Albinterferon α2b Adsorption to Silicone Oil–Water Interfaces: Effects on Protein Conformation,Aggregation, and Subvisible Particle Formation

Silicone oil used as a lubricant in prefilled syringes has the potential to induce formation of particles in protein formulations. In the current study, we used a therapeutic fusion protein, albinterferon α_2b, to evaluate protein aggregation and particle formation in the presence of silicone oil microdroplets or immobilized silicone interfaces. Tertiary structure of albinterferon α_2b adsorbed on silicone oil microdroplets was perturbed in a formulation containing only buffer. In contrast, native-like tertiary structure was retained for albinterferon α_2b adsorbed on silicone oil microdroplets in 300 mM sodium chloride or 300 mM sucrose formulations. Agitation of albinterferon α_2b samples in the presence of silicone oil droplets or siliconized beads, respectively, caused albinterferon α_2b aggregation and subvisible particle formation in formulations containing buffer or 300 mM sucrose. Adsorption of albinterferon α_2b onto silicone oil was inhibited by addition of 0.01% (w/v) polysorbate 80, and this excipient prevented aggregation during agitation in the presence of silicone oil microdroplets. Aggregation was also reduced in the presence of 300 mM sodium chloride during agitation at least in part because of the increased conformational stability of the protein.     

Holographic Characterization of Protein Aggregates in the Presence of Silicone Oil and Surfactants

Characterizing protein aggregates in the presence of silicone oil is a long standing challenge for the pharmaceutical industry. Silicone oil is often used as a lubricant in devices that deliver and store therapeutic protein products and has been linked to protein aggregation, which can compromise a drug’s effectiveness or cause autoimmune responses in patients. Most traditional technologies cannot quantitatively distinguish protein aggregates and silicone oil in their native formulations for sizes less than 5 μm. We use holographic video microscopy to study protein aggregation to demonstrate its capability to quantitatively distinguish protein aggregates and silicone oil in the presence of varying amounts of the surfactants SDS and polysorbate 80 in the size range of 0.5-10 μm without the need for dilution or special sample preparation. We show that SDS denatures proteins and stabilizes silicone oil. We also show that polysorbate 80 may limit protein aggregate formation if it is added to an IgG solution before introducing silicone oil.     

Protein adsorption and excipient effects on kinetic stability of silicone oil emulsions

The effect of silicone oil on the stability of therapeutic protein formulations is of concern in the biopharmaceutical industry as more proteins are stored and delivered in prefilled syringes. Prefilled syringes provide convenience for medical professionals and patients, but prolonged exposure of proteins to silicone oil within prefilled syringes may be problematic. In this study, we characterize systems of silicone oil‐in‐aqueous buffer emulsions and model proteins in formulations containing surfactant, sodium chloride, or sucrose. For each of the formulations studied, silicone oil‐induced loss of soluble protein, likely through protein adsorption onto the silicone oil droplet surface. Excipient addition affected both protein adsorption and emulsion stability. Addition of surfactant stabilized emulsions but decreased protein adsorption to silicone oil microdroplets. In contrast, addition of sodium chloride increased protein adsorption and decreased emulsion stability. Silicone oil droplets with adsorbed lysozyme rapidly agglomerated and creamed out of suspension. This decrease in the kinetic stability of the emulsion is ascribed to surface charge neutralization and a bridging flocculation phenomenon and illustrates the need to investigate not only the effects of silicone oil on protein stability, but also the effects of protein formulation variables on emulsion stability. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1721–1733, 2010     

Influence of Protein Adsorption on Aggregation in Prefilled Syringes

Protein aggregate formation in prefilled syringes (PFSs) can be influenced by protein adsorption and desorption at the solid–liquid interface. Although inhibition of protein adsorption on the PFS surface can lead to a decrease in the amount of aggregation, the mechanism underlying protein adsorption-mediated aggregation in PFSs is unclear. This study investigated protein aggregation caused by protein adsorption on silicone oil-free PFS surfaces [borosilicate glass (GLS) and cycloolefin polymer (COP)] and the factors affecting the protein adsorption on the PFS surfaces. The adsorbed proteins formed multilayered structures that consisted of two distinct types of layers: proteins adsorbed on the surface of the material and proteins adsorbed on top of the proteins on the surface. A pH-dependent electrostatic interaction was the dominant force for protein adsorption on the GLS surface, while hydrophobic effects were dominant for protein adsorption on the COP surface. When the repulsion force between proteins was weak, protein adsorption on the adsorbed protein layer was increased for both materials and as a result, protein aggregation increased. Therefore, a formulation with high colloidal stability can minimize protein adsorption on the COP surface, leading to reduced protein aggregation.     

