Protein Characterization by MS in the Pharmaceutical Industry

Abstract

SummaryLC-MS based methodology for the rapid identification of congeners in preparations of the protein r-hirudin sequence variant 1 (rHV1, structure I) was developed. On-line coupling of reversed phase HPLC with electrospray ionization mass spectrometry (LC-ESMS) was found sufficiently sensitive to allow direct detection of such congeners at low concentrations in batch samples despite the presence of the large bulk of r-HV1 and the relatively high concentration of ammonium acetate necessary as a buffer system.Routine fingerprinting of congeners by LC-MS required full structural characterization of the modified site, carried out beforehand on sufficient amounts of off-line HPLC-isolated material. For this task, smaller peptides that better exposed their structural detail were produced by combining chemical reactions (reduction of disulfide bonds, pyridylethylation of free SH groups) with enzymatic digestion (Glu-C, Asp-N or trypsin). Peptides carrying structural modifications were either identified in ESIvIS ‘maps’ by changes in mass relative to unmodified peptides in reference digests or, when isobaric, by changes in retention behavior in HPLC. In general, the modified peptides were subsequently sequenced by tandem mass spectrometry (ES-MSMS) in order to define and locate the sites in question. In a few cases MSMS was ineffective and sequencing by automated Edman degradation was employed.LC-ESMS on representative batches established the presence of low concentrations of two isoforms of rHV1 in addition to the bulk component, of at least two monodehydration products, and of several mono-and dihexosyl derivatives. As illustrated for the off-line isolated Asp33 isoform of rHV1 (r[iso-Asp33]-HV1) α→β conversion at the isomerized site can be deduced from the formation of a highly diagnostic β-specific sequence ion in low-energy collision induced dissociation. The two dehydration products were characterized as r[succinimide33]-and r[succinimide53]-HV1 after ring opening at the respective anhydro-positions with H2 18O (discussed in detail for the 53-isomer). For two of the monohexosylated congeners O-mannosylation at Thr4 (r[Man-Thr4]-HV1) or, alternatively, Thr7 (r[ManThr7]-HV1) was established by consecutive enzymatic digestions using Glu-C and Asp-N, followed by ESMS, ESMSMS, Edman degradation and monosaccharide analysis. The third isomer was O-hexosylated at Thr45 (r[Man-Thr45]-HV1). In one of the dihexosylated homologues (r[ManThr4, Man-Thr7]-HV1) both Thr4 and Thr7 carried separate monosaccharide residues in contrast to its isomer (r[Man 2 -Thr4]-HV1) substituted only at the Thr4 position, but carrying a disaccharide residue.