d(GC) - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 6 (2) 229-240 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)
Chemical Modification of Recombinant Human Interferon
Beta-1a Using Linear and Branched mPEGs
Ahmad Abolhasani 1 , Sameereh Hashemi-Najafabadi 1* , Mahvash Khodabandeh Shahraki *2
and Zohreh-Azita Sadigh 3
1 Biotechnology Group, Department of Chemical Engineering, Tarbiat Modares University,
P. O. Box: 14115/114, Tehran, I. R. Iran
2 National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, I. R. Iran
3 Razi Vaccine and Serum Research Institute, Tehran, I. R. Iran
* For correspondence - s.hashemi@modares.ac.ir
Abstract
Interferon-beta-1a (IFN-β-1a) is used
clinically in the treatment of multiple sclerosis.
Similar to other biological molecules, IFN-β-1a
has a relatively short serum half-life and is rapidly
detected by the host’s immune system.
PEGylation is a common approach to increase
the blood circulation time of therapeutic proteins.
In the present study, IFN-β-1a was PEGylated
using linear methoxy polyethylene glycols
(mPEGs) with molecular weights of 5 and 20 kDa
and also 40 kDa branched mPEG-SPA. Prior to
PEGylation, the mPEGs were activated by
succinimidyl propionic acid (SPA). PEGylation
was evaluated by size-exclusion HPLC (SE-
HPLC) and Ninhydrin method. In the designed
experiments, the factors of mPEG molecular
weight, pH, and the molecular ratio of protein to
mPEG fractions were studied. The results were
analyzed using Design-Expert statistical software
and the significant factors were determined.
Then, in order to find the optimum conditions,
Taguchi method (L 9 array) was used by
considering the significant factors. Consequently,
the optimum conditions for PEGylation, using 20
kDa linear mPEG-SPA was found to be at pH 8
and the protein to mPEG molar ratio of 1/40. The
extents of protein modification were obtained
45.5% and 46.8% by HPLC and Ninhydrin
methods, respectively. Optimum PEGylation with
40 kDa branched mPEG-SPA was obtained at
pH 8 and protein/mPEG of 1/40. In this case, the
Interferon Beta-1a PEGylation
229
extents of protein modification were obtained
46.5% and 47.7% by HPLC and Ninhydrin
methods, respectively. The biological activity test
showed that the PEGylated protein retained
about 80% of its activity, were compared to that
of the unmodified protein.
Key words: Beta interferon, Biological activity,
mPEG-SPA, PEGylation, Taguchi method.
Introduction
Interferons (IFNs) are a family of cytokines
that mediate antiviral, antiproliferative and
immunomodulatory effects in response to
biological and chemical stimuli. Two types of IFNs
are recognized based on their physical and
biological properties; type I which contains IFNsα,
-β, -κ, -τ, and -ω, and type II the only member
of which is IFN-γ (1-5). Recombinant forms of
IFN-β-1a (Avonex, Biogen Idec, Rebif and
Serono) and IFN-β-1b (Betaferon, Schering AG)
have been approved for the treatment of multiple
sclerosis (MS), while non-recombinant forms of
IFN-β (e.g., Feron and Toray) have been
approved in Japan for the treatment of chronic
viral hepatitis C (HCV). Therapeutic proteins have
poor pharmacokinetic profiles because of their
rapid clearance from blood circulation.
Polyethylene glycol (PEG) attachment to the
proteins offers a solution to these problems. The
antiviral and immunomodulator drug, IFN-β-1a,
is a common example (6).