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Characterization of recombinant human C1 inhibitor secreted in milk of transgenic rabbits

Paper ID Volume ID Publish Year Pages File Format Full-Text
23641 43459 2012 8 PDF Available
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 αS1 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 (Mr) of 67,200. Differences in Mr 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.

► Transgenic rabbits expressed high levels of recombinant human C1 inhibitor in milk. ► A highly purified rhC1INH product was obtained from transgenic rabbit milk. ► Milk- and human plasma-derived C1INH displaced similar protease inhibition kinetics. ► rhC1INH is approved by the EMA for treatment of hereditary angioedema attacks.

Keywords
C1INH, C1 inhibitor; pd-hC1INH, human plasma-derived C1INH; rhC1INH, recombinant human C1INH; rhC1INH-Thr85 and -Thr97, rhC1INH lacking Asn1-Thr84 and Asn1-Thr96, respectively; HAE, hereditary angioedema; Ruconest®, rhC1INH approved in the EU for treatmen
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 162, Issues 2–3, 31 December 2012, Pages 319–326
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us