Demonstrating the stability of albinterferon alfa-2b in the presence of silicone oil

Silicone oil is often used to decrease glide forces in prefilled syringes and cartridges, common primary container closures for biopharmaceutical products. Silicone oil has been linked to inducing protein aggregation (Diabet Med 1989;6:278; Diabet Care 1987;10:786-790), leading to patient safety and immunogenicity concerns. Because of the silicone oil application process (Biotech Adv 2007;25:318-324), silicone oil levels tend to vary between individual container closures. Various silicone oil levels were applied to a container closure prior to filling and lyophilization of an albumin and interferon alfa-2b fusion protein (albinterferon alfa-2b). Data demonstrated that high silicone oil levels in combination with intended and stress storage conditions had no impact on protein purity, higher order structure, stability trajectory, or biological activity. Subvisible particulate analysis (1-10 μm range) from active and placebo samples from siliconized glass barrels showed similar particle counts. Increases in solution turbidity readings for both active and placebo samples correlated well with increases in silicone oil levels, suggesting that the particles in solution are related to the presence of silicone oil and not large protein aggregates. Results from this study demonstrate that silicone oil is not always detrimental to proteins; nevertheless, assessing the impact of silicone oil on a product case-by-case basis is still recommended.     

Understanding Protein-Interface Interactions of a Fusion Protein at Silicone Oil-Water Interface Probed by Sum Frequency Generation Vibrational Spectroscopy

Protein adsorbed at the silicone oil-water interface can undergo a conformational change that has the potential to induce protein aggregation on storage. Characterization of the protein structures at interface is therefore critical for understanding the protein-interface interactions. In this article, we have applied sum frequency generation (SFG) spectroscopy for studying the secondary structures of a fusion protein at interface and the surfactant effect on protein adsorption to silicone oil-water interface. SFG and chiral SFG spectra from adsorbed protein in the amide I region were analyzed. The presence of beta-sheet vibrational band at 1635 cm^?1 implies the protein secondary structure was likely perturbed when protein adsorbed at silicone oil interface. The time-dependent SFG study showed a significant reduction in the SFG signal of preadsorbed protein when polysorbate 20 was introduced, suggesting surfactant has stronger interaction with the interface leading to desorption of protein from the interface. In the preadsorbed surfactant and a mixture of protein/polysorbate 20, SFG analysis confirmed that surfactant can dramatically prevent the protein adsorption to silicone oil surface. This study has demonstrated the potential of SFG for providing the detailed molecular level understanding of protein conformation at interface and assessing the influence of surfactant on protein adsorption behavior.     

Ionic Strength Affects Tertiary Structure and Aggregation Propensity of a Monoclonal Antibody Adsorbed to Silicone Oil–Water Interfaces

Therapeutic proteins formulated in prefilled syringes lubricated with silicone oil come in contact with silicone oil–water interfaces for their entire shelf lives. Thus, the interactions between protein and silicone oil were studied to determine the effect of silicone oil on a monoclonal antibody's stability, both at the interface and in the bulk solution. The influence of ionic strength on these interactions was also investigated through the addition of various monovalent and divalent salts to sample formulations. The tertiary structure of the antibody was perturbed when it adsorbed to the silicone oil–water interface in solutions at low ionic strength. However, the tertiary structure of the antibody at the interface was not perturbed when the ionic strength of the formulation was increased. Even at low ionic strength, the secondary structure of the antibody adsorbed to the silicone oil–water interface was retained, suggesting that at low ionic strength, the adsorbed antibody assumes a molten globule-like conformation. This partially unfolded species was aggregation-prone, especially during agitation. Silicone oil-induced aggregation of the antibody was inhibited at higher ionic strength.     

