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Fast QC of Intact Monoclonal Antibodies Using MALDI-Top-Down Sequencing

By R. Paape, L. Vorwerg, D. Suckau and A. Resemann

Abstract

Antibodies are playing an important role in drug development in the last years. The function of therapeutic monoclonal antibodies (mAbs) is directly depending on their structure including terminal modifications like C-terminal lysine excision and N-terminal pyroglutamylation of the heavy chain. Here, we describe a method for very fast and simple validation of the terminal sequences of heavy and light chain of intact monoclonal antibodies using MALDI-Top-Down-Sequencing (MALDITDS) using in-source decay (ISD). Intact mAb (IgG) in a concentration of 1 mg to 5 mg/ml was directly mixed with matrix solution without prior reduction and alkylation or separation of the different chains. We used 1,5-diaminonaphtalene (DAN) as matrix because of its reductive and ISD enhancing properties. The samples were mixed with matrix solution and dried at ambient air on the MALDI sample plate. In a second experiment, the same samples were prepared using sDHB (super DHB), which is known to be a good ISD matrix but without reduction capacity. The samples in sDHB didn't generate any ISD fragmentation due to the intact disulfide crosslinks in IgG. In DAN, the same samples generated rich ISD spectra containing fragments from both the heavy and the light chain of the antibodies. The ISD spectra permitted reading through the cysteine residues in the sequences implicating the disulfide bridges were reduced by DAN. Up to 60 residues of the different chains of various mAbs were matched and validated, respectively. A dedicated software tool allowed a very fast interpretation of the spectra. Overall the method takes only a few minutes from sample preparation, spectrum acquisition and processing to the result in form of sequence annotations in the ISD spectra

Topics: Poster Session Abstracts
Publisher: Association of Biomolecular Resource Facilities
OAI identifier: oai:pubmedcentral.nih.gov:3186624
Provided by: PubMed Central
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