Dynamic light scattering and second derivative FTIR spectra of non-pegylated Met-G-CSF and of the two isomeric Met-G-CSF-Met1-PEG and Met-G-CSF-Gln135-PEG.

<p>A) Light scattering intensity, in arbitrary units, as a function of the incubation time at 55°C. At the zero time the bath temperature was changed from 37°C to 55°C. B) Polydispersity index (Eq.3) of the non-pegylated Met-G-CSF and of the two isomeric pegylated proteins as a function of the incubation time. C) Distribution of the hydrodynamic radii corresponding to the most abundant component in the DLS decay. The solid and dashed lines are best fit Gaussian function to the data that corresponds to Rh = 39.4±4 nm and Rh = 70±20 nm for the pegylated and non-pegylated samples respectively. D) Met-G-CSF, Met-G-CSF-Met1-PEG and Met-G-CSF-Gln135-PEG second derivative FTIR spectra measured after 7 hours of incubation at 55°C.Concerning the size of the protein aggregates, the diffusion coefficients (Eq.1) were used to evaluate the protein aggregate hydrodynamic radii through Eq.2. Non-pegylated Met-G-CSF displayed an average hydrodynamic radius of 1.4±0.4 nm at 37°C with negligible presence of protein aggregates. At 55°C, after thermal equilibrium was reached, we found instead a prevalent component with R_h = 70±20 nm. It should be noted that the value of 70 nm represents the average size of protein aggregates still in solution after 7 hours of incubation at 55^oC.</p

Thermal stability of Met-G-CSF and of the two pegylated forms by CD and fluorescence spectroscopies.

<p>A) Temperature dependence of the CD intensity at 222 nm. B) Temperature dependence of the HT[V] intensity at 222 nm. C) Temperature dependence of the fluorescence emission at 330 nm (excitation at 295 nm). D) Temperature dependence of the ratio between the fluorescence emission at 330 nm and at 350 nm (excitation at 295 nm).</p

FTIR spectroscopy analysis of Met-G-CSF and of the two pegylated forms.

<p>A) FTIR spectra of non-pegylated Met-G-CSF and of the two isomeric Met-G-CSF-Met1-PEG and Met-G-CSF-Gln135-PEG. B) Second derivative spectra in the Amide I band of the three proteins as in A).</p

Thermal stability of Met-G-CSF and of the two pegylated forms by CD spectroscopy.

<p>CD spectra of non-pegylated Met-G-CSF and of the two isomeric Met-G-CSF-Met1-PEG and Met-G-CSF-Gln135-PEG at 20°C (continuous line), 95°C (dashed line), and 20°C after heating up to 95°C (dotted line).</p