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Purposes: We previously reported an unexpected phenomenon that shaking stress could cause more protein degradation in freeze-dried monoclonal antibody (mAb) formulations than liquid ones (J Pharm Sci, 2022, 2134). The main purposes of the present study were to investigate the effects of shaking stress on protein degradation and sub-visible particle (SbVP) formation in freeze-dried mAb formulations, and to analyze the factors influencing protein degradation during production and transportation. Methods: The aggregation behavior of mAb-X formulations during production and transportation was simulated by shaking at a rate of 300 rpm at 25°C for 24 h. The contents of particles and monomers were analyzed by micro-flow imaging, dynamic light scattering, size exclusion chromatography, and ultraviolet − visible (UV–Vis) spectroscopy to compare the protective effects of excipients on the aggregation of mAb-X. Results: Shaking stress could cause protein degradation in freeze-dried mAb-X formulations, while surfactant, appropriate pH, polyol mannitol, and high protein concentration could impact SbVP generation. Water content had little effect on freeze-dried protein degradation during shaking, as far as the water content was controlled in the acceptable range as recommended by mainstream pharmacopoeias (i.e., less than 3%). Conclusions: Shaking stress can reduce the physical stability of freeze-dried mAb formulations, and the addition of surfactants, polyol mannitol, and a high protein concentration have protective effects against the degradation of model mAb formulations induced by shaking stress. The experimental results provide new insight for the development of freeze-dried mAb formulations. [ABSTRACT FROM AUTHOR]

Purposes: In reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) analysis of a monoclonal antibody (mAb-1), the peak area ratio of heavy chain (HC) to light chain (LC) was out of balance, while multiple artifact peaks were observed following the migration of HC. The main purposes of this study were to describe the techniques utilized to eliminate this artifact and clarify the root cause for this interesting phenomenon. Methods: We optimized the CE-SDS analysis of mAb-1 by a vairety of techniques including changing the concentration of protein or replacing SDS with a more hydrophobic surfactant (i.e., sodium hexadecyl sulfate (SHS) or sodium tetradecyl sulfate (STS) instead of SDS) in sample and/or the sieving gel buffer. Dynamic light scattering (DLS) and reversed phase high-performance liquid chromatography (RP-HPLC) were used to study the protein-surfactant complex. Results: The artifact could be partially mitigated by reducing the protein concentration and replacing SDS with SHS or STS in the sample and/or the sieving gel buffer solutions. Due to replacing a more hydrophobic surfactant, the HC-surfactant complex formed was more resistant to dissociation, preventing additional hydrophobic HC-HC interaction and aggregation, thus eliminating the artifact problem. Conclusions: DLS and RP-HPLC are powerful supplementary techniques in characterizing the protein-surfactant complex, and hydrophobic surfactants such as SHS and STS could afford more normal electropherograms during the analysis of mAbs. [ABSTRACT FROM AUTHOR]

Purposes: This article describes an interesting phenomenon in which optimized freeze-dried (FD) biopharmaceutical formulations are generally more prone to degradation than their liquid counterparts during dropping and proposes an underlying cause for this surprising phenomenon. Methods: Two monoclonal antibodies (mAbs) and a fusion protein (FP) were used as model biopharmaceuticals. The stability after dropping stress was determined by ultraviolet–visible (UV–Vis), size exclusion high-performance liquid chromatography (SE–HPLC), micro-flow imaging (MFI), and dynamic light scattering (DLS). Results: Contrary to what we would normally assume, the FD formulations of the three biopharmaceuticals studied here generally showed much higher amounts of protein sub-visible particles (SbVPs) than liquid formulations after applying the same dropping stress as determined by MFI and DLS. Traditional techniques, such as UV–Vis and SE–HPLC, could hardly detect such degradation. Conclusions: We propose that the higher temperature caused by dropping for the FD powders than the liquid formulations was probably one of the root causes for the higher amount of particles formed for the FD powders. We also recommend that dropping stress should be included for early-stage screening and choosing liquid versus FD biopharmaceutical formulations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Previously our laboratory first reported that dropping of freeze-dried monoclonal antibody (mAb) formulations could cause protein degradation and aggregation (J Pharm Sci, 2021, 1625). In this manuscript, we evaluated effects of secondary package on stability of several freeze-dried biopharmaceutical formulations during dropping. The degradation of mAb-Y during dropping with different secondary packages was determined by the sensitive particle analyzing techniques micro-flow imaging (MFI) and dynamic light scattering (DLS). Electron paramagnetic resonance (EPR) was used to detect free radicals after repeated dropping in different secondary packages. The amount of free radicals and SbVPs was correlated to the sample temperature as well as the secondary package during dropping. Our observations suggest that secondary packaging has significant effect on freeze-dried biopharmaceutical stability during dropping and therefore should be thoroughly screened and optimized to assure high product quality even for the presumed highly stable freeze-dried biopharmaceuticals. [ABSTRACT FROM AUTHOR]

Purposes: Liquid protein-based biopharmaceutical formulations have been reported to form aggregation and protein sub-visible particles (SbVPs) during dropping (Randolph et al., J Pharm Sci 2015, 104, 602). However, effects of secondary package on liquid biopharmaceutical formulation stability during dropping are overlooked and have not been reported so far. This study reports the first real-world evaluation on effects of secondary package on liquid biopharmaceutical formulation stability during dropping, using two monoclonal antibodies (mAb-1 and mAb-2) and one fusion protein (FP-1) as model biopharmaceuticals. Methods: The potential protective effects of secondary package and formulation composition on liquid biopharmaceutical formulations during dropping were evaluated with micro-flow imaging (MFI) and dynamic light scattering (DLS). Results: The dropping-induced degradation could be detected with the two sensitive particle analyzing techniques MFI and DLS. Formulation compositions have dramatic impact on biopharmaceutical stability during dropping. Surprisingly, unlike the primary packages that have been reported to impact liquid biopharmaceutical stability, the secondary packaging system as described in our current preliminary design has little or no protective effect during dropping. Conclusions: Our study is the first real-world data showing that the secondary package system has little to no effect on the liquid biopharmaceutical formulation quality during dropping. On the contrary, the stability of liquid biopharmaceutical formulations during dropping is more relevant to formulation compositions and primary packages. [ABSTRACT FROM AUTHOR]