Expression, purification, and characterization of rat interferon-beta, and preparation of an N-terminally PEGylated form with improved pharmacokinetic parameters.
To identify potential new clinical uses and routes of administration for human interferon-beta-1a (IFN-beta-1a), we have developed an expression and purification procedure for the preparation of highly purified rat interferon-beta (IFN-beta) suitable for testing in rat models of human disease. An expression vector containing the rat IFN-beta signal sequence and structural gene was constructed and transfected into Chinese hamster ovary (CHO) cells. The protein was purified from CHO cell conditioned medium and purified to > 99.5% purity using standard chromatographic techniques. Analytical characterization indicated that the protein was a heavily glycosylated monomeric protein, with two of the four predicted N-glycosylation sites occupied. Analysis of the attached oligosaccharides showed them to be a complex mixture of bi-antennary, tri-antennary, and tetra-antennary structures with a predominance of sialylated tri-antennary and tetra-antennary structures. Peptide mapping, N-terminal sequencing, and mass spectrometry confirmed the identity and integrity of the purified protein. The purified protein had a specific activity of 2.1x10(8)U/mg when assayed on rat RATEC cells, which is similar in magnitude to the potencies observed for murine IFN-beta and human IFN-beta-1a assayed on murine and human cells, respectively. We also prepared an N-terminally PEGylated form of rat IFN-beta in which a 20 kDa methoxy polyethylene glycol (PEG)-propionaldehyde was attached to the N-terminal alpha-amino group of Ile-1. The PEGylated protein, which retained essentially full in vitro antiviral activity, had improved pharmacokinetic parameters in rats as compared to the unmodified protein. Both the unmodified and PEGylated forms of rat IFN-beta will be useful for testing in rat models of human disease.
Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA 02142, USA.
Robert M
RM
Arduini
Zhifang
Z
Li
Alan
A
Rapoza
Robert
R
Gronke
Donna M
DM
Hess
Dingyi
D
Wen
Konrad
K
Miatkowski
Caroline
C
Coots
Azita
A
Kaffashan
Nelly
N
Viseux
Jeannine
J
Delaney
Bruno
B
Domon
Carmen N
CN
Young
Raymond
R
Boynton
Ling Ling
LL
Chen
Liqing
L
Chen
Matthew
M
Betzenhauser
Stephan
S
Miller
Alan
A
Gill
R Blake
RB
Pepinsky
Paula S
PS
Hochman
Darren P
DP
Baker
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D007370:Interferon Type I / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000068556:Interferon beta-1a; D016899:Interferon-beta / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry*; D051381:Rats; D011994:Recombinant Proteins
D007370:Interferon Type I; D009844:Oligosaccharides; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D016899:Interferon-beta; D000068556:Interferon beta-1a