IMPURITY ANALYSIS
Table II: Comparison of impurity peaks detected in model PEGylated protein samples.
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A process intermediate and several forced degradation samples were analyzed by the tetra detection system (see Figure 2).
Generally, two primary peaks were detected, a soluble aggregate peak near the void of the column eluting near 8 mL and a soluble
oligomeric peak eluting at 10 mL, near the main peak. For the oligomer and aggregate peaks Mw information was obtained from static light scattering and the copolymer method in the OmniSEC software. Analysis of the integrated
peaks is shown in Table II, along with the repeatability and precision of the measurement for the oligomer peak. Data are
tight across two days of testing for a peak at 0.34% (peak area by RI). Stress by agitation, heat or extended room temperature
incubation produces samples with HMW impurities with different molecular weights. The agitated sample contains a large oligomeric
peak whose molecular weight is indicative of a dimer of the PEGylated protein. In addition to molecular weight, the hydrodynamic
radii can be determined by this analysis.
The oligomer and aggregate peaks were less homogeneous than the native conjugate. The heterogeneity is numerically depicted
in the polydispersity value (Mw/Mn). Visual examination of the light-scattering signal for the HMW impurities indicates differences between samples and heterogeneity
in this peak. The aggregate peaks demonstrated greater variability in molecular weight and polydispersity. The intrinsic viscosity
of the aggregate was unable to be reliably measured by this method. This is likely due to the small weight fraction present
of these species and the inability to produce adequate viscosity data.
Interpretation of the results depends on many variables. These measurements used the same dn/dC inputs for protein and polymer. The assumption is that the dn/dC values of the HMW impurities are the same as that of the monomeric PEGylated protein. Confirming the dn/dC values would require purifying individual HMW impurities. This would be challenging, considering the transient nature of
certain HMW impurities described elsewhere for a protein of similar Mw (10, 11). We noticed a qualitative loss of aggregate when testing several stressed samples. In addition, confirming the homogeneity
of the peaks themselves would require thorough orthogonal testing. The chromatography could be further optimized using an
alternate column or mobile phase to improve separation between different HMW components (12).
CONCLUSION
A tetra detection system was used to analyze a model PEGylated protein and its associated HMW impurities. To our knowledge,
this study is the first comparison of impurity peaks in a PEGylated protein by this methodology. The sensitivity of this system
is high and permits detection of HMW impurities as low as 0.34%. The outputs of this type of analyses can be used for comparison
of batches during process development, stressed samples and assessing mechanisms of aggregation.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Alok Sharma and Dr. Stan Bastiras for helpful comments on the manuscript and Dr. Thomas
Vanden Boom and Dr. Dave Schwinke for suggestions and support of this work.
BRYAN A. BERNAT is senior group leader at Hospira, Inc., Global Biologics R&D, 275 North Field Drive, Lake Forest, IL 60045, tel. 224.212.6363,
fax 224.212.7905, bryan.bernat@hospira.com
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MARK A. LEFERS is an associate research ccientist at Hospira, Inc. JASON SANCHEZ is a product manager at Malvern Instruments Inc.
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