ABSTRACT
The glycan moiety of human recombinant gonadotrophins (r-hFSH, r-hLH, and r-hCG) produced in CHO cell lines has been characterized by a combination of chromatographic and mass spectrometric techniques, including both matrix-assisted laser desorption ionization and electrospray. Two glycan mapping methods have been developed for the three gonadotrophins that allow separation of the glycans according to either their charge or sialylation level or their antennarity. A method was also developed for r-hCG that permits the complete resolution of the N-glycan from the O-glycan species. Whereas the structure found for the N-glycans of the gonadotrophins was in agreement with the complex type model, the structure for an O-glycan of r-hCG, not yet described, has been unambiguously determined using nanoelectrospray ion trap mass spectrometry. Using these two glycan mapping methods, the high level of batch-to-batch consistency achieved for the glycosylation of the three recombinant gonadotrophins in commercial production has been shown. These data demonstrate the tight control that can be achieved in the manufacturing of complex recombinant therapeutic glycoproteins, which is a prerequisite to the delivering of a guaranteed dose of drug from vial to vial, and in turn to ensuring the clinical efficacy of the product.
Subject(s)
Gonadotropins/biosynthesis , Gonadotropins/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Cricetinae , Glycosylation , Gonadotropins/metabolism , Humans , Molecular Conformation , Molecular Sequence Data , Polysaccharides/chemistry , Polysaccharides/isolation & purification , Recombinant Proteins/metabolism , Reproducibility of Results , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Static ElectricityABSTRACT
Divercin V41 is a new bacteriocin produced by Carnobacterium divergens V41, a lactic acid bacterium isolated from fish viscera. The amino acid sequence of divercin V41 showed high homologies with pediocin PA-1 and enterocin A. Two disulphide bonds were present in the hydrophilic N-terminal domain and in the highly variable hydrophobic C-terminal domain, respectively. A DNA probe designed from the N-terminal sequence of the purified peptide was used to locate the structural gene of divercin V41. A 6 kb chromosomal fragment containing the divercin V41 structural gene (dvnA) was cloned and sequenced. The results indicate that divercin V41 is synthesized as a pre-bacteriocin of 66 amino acids. The 23-residue N-terminal extension is cleaved off to yield the mature 43-amino-acid divercin V41. In addition, the fragment encodes putative proteins commonly found within bacteriocin operons, including an ATP-dependent transporter, two immunity-like proteins and the two components of a lantibiotic-type signal-transducing system. The genetic organization of the fragment suggested important gene rearrangements.