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)
Figure 3 (b-e) show intact HeLa cells which
have normal shapes and sizes. Figure 3f shows
the biological activity of HeLa cells in the
presence of virus and absence of IFN-β. In the
absence of IFN-β the cells were detected at the
bottom of the microplate (indicating the cytopathic
effect of virus). In Table 7, by considering image
b as representing 100% protein biological
activity and addition of the virus to the growth
medium, IFN-β biological activity decreases by
about 70% (image c).
By PEGylating the protein, its biological
activity, as compared to image b, has decreased
by about 40% (image d). Then, by adding virus
to the growth medium it is obvious that the IFNβ
biological activity in image e, compare to image
c, has calculated about 80%. The studies that
have been published regarding PEGylated IFNα-2b
(PEG-Intron) and PEGylated IFN-α-2a
(Pegasys) support our observation that nontargeted
PEGylation has a deleterious effect on
the activity of interferons. In the case of PEG-
Intron, reaction involving succinimidyl carbonate
is used to attach the linear 12 kDa PEG (35),
while for Pegasys an N-hydroxysuccinimide ester
derivative of the branched 40 kDa PEG was used
to modify the protein [13]. The use of such
chemical reactors has resulted in the modification
of numerous individual sites as well as a
significant reduction in the in vitro specific antiviral
activity.
With regard to the PEG-Intron, lysine,
tyrosine, histidine, serine, and cysteine residues
are modified, resulting in 14 different
monoPEGylated positional isomers. The antiviral
activity of the mixture is 28% of that of the
unmodified protein and ranges from 6 to 37%
for the individual species (36). In the case of
Pegasys, only lysine residues are modified,
resulting in 6 different monoPEGylated positional
isomers, with the mixture having an antiviral
activity of only 7% of the unmodified protein (13).
For rat IFN-β, the PEGylated protein, which
retains essentially full in vitro antiviral activity, had
improved pharmacokinetic parameters as
Interferon Beta-1a PEGylation
237
compared to the unmodified protein (23). Baker
et al. have shown that PEGylated IFN-β-1a
retains approximately 50% of its activity when
compared to the unmodified protein (1).
Relative to native IFN-β-1b, the
monoPEGylated compounds possess excellent
activity. The retention of about 20-70% of antiviral
activity in these derivatives compares favorably
to the marketed PEGylated IFN-R drugs, PEG-
INTRON and Pegasys, where in vitro antiviral
activities are about 28% or 7% of the unmodified
protein, respectively (13,14). It is apparent that
the conjugates with the highest molecular weight,
such as the 40 kDa PEG polymers have lower
antiviral activity in vitro (25).
Conclusions
mPEG-SPAs of 5, 20 and 40 kDa were
covalently attached to IFN-β-1a and the optimum
conditions for this reaction were determined by
different methods of experimental design. For 5
and 20 kDa mPEGs, the optimum molecular
weight, pH and the ratio of protein to mPEG were
found to be 20 kDa, 8 and 1:40, respectively.
Under optimum conditions, the percentages of
protein modifications were obtained 45.5% and
46.8% by HPLC and Ninhydrin methods,
respectively. For 40 kDa mPEG reactions, the
optimum molecular weight, pH and protein to
mPEG molar ratio were found to be 40 kDa, 8
and 1:40, respectively. Under optimum
conditions, the percentage protein modifications
were obtained 46.5% and 47.7% HPLC and
Ninhydrin methods, respectively. Further
increases in polymer concentration, may
increase the extent of protein coating. Based on
the results of the present and previous studies
(1,24,30), it can be concluded that the optimum
conditions for PEGylation of IFNs vary with
changing molecular weight of mPEG or using
different activating agents. The biological activity
test showed that the PEGylated protein retained
about 80% of its activity, were compared to that
of the unmodified protein, While, in other studies
the maximum biological activity were obtained
about 50%.