Louisette J. Basa

Identification and structural analysis of the tetrasaccharide NeuAc.alpha.(2.fwdarw.6)Gal.beta.(1.fwdarw.4)GlcNAc.beta.(1.fwdarw.3)Fuc.alpha.1.fwdarw.O-linked to serine 61 of human factor IX

O-Linked fucose has been found attached to Thr/Ser residues within the sequence Cys-X-X-Gly-Gly-Thr/Ser-Cys in the N-terminal EGF domains of several coagulation/fibrinolytic proteins. Carbohydrate composition and mass spectrometric analyses of tryptic and thermolytic peptides containing the corresponding site (Ser-61) in the first EGF domain of human factor IX indicated the presence of a tetrasaccharide containing one residue each of sialic acid, galactose, N-acetylglucosamine, and fucose. The Ser-61 tetrasaccharide was not susceptible to alpha-fucosidase digestion. Fragments generated during mass spectrometric analysis indicated that fucose was the attachment sugar residue. The involvement of fucose in the carbohydrate-peptide linkage was confirmed by two-dimensional 1H NMR spectroscopic analysis of the glycopeptide containing factor IX residues 57-65. The complete structure of the tetrasaccharide was obtained by methylation analysis and two-dimensional 1H TOCSY and ROESY experiments as NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Fuc alpha 1-->O-Ser61.

Drug-to-antibody ratio (DAR) and drug load distribution by LC-ESI-MS.

This chapter describes an LC-ESI-MS method for the DAR and drug load distribution analysis that is suitable for lysine-linked ADCs. The ADC sample is desalted using a reversed-phase LC column with an acetonitrile gradient prior to online MS analysis. The MS spectrum is processed (deconvoluted) and converted to a series of zero charge state masses that corresponds to the increasing number of drugs in the ADC. Integration of the mass peak area allows the calculation of the DAR and drug load distribution of ADCs.

Metal ion interactions with mAbs: Part 1

Fragmentation in the hinge region of an IgG1 monoclonal antibody (mAb) can affect product stability, potentially causing changes in potency and efficacy. Metals ions, such as Cu2+, can bind to the mAb and undergo hydrolysis or oxidation, which can lead to cleavage of the molecule. To better understand the mechanism of Cu2+-mediated mAb fragmentation, hinge region cleavage products and their rates of formation were studied as a function of pH with and without Cu2+. More detailed analysis of the chemical changes was investigated using model linear and cyclic peptides (with the sequence of SCDKTHTC) derived from the upper hinge region of the mAb. Cu2+ mediated fragmentation was determined to be predominantly via a hydrolytic pathway in solution. The sites and products of hydrolytic cleavage are pH and strain dependent. In more acidic environments, rates of Cu2+ induced hinge fragmentation are significantly slower than at higher pH. Although the degradation reaction rates between the linear and cyclic peptides are not significantly different, the products of degradation vary. mAb fragmentation can be reduced by modifying His, which is a potential metal binding site and a known ligand in other metalloproteins. These results suggest that a charge may contribute to stabilization of a specific molecular structure involved in hydrolysis, leading to the possible formation of a copper binding pocket that causes increased susceptibility of the hinge region to degradation.

IDENTIFICATION AND CHARACTERIZATION OF GLYCOPEPTIDES IN TRYPTIC MAPS BY HIGH-PH ANION-EXCHANGE CHROMATOGRAPHY

Publisher Summary This chapter describes the application of high-pH anion-exchange chromatography, with pulsed amperometric detection (HPAEC-PAD) to the identification and partial characterization of glycopeptides, within a reversed-phase high-performance liquid chromatography (HPLC) peptide map. HPAEC is a versatile chromatographic method that can be used for the detection and quantitation of monosaccharides, released by acid hydrolysis and for the analysis of intact oligosaccharides released enzymatically or chemically. A large number of other analytical methods, including mass spectrometry (MS), enzymatic microsequencing, nuclear magnetic resonance (NMR), and capillary electrophoresis, can also be applied to the characterization of glycopeptides; in general, a combination of several complementary analytical methods are needed to achieve a definitive carbohydrate structure determination. This chapter discusses the type of information that can be obtained, using a single, comparatively simple, analytical method.

Structural Characterization of a Novel Tetrasaccharide Attached to Ser-61 of Human Factor IX by Mass Spectrometry and 1H NMR Spectroscopy

Publisher Summary This chapter explores the structural characterization of a novel tetrasaccharide attached to ser-61 of human factor ix by mass spectrometry and 1H NMR spectroscopy. In a study described in the chapter, trypsin-digested S-carboxymethylated human plasma factor IX was prepared. No mass was observed that corresponded to any of the expected forms of the factor IX 44-80 tryptic peptide. The 44-80 peptide sequence is QYVDGDQCE S NPCLNGG S CK D DINSYECWCPFGFEGK. Tryptic RP-HPLC fractions were then subjected to amino acid analysis to identify a doublet peak containing the 44–80 peptides. The mass obtained for this fraction indicated the presence of some unknown modification that added 801.2 amu to the expected glycopeptide mass, far in excess of the 146.2 amu indicative of O-linked fucose. Digestion of the 44–80 peptides with thermolysin produced fragments demonstrating that the unknown -802 amu modification was found within residues 57–65. Digestion with α-fucosidase had no effect on the 57–65 glycopeptide, and the ES–MS fragmentation pattern indicated that fucose was present at the reducing end.