title: The effect of posttranslational modifications on the in vitro activity of recombinant human thyroid-stimulating hormone.
abstract: Posttranslational modification can influence the biologic activity of recombinant proteins. The effects of beta-subunit C-terminal truncation, oligosaccharide heterogeneity, and chemical oxidation on the in vitro activity of recombinant human thyroid-stimulating hormone (rhTSH) were investigated. beta-Subunit C-terminal truncation up to residue 113 did not effect the in vitro activity of the hormone. The relationship between the heterogeneity of oligosaccharide structures on rhTSH and specific activity of the glycoprotein hormone was also examined. Oligosaccharide profiles were generated for preparations of rhTSH containing similar sialic acid levels. A weak correlation was observed between relative levels of monosialylated biantennary, bisialylated biantennary, and trisialylated triantennary oligosaccharide species and in vitro activity of the recombinant hormone (p < 0.05). To examine the effect of chemically induced methionine oxidation on the activity of rhTSH, the hormone was treated with tert-butyl hydroperoxide and then characterized. Using peptide mapping and mass spectrometry, the degree of oxidation of the five methionine residues within rhTSH was measured. Met-71 in the alpha-subunit was the most susceptible to oxidation whereas Met-9 in the beta-subunit was the most resistant. Also, after tert-butyl hydroperoxide treatment, levels of oxidation of Met-32 in the beta-subunit, and Met-29 and Met-47 in the alpha-subunit were less than half of that observed for Met-71. The in vitro activity of rhTSH initially declined with increasing oxidation; however, the loss in activity plateaued at approximately 50% of the control sample activity. In summary, despite the possible effects that posttranslational modifications may have on the bioactivity of a protein, a limited degree of variation in bioactivity was observed for the rhTSH preparations described in this study.
authors.affiliation: Therapeutic Protein Research and Development, Genzyme Corporation, Framingham, Massachusetts 01701, USA. rebecca.sendak@genyme.com
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Sendak
authors.forename: Chandrashekar
authors.initials: C
authors.lastname: Ganesa
authors.forename: Karen L
authors.initials: KL
authors.lastname: Lee
authors.forename: John J
authors.initials: JJ
authors.lastname: Harrahy
authors.forename: Roger
authors.initials: R
authors.lastname: Théberge
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Morgan
authors.forename: Edward S
authors.initials: ES
authors.lastname: Cole
authors.forename: Leonard D
authors.initials: LD
authors.lastname: Kohn
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Mattaliano
mesh_terms: D002853:Chromatography, Liquid; D002942:Circular Dichroism; D005453:Fluorescence; D006801:Humans; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000032:analysis; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013972:Thyrotropin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D013972:Thyrotropin; D020122:tert-Butylhydroperoxide