Effects of polyol excipient stability during storage and use on the quality of biopharmaceutical formulations.

Biopharmaceuticals are formulated using a variety of excipients to maintain their storage stability. However, some excipients are prone to degradation during repeated use and/or improper storage, and the impurities generated by their degradation are easily overlooked by end users and are usually not strictly monitored, affecting the stability of biopharmaceuticals. In this study, we evaluated the degradation profile of polyol excipient glycerol during repeated use and improper storage and identified an unprecedented cyclic ketal impurity using gas chromatography with mass spectrometry (GC-MS). The other polyol excipient, mannitol, was much more stable than glycerol. The effects of degraded glycerol and mannitol on the stability of the model biopharmaceutical pentapeptide, thymopentin, were also evaluated. The thymopentin content was only 66.4% in the thymopentin formulations with degraded glycerol, compared to 95.8% in other formulations after the stress test. Most glycerol impurities (i.e., aldehydes and ketones) reacted with thymopentin, affecting the stability of thymopentin formulations. In conclusion, this work suggests that more attention should be paid to the quality changes of excipients during repeated use and storage. Additional testing of excipient stability under real or accelerated conditions by manufacturers would help avoid unexpected and painful results.

Characterization of Grinding-Induced Subvisible Particles and Free Radicals in a Freeze-Dried Monoclonal Antibody Formulation.

PURPOSES: The primary objectives of this study were to investigate the degradation mechanisms of freeze-dried monoclonal antibody (mAb) formulations under mechanical grinding, assess the sensitivity and suitability of various particle analysis techniques, analyze the structure of the collected subvisible particles (SbVPs), and analyze the antioxidant mechanism of methionine (Met) under degradation process to gain a thorough understanding of the phenomenon. METHODS: The freeze-dried mAb-X formulations underwent grinding, and the resultant SbVPs were characterized through visual inspection, flow imaging microscopy, dynamic light scattering, ultraviolet-visible spectroscopy, and size-exclusion high-performance liquid chromatography. We further evaluated the effect of different temperatures and the free radical scavenger Met on SbVP formation. The produced free radicals were detected using electron paramagnetic resonance, and Met S-oxide formation was detected using liquid chromatography-mass spectrometry. In addition, we analyzed the obtained SbVPs using capillary electrophoresis sodium dodecyl sulfate and Fourier transform infrared spectroscopy. RESULTS: Grinding leads to SbVP formation under high temperature and free radical formation. Free radicals produced during grinding require the participation of a macromolecule. Met could then bind to the produced free radicals, thus partially protecting mAb-X from degradation while itself undergoing oxidation to form Met(O). Sensitivity differences between different particle analysis techniques were evaluated, and the obtained SbVPs showed significant changes in secondary structure and the formation of covalent aggregates and fragments. CONCLUSIONS: Met plays the role of an antioxidant in protecting macromolecules by quenching the free radicals produced during grinding. To thoroughly characterize SbVPs, multiple and orthogonal particle analysis techniques should be used, and if necessary, SbVPs should be processed by enrichment to accurately analyze primary and high order structures.

Protein Sub-Visible Particle and Free Radical formation of a Freeze-Dried Monoclonal Antibody Formulation During Dropping.

Dropping during shipping and handling of liquid biopharmaceutical formulations has long been known to cause protein degradation and aggregation. On the other hand, accidental dropping of freeze-dried protein formulations is generally considered not a major issue for biopharmaceutical quality. Reports of stability and especially the underling degradation mechanism(s) during shipping and handling of freeze-dried protein formulations were rarely seen in literature. In this manuscript, we report an interesting phenomenon in which repeated dropping of freeze-dried monoclonal antibody X (mAb-X) formulation powder resulted in significant protein sub-visible particles (SbVPs) in the reconstituted liquid as determined by the sensitive particle analyzing technique micro-flow imaging (MFI). Free radicals were observed after repeated dropping by electron paramagnetic resonance (EPR). Formation of SbVPs could be partially inhibited by the free radical scavengers methionine and 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidin-yloxy free radical (CTPO). The amount of free radicals and SbVPs was correlated to the sample temperature during dropping. Therefore we propose that the high temperature formed during dropping was probably the root cause for protein aggregation and free radical formation, which could further cause protein aggregation. Our observations suggest that similar to liquid protein formulations, dropping of freeze-dried protein formulations should also be avoided or mitigated.