Rapid, Automated Sample Preparation for Peptide Mapping of Antibodies and Recombinant Proteins
an abstract for the poster to be presented at the BioProcess International Conference & Exhibition (October 31–November 4, 2011) in Long Beach, CA
Abstract
The recent dramatic improvements in cell culture expression levels have a potential consequence of significant changes in post-translational modification (PTM) patterns of the product. Peptide mapping (using HPLC or LC/MS for analysis) is the core method for monitoring changes in PTM, and demand for this type of product characterization is increasing. The complex sample preparation process for peptide mapping includes purification of the target protein, denaturation, reduction and alkylation, enzymatic digestion and peptide cleanup, which can become a major bottleneck and source of variability.
The AssayMAP® high throughput microchromatography platform enables full automation of the complex, multi-step, peptide mapping workflow. The system utilizes 5 µL packed bed cartridges run on a 96-channel liquid handler (Agilent AssayMAP Bravo), which provides precise positive displacement flow control through a special probe syringe head. Cartridge types include affinity purification (using protein A or immobilized antibodies as affinity ligands), immobilized trypsin (or other enzymes) and peptide cleanup (by reversed-phase, ion exchange or other resins). Because of the flow rate control, binding, elution and digestion reactions are highly quantitative and reproducible.
This poster will demonstrate the use of the system with both monoclonal antibodies and non-antibody proteins, showing high throughput and fully automated purification from complex feedstreams, digestion and cleanup.
Take-home Message
A complete sample preparation workflow for peptide mapping, (including purification, digestion and cleanup) can be fully automated in a high throughput format.