title: Characterization of recombinant human C1 inhibitor secreted in milk of transgenic rabbits.
abstract: C1 inhibitor (C1INH) is a single-chain glycoprotein that inhibits activation of the contact system of coagulation and the complement system. C1INH isolated from human blood plasma (pd-hC1INH) is used for the management of hereditary angioedema (HAE), a disease caused by heterozygous deficiency of C1INH, and is a promise for treatment of ischemia-reperfusion injuries like acute myocardial or cerebral infarction. To obtain large quantities of C1INH, recombinant human C1INH (rhC1INH) was expressed in the milk of transgenic rabbits (12 g/l) harboring genomic human C1INH sequences fused to 5' bovine αS(1) casein promoter sequences. Recombinant hC1INH was isolated from milk to a specific activity of 6.1 U/mg and a purity of 99%; by size-exclusion chromatography the 1% impurities consisted of multimers and N-terminal cleaved C1INH species. Mass spectrometric analysis of purified rhC1INH revealed a relative molecular mass (M(r)) of 67,200. Differences in M(r) on SDS PAGE and mass spectrometric analysis between rhC1INH and pd-hC1INH are explained by differential glycosylation (calculated carbohydrate contents of 21% and 28%, respectively), since protein sequencing analysis of rhC1INH revealed intact N- and C-termini. Host-related impurity analysis by ELISA revealed trace amounts of rabbit protein (approximately 10 ppm) in purified batches, but not endogenous rabbit C1INH. The kinetics of inhibition of the target proteases C1s, Factor XIIa, kallikrein and Factor XIa by rhC1INH and pd-hC1INH, indicated comparable inhibitory potency and specificity. Recently, rhC1INH (Ruconest(®)) has been approved by the European Medicines Agency for the treatment of acute attacks of HAE.
authors.affiliation: Pharming Technologies B.V., Leiden, The Netherlands. h.veen@pharming.com
authors.forename: Harrie A
authors.initials: HA
authors.lastname: van Veen
authors.forename: Jaco
authors.initials: J
authors.lastname: Koiter
authors.forename: Carla J M
authors.initials: CJ
authors.lastname: Vogelezang
authors.forename: Noucha
authors.initials: N
authors.lastname: van Wessel
authors.forename: Tijtje
authors.initials: T
authors.lastname: van Dam
authors.forename: Ingeborg
authors.initials: I
authors.lastname: Velterop
authors.forename: Kristina
authors.initials: K
authors.lastname: van Houdt
authors.forename: Luc
authors.initials: L
authors.lastname: Kupers
authors.forename: Danielle
authors.initials: D
authors.lastname: Horbach
authors.forename: Mourad
authors.initials: M
authors.lastname: Salaheddine
authors.forename: Jan H
authors.initials: JH
authors.lastname: Nuijens
authors.forename: Maurice L M
authors.initials: ML
authors.lastname: Mannesse
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle; D003174:Complement C1 Inactivator Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D050718:Complement C1 Inhibitor Protein; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007700:Kinetics; D007774:Lactation; D008892:Milk / Q000737:chemistry*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D003174:Complement C1 Inactivator Proteins; D050718:Complement C1 Inhibitor Protein; D011994:Recombinant Proteins; C469952:SERPING1 protein, human