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Polysorbates are amphiphilic, non-ionic surfactants, and they represent one of the key components of biopharmaceuticals. They serve as stabilisers, and their degradation can cause ...particle formation, which has been an industry-wide issue over the past decade. To determine the influence of the buffers most frequently used in biopharmaceuticals on polysorbate degradation, an accelerated stability study was carried out using placebo formulations containing 0.02% polysorbates and 20 mM buffers (pH 5.5, 6.5). These included histidine chloride, sodium citrate, sodium succinate and sodium phosphate buffers. The rate of polysorbate degradation was highest in histidine chloride buffer, and therefore we further focused on the mechanism here. The predominant degradation pathway of polysorbates in this buffer was ester hydrolysis, catalysed by the imidazole moiety of the histidine. Interestingly, the presence of therapeutic proteins in the formulations slowed histidine-catalysed degradation of polysorbates in 50% of cases, with negligible degradation seen otherwise. This emphasises the complex nature of the interactions between the components of biopharmaceutical drug products. Nonetheless, there are disadvantages of using histidine chloride buffers in biopharmaceuticals that contain polysorbates. Careful consideration should be given to selection of excipients used in parenteral formulations, whereby compatibility between buffer and surfactant is of key importance.
A study on the polysorbate 80 stability in various formulation buffers commonly used in biopharmaceuticals was performed, to investigate the excipients influence on polysorbate 80 degradation. ...Polysorbate 80 is a common excipient in biopharmaceutical products. However, its degradation will potentially impact the drug product quality, and may trigger protein aggregation and particles formation. Due to the heterogeneity of the polysorbates and the mutual effects with other formulation compositions, the study of polysorbate degradation is challenging. Herein, a real-time stability study was designed and performed. The polysorbate 80 degradation trend was monitored by fluorescence micelle-based assay (FMA), reversed-phase-ultra-performance liquid chromatography-evaporative light scattering detector (RP-UPLC-ELSD) assay, and LC-MS assay. These assays provide orthogonal results to reveal both the micelle-forming capability and the compositional changes of polysorbate 80 in different buffer systems. The degradation occurred after a period of storage under 25 °C in different trend, which indicates the excipients could impact the degradation kinetics. Upon comparison, the degradation is prone to happen in histidine buffer than in acetate, phosphate or citrate buffers. LC-MS confirms oxidation as an independent degradation pathway with detection of the oxidative aldehyde. Thus, it is necessary to pay more attention to the excipients selection and their potential impact on polysorbate 80 stability to achieve longer shelf life for the biopharmaceuticals. Besides, the protective roles of several additives were figured out, which could be applied as potential industrial solutions to the polysorbate 80 degradation issues.
•Investigate excipients impact on PS80 stability in various formulation buffers.•Use orthogonal assays to monitor PS80 change.•Propose a degradation mechanism and mitigation strategies.
Polysorbate 20 (PS20), a commonly used surfactant in biopharmaceuticals, showed degradation upon long-term (∼18–36 months) storage of two monoclonal antibody (mAb, mAb-A, and mAb-B) drug products at ...2–8 °C. The PS20 degradation resulted in the accumulation of free fatty acids (FFA), which ultimately precipitated to form particles upon long-term storage. This study documents the development, qualification, and application of a method for FFA quantification in soluble and insoluble fraction of protein formulation. The method was applied to the quantification of capric acid, lauric acid, myristic acid, palmitic/oleic acid, and stearic acid in placebo as well as active protein formulations on stability. Quantification of FFA in both the soluble and insoluble fraction of mAb-A and mAb-B provided a better mechanistic understanding of PS20 degradation and the dynamics of subsequent fatty acid particle formation. Additionally, the use of this method for monitoring and quantitation of the FFA on real time storage stability appears to aid in identifying batches with higher probability for particulate formation upon extended storage at 5 °C.
The coformulation of monoclonal antibody (mAb) mixtures provides an attractive route to achieving therapeutic efficacy where the targeting of multiple epitopes is necessary. Controlling and ...predicting the behavior of such mixtures requires elucidating the molecular basis for the self- and cross-protein–protein interactions and how they depend on solution variables. While self-interactions are now beginning to be well understood, systematic studies of cross-interactions between mAbs in solution do not exist. Here, we have used static light scattering to measure the set of self- and cross-osmotic second virial coefficients in a solution containing a mixture of two mAbs, mAbA and mAbB, as a function of ionic strength and pH. mAbB exhibits strong association at a low ionic strength, which is attributed to an electrostatic attraction that is enhanced by the presence of a strong short-ranged attraction of nonelectrostatic origin. Under all solution conditions, the measured cross-interactions are intermediate self-interactions and follow similar patterns of behavior. There is a strong electrostatic attraction at higher pH values, reflecting the behavior of mAbB. Protein–protein interactions become more attractive with an increasing pH due to reducing the overall protein net charges, an effect that is attenuated with an increasing ionic strength due to the screening of electrostatic interactions. Under moderate ionic strength conditions, the reduced cross-virial coefficient, which reflects only the energetic contribution to protein–protein interactions, is given by a geometric average of the corresponding self-coefficients. We show the relationship can be rationalized using a patchy sphere model, where the interaction energy between sites i and j is given by the arithmetic mean of the i–i and j–j interactions. The geometric mean does not necessarily apply to all mAb mixtures and is expected to break down at a lower ionic strength due to the nonadditivity of electrostatic interactions.