Protein Aggregation and Particle Formation in Prefilled Glass Syringes

The stability of therapeutic proteins formulated in prefilled syringes (PFS) may be negatively impacted by the exposure of protein molecules to silicone oil–water interfaces and air–water interfaces. In addition, agitation, such as that experienced during transportation, may increase the detrimental effects (i.e., protein aggregation and particle formation) of protein interactions with interfaces. In this study, surfactant-free formulations containing either a monoclonal antibody or lysozyme were incubated in PFS, where they were exposed to silicone oil–water interfaces (siliconized syringe walls), air–water interfaces (air bubbles), and agitation stress (occurring during end-over-end rotation). Using flow microscopy, particles (≥2 μm diameter) were detected under all conditions. The highest particle concentrations were found in agitated, siliconized syringes containing an air bubble. The particles formed in this condition consisted of silicone oil droplets and aggregated protein, as well as agglomerates of protein aggregates and silicone oil. We propose an interfacial mechanism of particle generation in PFS in which capillary forces at the three-phase (silicone oil–water–air) contact line remove silicone oil and gelled protein aggregates from the interface and transport them into the bulk. This mechanism explains the synergistic effects of silicone oil–water interfaces, air–water interfaces, and agitation in the generation of particles in protein formulations. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1601–1612, 2014     

Particle Characterization for a Protein Drug Product Stored in Pre-Filled Syringes Using Micro-Flow Imaging,Archimedes, and Quartz Crystal Microbalance with Dissipation

Micro-flow imaging (MFI) has been used for formulation development for analyzing sub-visible particles. Archimedes, a novel technique for analyzing sub-micron particles, has been considered as an orthogonal method to currently existing techniques. This study utilized these two techniques to investigate the effectiveness of polysorbate (PS-80) in mitigating the particle formation of a therapeutic protein formulation stored in silicone oil-coated pre-filled syringes. The results indicated that PS-80 prevented the formation of both protein and silicone oil particles. In the case of protein particles, PS-80 might involve in the interactions with the hydrophobic patches of protein, air bubbles, and the stressed surfaces of silicone oil-coated pre-filled syringes. Such interactions played a role in mitigating the formation of protein particles. Subsequently, quartz crystal microbalance with dissipation (QCM-D) was utilized to characterize the interactions associated with silicone oil, protein, and PS-80 in the solutions. Based on QCM-D results, we proposed that PS-80 likely formed a layer on the interior surfaces of syringes. As a result, the adsorbed PS-80 might block the leakage of silicone oil from the surfaces to solution so that the silicone oil particles were mitigated at the presence of PS-80. Overall, this study demonstrated the necessary of utilizing these three techniques cooperatively in order to better understand the interfacial role of PS-80 in mitigating the formation of protein and silicone oil particles.     

Steric Repulsion Forces Contributed by PEGylation of Interleukin-1 Receptor Antagonist Reduce Gelation and Aggregation at the Silicone Oil-Water Interface

Silicone oil, used as a lubricating coating in pharmaceutical containers, has been implicated as a cause of therapeutic protein aggregation. After adsorbing to silicone oil-water interfaces, proteins may form interfacial gels, which can be transported into solution as insoluble aggregates if the interfaces are perturbed. Mechanical interfacial perturbation of both monomeric recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and PEGylated rhIL-1ra (PEG rhIL-1ra) in siliconized syringes resulted in losses of soluble monomeric protein. However, the loss of rhIL-1ra was twice that for PEG rhIL-1ra; even though in solution, PEG rhIL-1ra had a lower ΔG_unf and exhibited a more perturbed tertiary structure at the interface. Net protein-protein interactions in solution for rhIL-1ra were attractive but increased steric repulsion because of PEGylation led to net repulsive interactions for PEG rhIL-1ra. Attractive interactions for rhIL-1ra were associated with increases in intermolecular β-sheet content at the interface, whereas no intermolecular β-sheet structures were observed for adsorbed PEG rhIL-1ra. rhIL-1ra formed interfacial gels that were 5 times stronger than those formed by PEG rhIL-1ra. Thus, the steric repulsion contributed by the PEGylation resulted in decreased interfacial gelation and in the reduction of aggregation, in spite of the destabilizing effects of PEGylation on the protein’s conformational stability.     

