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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: MoP-071
Session: Glycomics and Oligosaccharides
Presentation date: Mon, Aug 28, 2006
Presentation time: 09:50 – 11:20

Quality Control of Intact Proteins and Antibodies by ESI-TOF

Kjell Andersson1, Matthias Pelzing2, Christian Neusuess2

1 Bruker Daltonics Scandinavia AB, Taby, Sweden
2 Bruker Daltonik, Bremen, Germany

Correspondence address: Kjell Andersson, Bruker Daltonics Scandinavia AB, Polygonvagen 79, Taby, SE-187 66 Sweden.

Web site: http://www.bdal.de

Keywords: Electrophoresis Capillary; Glycoproteins; Mass Spectrometry, Time of Flight; Protein.

Novel aspect: Fast and information-rich glycoprotein characterization by ESI-TOF MS after appropriate separation by LC or CZE is presented.

 

An increasing number of drugs are based on recombinant proteins. Particularly antibodies are expected to be of significant importance in future biopharmaceutical developments. However, there is a lack of fast methods, which are appropriate for both quality control and for development support of these complex molecules. This is primarily due to the often observed microheterogeneity of posttranslational modified proteins, especially glycosylation. These complexities in addition to the observed charge envelope in ESI MS prevent often a direct analysis by MS. Thus, separation is required, though difficult to achieve due to variable properties of proteins.

An orthogonal-accelerated ESI-TOF MS at a resolution of 15000 is used for obtaining spectra of different glycoproteins as well as intact antibodies, applied both intact and reduced.
Separation is achieved in various matters:
• An LC-based approach using a C8 column at 60°C in order to obtain clear spectra for intact antibodies as well as for the separation of light and heavy chain after reduction.
• A CE approach using acetic acid as background electrolyte and a new dynamic polyacrylamide-based coating to separate glycoforms of proteins like Ribonuclease B, fetuin, alpha-acid glycoprotein or erythropoietin.
• A fast CE-MS method for the characterization of non-derivatized glycans has been developed to support the protein data.

The mass accuracy for the isotopically resolved small proteins (≤ 15-17kDa) is better than 5 ppm, for non-isotopically resolved mass spectra better than 1 Da. Even intact antibodies (140-150kDa) could be characterized with a reproducibility of a few Dalton allowing the determination of glycosylated isoforms. The LC separation enabled both the sensitive characterization of intact antibodies as well as of the light and heavy chains. The CE-MS approach allows the separation of glycoforms, differing in its sialic acid content. Moreover, even small mass/size changes without introducing a charge (as repeats of hexose-N-acetylhexosamines) could be separated. Supplementary analysis of the enzymatically released unmodified glycans was performed by a newly developed method by CE-MS. In this way, the amount and composition (sialic acids, HexHexNAc repeats) of the N-glycans have been obtained. Moreover, common glycan modifications as acetylations, NeuAc-NeuGc exchanges, sulfations and OH-NH2 exchanges have been successfully characterized. The method has been proven to be simple and sensitive since glycans that were not described previously (4Ant 5SiA) have been detected for erythropoietin. Based on the carbohydrate information on the glycan level an integral carbohydrate composition for each intact glycoform can be assigned with high confidence.