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•Oxidative degradation byproducts of polysorbates were isolated.•For the first time Aldehyde derivatives of fatty acid esters were isolated and identified as novel markers of ...oxidative polysorbate degradation in sterile formulations.•A promising strategy to track polysorbate oxidation in biopharmaceutical formulation is proposed.
Polysorbates can undergo oxidative degradation in pharmaceutical formulations resulting in both soluble and insoluble degradation products. The insoluble degradants may precipitate to form subvisible and visible particulates, which are undesirable in liquid parenteral products. To date, no oxidation byproduct has been identified as an established marker to track Polysorbate 20 oxidation. Herein, we identified the aldehyde derivative of free fatty acid esters as a byproduct of polysorbate oxidation that can be derivatized using 2,4-dinitrophenylhydrazine and tracked analytically to monitor oxidative polysorbate degradation in pharmaceutical formulations.
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Surfactants are used to stabilize biologics. Particularly, polysorbates (Tween® 20 and Tween® 80) dominate the group of surfactants in protein and especially antibody drug products. ...Since decades drug developers rely on the ethoxylated sorbitan fatty acid ester mixtures to stabilize sensitive molecules such as proteins. Reasons are (i) excellent stabilizing properties, and (ii) well recognized safety and tolerability profile of these polysorbates in humans, especially for parenteral applications. However, over the past decade concerns regarding the stability of these two polysorbates were raised. The search of alternatives with preferably less reservations concerning degradation and product quality reducing issues leads, among others, to poloxamer 188 (e.g. Kolliphor® P188), a nonionic triblock-copolymer surfactant. This review sums up our current knowledge related to the characterization and physico-chemical properties of poloxamer 188, its analytics and stability properties for biological formulations. Furthermore, the advantages and disadvantages as a suitable polysorbate-alternative for the stabilization of biologics are discussed.
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.
The aim of this study was to develop antisense oligonucleotide tablet formulations using high-speed electrospinning. Hydroxypropyl-beta-cyclodextrin (HPβCD) was used as a stabilizer and as an ...electrospinning matrix. In order to optimize the morphology of the fibers, electrospinning of various formulations was carried out using water, methanol/water (1:1), and methanol as solvents. The results showed that using methanol could be advantageous due to the lower viscosity threshold for fiber formation enabling higher potential drug loadings by using less excipient. To increase the productivity of electrospinning, high-speed electrospinning technology was utilized and HPβCD fibers containing 9.1% antisense oligonucleotide were prepared at a rate of ~330 g/h. Furthermore, to increase the drug content of the fibers, a formulation with a 50% drug loading was developed. The fibers had excellent grindability but poor flowability. The ground fibrous powder was mixed with excipients to improve its flowability, which enabled the automatic tableting of the mixture by direct compression. The fibrous HPβCD-antisense oligonucleotide formulations showed no sign of physical or chemical degradation over the 1-year stability study, which also shows the suitability of the HPβCD matrix for the formulation of biopharmaceuticals. The obtained results demonstrate possible solutions for the challenges of electrospinning such as scale-up and downstream processing of the fibers.
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Triton X-100 (TX-100) is the most common surfactant used to split viruses during the production of influenza split-virus vaccines. It is a mild surfactant not known to denature the ...viral proteins; this property makes TX-100 useful for maintaining antigen conformational structure, and, as an added benefit, for partially stabilizing vaccine formulations against protein aggregation. Despite its benefits, TX-100 needs to be filtered out after virus splitting has been achieved, due to its toxicity in large quantities. Accordingly, residual TX-100 presence in vaccine formulations has implications for both formulation stability and safety, necessitating both accurate screening during processing to guide decision-making about filtration repeats and accurate quantitation in the final product. Accurate HPLC-based methods are used successfully for the latter but their use for routine screening during processing is far from ideal because they often require extensive sample preparation and are fairly slow, complicated and costly. Here, “deconstruction” of UV–Vis absorption spectra into components corresponding to different absorbing “species” is demonstrated as a novel and viable method for routine TX-100 screening in vaccine samples from different industrial processing steps. This method is fairly accurate and, more importantly, preparation-free, rapid, simple/user-friendly and comparatively inexpensive. It is evaluated in depth in terms of applicability conditions, limitations and potential for high-throughput adaptation as well as generalization to other complex biopharmaceutical formulations.
Highlights • Flu pre-vaccine samples from various strains/processing steps analyzed orthogonally. • Large protein aggregates seen after splitting cause shouldering in Nile Red spectra. • Novel ...correlation of insights from orthogonal methods with spectrum decomposition. • Development of a rapid, reliable, user-friendly large aggregate screening test. • Potential for high-throughput screening and adaptation to other bioformulations.