Protein conformational diseases: from mechanisms to drug designs

Amyloidosis comprises a group of diseases characterized by the deposition of insoluble protein fibrils in specific organs and includes several serious medical disorders, such as Alzheimer's disease, prion-associated transmissible spongiform encephalitis, and type II diabetes. Despite the structural dissimilarity between the soluble proteins and peptides, these fibrils exhibit similar morphologies under electron microscopy with a characteristic "cross beta-sheet" pattern examined by x-ray fiber diffraction experiments. Many studies have revealed that each of these diseases is associated to a specific protein that is partially unfolded, misfolded, and aggregated. However, the detailed structures of the causative agents and the toxicity mechanisms are less known. This review summarizes recent studies in the conformational disorders leading to aggregation; including which proteins potentially cause conformational diseases, the aggregation mechanisms of these proteins, and recent researches on the conformational changes using advanced experiments or molecular dynamics simulations. Finally, current drug designs towards these protein conformational diseases are also discussed. It is believed that the advances in basic understanding of the mechanisms of conformational changes as well as biological functions of these proteins will shed light on the development and design of potential interfering compounds against amyloid formation associated with protein conformational diseases.     

Mechanistic Understanding of Protein-Silicone Oil Interactions

Pharmaceutical Research - To investigate interactions between protein and silicone oil so that we can provide some mechanistic understanding of protein aggregation in silicone oil lubricated...     

Quantification of Silicone Oil and Its Degradation Products in Aqueous Pharmaceutical Formulations by 1H-NMR Spectroscopy

During the past years, there has been an increasing focus on the presence of silicone oil as a contaminant in pharmaceutical formulations kept in prefilled syringes (PFSs). As the PFSs are coated on the inner wall with silicone oil (polydimethylsiloxane), there is a potential risk that the oil can migrate from the inner surface of the primary packing material into the aqueous solution. Several studies have demonstrated that presence of silicone oil as droplets in a high-concentrated protein formulation can cause protein aggregation. Hence, because the use of silicone-coated primary packing material for protein formulations are increasing, the call for an easy and quantitative method for determination of silicone oil and its degradation products in pharmaceutical formulations is therefore needed. Several analytical techniques have in the past been developed with the aim of detecting the presence of silicone oil and degradation products hereof. Most of these methods require hydrolyzation, derivatization, and extraction steps followed by, for example, gas chromatography-mass spectrometry analysis. Applying these methods can cause a loss in detection or an overestimation of the hydrolytic degradation products of silicone oil, that is, trimethylsilanol and dimethylsilanediol. The 2 silanols are highly hydrophilic and prefers the aqueous environment. Analysis of an aqueous formulation obtained from a PFS by ¹H-NMR spectroscopy provides data about the content and levels of silicone oil and the 2 silanols even in levels below 10 ppm. The ¹H-NMR method offers an easy and direct, quantitative measurement of samples intended for clinical use and samples kept at elevated temperature for a prolonged time (i.e., stability studies). The result of the study presented here showed dimethylsilanediol to be the main silicone compound present in the aqueous formulation when kept in baked-on PFSs. The degradation product dimethylsilanediol, in full accordance with expected hydrolytic degradation of silicone oil, increased during storage and with elevated temperature. In addition, the method can be applied to aqueous samples where polydimethylsiloxane has been added as, for example, the major constituent of antifoam.     

