Figure 1: Tetra detector chromatogram of the PEGylated protein main peak. Detector signals shown include refractive index
(red), UV at 280 nm (purple), viscometer (blue), and right angle static light scattering (green). All signals were normalized
and the logMW data is shown in black to highlight the homogeneity of this native peak.
Samples prepared with different lots of mPEG were analyzed by the tetra detector system. The unmodified model protein was
also tested for comparison. Figure 1 contains a representative chromatogram noting the signal from all four detectors. Table
I lists parameters obtained for the main peak. Small shifts in the Mw can be detected between lots. As noted in the table, the relative difference in Mw for the mPEG accounts for the variability in the Mw of the PEGylated protein. While the mPEG is labeled as 20 kDa, this represents a polydisperse polymer with a broad population
of mPEG molecules with different chain lengths. Orthogonal testing by mass spectrometry and nuclear magnetic resonance (NMR)
indicates the Mw of the material used for PEGylation ranges from approximately 20100 Da to 21500 Da (7, 8, 9). The protein content percentage
for each main peak is approximately 50% as would be expected for this protein modified primarily by a single molecule of 20
Table I: Comparison of model PEGylated proteins modified with different lots of mPEG1.
The intrinsic viscosity is measured directly whereas the hydrodynamic size is calculated from Einstein's viscosity equation,
which relates intrinsic viscosity and Mw to the hydrodynamic size of an equivalent sphere. The intrinsic viscosity for the model PEGylated protein (0.228–0.240 dL/g)
is similar to free PEG (0.38 dL/g) and greater than protein alone (0.034 dL/g), as expected. The measured intrinsic viscosity
for BSA (0.039 dL/g) is comparable to the published value (0.037 dL/g) (5). The hydrodynamic size of the PEGylated protein
increases as the mPEG of larger size was used for modification. The hydrodynamic radius for the main peaks (5.17–5.35 nm)
are similar to those measured (by SEC with calibrated PEG standards) for proteins with a similar Mw modified with a single 20 kDa mPEG (6).
Figure 2: Staggered overlay of the refractive index signal (a) and right angle static light scattering (RALS) signal (b) for
the five stressed samples. It is clear that different applied stress conditions will impact both the concentration of oligomer
and aggregate species, as well as their distribution. Note that the light scattering signal is a function of size, not necessarily
abundance, especially for large aggregates.
Repeat injections differ by less than 1% for all parameters noted in Table I. These highly reproducible results were obtained
with separate calibrations using BSA or the unmodified protein.