analytical chemist is that conventional methods utilised are complex and time consuming. For instance, a typical workflow could involve multiple sample preparation steps: Enzymatic deglycosylation of mAbs, hydrolysis of glycosylamines to free reducing end glycans, and (dependent on the detection method) the labelling of glycans with fluorescent tags. Glycan analyses apply capillary electrophoresis or HPLC with fluorescence and/or MS detection, or MALDI-TOF-MS. Together with data acquisition and processing the chemist is looking at one half to several days to complete the analysis [2]. In addition, if sample preparation is done manually, each step exhibits a potential source of error.
With this in mind the mAb-Glyco Chip Kit was developed. It is a complete workflow solution designed to characterise N-linked glycans on mAbs. Major development goals were to fully automate the workflow, to reduce the complexity of sample preparation, data acquisition and data processing, and to substantially increase sample throughput. This article provides a technical description of this solution and explains how it can aid in removing a major bottleneck during the development phase of these biological drugs.
Figure 1: Rotor-in-rotor valve and chip design of the mAb-Glyco Chip incorporating (a) enzyme reactor, (b) glycan enrichment column, (c) glycan separation column, and (d) nano electrospray tip. OR = outer rotor, IR = inner rotor
mAb-Glyco Chip Kit
The Kit contains mAb-Glyco Chip, reagent pack and a content disk. mAb-Glyco Chip and its function will be described below. The reagent pack provides chemicals needed for ready chip operation: System Conditioning Reagent for flow path coating and carry over minimisation, Glycan Standards for chromatographic checkout and method development, Antibody Standard (IgG type mAb spiked with stable internal standard, a free reducing end glycan) for functional checkout of the chip, and Deglycosylation Buffer for dilution of standards, samples and for loading the mAb samples onto the chip’s
PNGase F enzyme reactor (see below). The content disk has optimised methods for glycan profiling analysis and data processing, including efficiency tools such as glycan accurate mass and structure database, and reporting templates. More details in [3].
Instrumental setup All analyses were run on the Agilent 1260 Infinity HPLC-Chip/MS system comprising micro autosampler with thermostat (set to 4°C), capillary and nanoflow pump with micro degasser, and the Chip-Cube that interfaces LC modules and the MS instrument [4-6]. HPLC-grade H2O [5mM formic acid (FA)] and
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