d(GC) - Association of Biotechnology and Pharmacy
d(GC) - Association of Biotechnology and Pharmacy
d(GC) - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 6 (2) 229-240 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)<br />
Table 1. The selected variables <strong>and</strong> their corresponding levels for full factorial design.<br />
Variable Low level High level<br />
(1) (2)<br />
A: Polymer molecular weight (KDa) 5 20<br />
B: pH 7.4 8.0<br />
C: The ratio <strong>of</strong> protein to mPEG fractions 1:10 1:40<br />
Table 2. Yates table design (full factorial design) for screening the factors.<br />
Treatment Polymer pH Protein/mPEG HPLC Ninhydrin<br />
Combination a MW (B) fractions (C) Response Response<br />
(kDa) (A)<br />
1 5 7.4 1:10 0.879 0.128<br />
a 20 7.4 1:10 0.868 0.135<br />
b 5 8.0 1:10 0.860 0.153<br />
ab 20 8.0 1:10 0.809 0.208<br />
c 5 7.4 1:40 0.655 0.352<br />
ac 20 7.4 1:40 0.588 0.415<br />
bc 5 8.0 1:40 0.612 0.405<br />
abc 20 8.0 1:40 0.545 0.468<br />
a The low level <strong>of</strong> any variable is denoted by l, <strong>and</strong> the high level <strong>of</strong> any variable is denoted by its<br />
lower-case letter.<br />
Table 3. The selected variables <strong>and</strong> their corresponding levels for Taguchi design.<br />
Variable First level (1) Second level (2) Third level (3)<br />
A:Polymer molecular weight (KDa) 5.0 12.5 20.0<br />
B: pH 7.4 7.7 8.0<br />
C: The ratio <strong>of</strong> protein to mPEG fractions 1:10 1:25 1:40<br />
Protein PEGylation: The degree <strong>of</strong> polymer<br />
attachment to IFN-β was assessed by HPLC for<br />
PEGylated proteins using linear polymers with<br />
molecular weights <strong>of</strong> 5 <strong>and</strong> 20 kDa, <strong>and</strong> also the<br />
branched polymer <strong>of</strong> 40 kDa. The samples were<br />
loaded on a SE-HPLC column to separate the<br />
PEGylated <strong>and</strong> non-PEGylated proteins on the<br />
basis <strong>of</strong> their molecular size, as evaluated by the<br />
Autochro 3000 s<strong>of</strong>tware. For each test, the ratio<br />
<strong>of</strong> the non-PEGylated protein peak to the total<br />
Interferon Beta-1a PEGylation<br />
233<br />
peaks represented the response. Therefore, in<br />
this procedure, a lowers ratio leads to higher<br />
levels <strong>of</strong> polymer attachment to the protein.<br />
Furthermore, Ninhydrin test was also done to<br />
obtain the total amount <strong>of</strong> polymer attached to<br />
the protein. In this test, the reaction <strong>of</strong> Ninhydrin<br />
with free amino groups on the protein surface<br />
showed that the PEGylated protein reacts with<br />
lower levels <strong>of</strong> Ninhydrin in comparison to the<br />
non-PEGylated protein. Consequently, in this test,