Analysis of PEGylated Protein by Tetra Detection Size Exclusion Chromatography - Reliably detecting low amounts of high molecular weight impurities during process development and characterization of b

ADVERTISEMENT

Analysis of PEGylated Protein by Tetra Detection Size Exclusion Chromatography
Reliably detecting low amounts of high molecular weight impurities during process development and characterization of biopharmaceutical products.


BioPharm International Supplements
Volume 24, Issue 8, pp. s10-s14

RESULTS AND DISCUSSION


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 kDa mPEG.


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.


blog comments powered by Disqus

ADVERTISEMENT

ADVERTISEMENT

GPhA Issues Statement on Generic Drug Costs
November 20, 2014
Amgen Opens Single-Use Manufacturing Plant in Singapore
November 20, 2014
Manufacturing Issues Crucial to Combating Ebola
November 20, 2014
FDA Requests Comments on Generic Drug Submission Criteria
November 20, 2014
USP Joins Chinese Pharmacopoeia Commission for Annual Science Meeting
November 20, 2014
Author Guidelines
Source: BioPharm International Supplements,
Click here