Evaluation of the effect of syringe surfaces on protein formulations

Packaging of drugs in prefillable syringes offers considerable advantages over conventional vials. Almost all major biotech molecules are available on the market today in prefilled syringes, and are safe and efficacious. Newer high-concentration liquid formulations, especially fusion proteins, however, can suffer from instability in prefilled syringes due to syringe components like silicone oil. To assess the effect of siliconized and modified syringe surfaces on protein formulations, the stability of the recombinant protective antigen (rPA) for anthrax, abatacept, a fusion protein formulation with known silicone oil sensitivity, and an antistaphylococcal enterotoxin B (anti-SEB) monoclonal antibody (mAb) was assessed in siliconized, uncoated, and BD-42-coated (a proprietary coating developed by BD Technologies) prefilled syringes under different conditions. Both the soluble protein content and the number of subvisible particles were followed over time. When filled in siliconized syringes, all three protein solutions showed increased number of subvisible particles relative to uncoated or BD-42-coated syringes; the abatacept formulation with known silicone sensitivity also developed visible particles. Although rPA and anti-SEB mAb formulations mainly showed individual droplets, presumably of silicone, the abatacept formulation also showed droplets entangled in a fibrous structure. Uncoated glass and BD-42-coated syringes considerably reduced the formation of both visible and subvisible particles after immediate contact and after agitation. The anti-SEB mAb also adhered as a thin layer to the siliconized surface after agitation, irrespective of storage temperature. The development of visible particles could not be correlated with the loss of soluble protein fraction at protein concentrations above 4 mg/mL. It appears that protein formulations interact differently with different surfaces. The BD-42 coating appears to be a promising solution for packaging silicone-sensitive proteins in prefillable syringes and needs to be investigated further. It is demonstrated that BD-42 provides an inert surface with adequate lubrication while limiting the formation of visible and subvisible particles. It is hypothesized that these particles are formed due to the release of silicone droplets in the solution and result in the formation of silicone-induced visible aggregates.     

DISSOCIATION,AGGREGATION OF SESAME α-GLOBULIN IN NONIONIC DETERGENT SOLUTION

Nonionic detergents Triton X-100 and Brij 36T induce dissociation and aggregation of the protein sesame α-globulin above the critical micelle concentrations (cmc) of the detergents. Spectrophotometric titration in Triton shows no change in the pK_Int value of the tyrosyl groups at 1 × 10^-3M detergent where both dissociation and aggregation of the protein are observed. Fluorescence measurement does not indicate any change in the environment of the tryptophan groups of the protein in Brij. Viscosity measurements show no major conformational change of the protein in the detergent solution. Binding measurements suggest that perhaps micelles of the detergent predominantly bind to the protein. The detergent micelles preferentially bind to the exposed hydrophobic surfaces of the protein subunits. The association of the protein detergent complex through electrostatic interaction is probably responsible for the formation of the aggregates.     

Neurodegenerative diseases caused by protein aggregation: a phenomenon at the borderline between molecular evolution and ageing.

A process of protein aggregation that causes intracellular or extracellular accumulation of insoluble protein deposits causes many important neurodegenerative diseases associated with the ageing. The recognition that protein aggregation plays a prominent role in pathogenesis of important pathologies such as Alzheimer's and Parkinson's diseases prompted the scientific community to focus on the molecular mechanism of protein aggregation. Many proteins with sophisticated functions can self-aggregate because their folding is complicate and abnormal intermolecular contacts can predominate over the normal intramolecular interactions. The review of biochemical functional and pathogenic implications attributed to alpha synuclein, A beta peptide, presenilin and apoE highlights for these proteins a common conformational plasticity and the capacity to adapt their secondary structure to surrounding solvent as well as to the contacted ligands. Their functions are not fully elucidated but there is an elevated number of metabolic pathways in which apparently they are involved as well as they generate functional contact with a remarkable number of other proteins. The mechanism by which alpha synuclein and A beta protein make fibrils is an example of conformational plasticity because both these polypeptides can visit a coil or helical structure, but otherwise they convert into a pathogenic beta sheet structure highly suitable for polymerisation and fibril formation. The emerging question in the puzzling pathogenic basis of these diseases is if protein aggregation associated with ageing has a role in molecular evolution of the species or if it just represents a calculated drawback.     

Surface-Induced Protein Aggregation and Particle Formation in Biologics: Current Understanding of Mechanisms,Detection and Mitigation Strategies

Protein stability against aggregation is a major quality concern for the production of safe and effective biopharmaceuticals. Amongst the different drivers of protein aggregation, increasing evidence indicates that interactions between proteins and interfaces represent a major risk factor for the formation of protein aggregates in aqueous solutions. Potentially harmful surfaces relevant to biologics manufacturing and storage include air-water and silicone oil-water interfaces as well as materials from different processing units, storage containers, and delivery devices. The impact of some of these surfaces, for instance originating from impurities, can be difficult to predict and control. Moreover, aggregate formation may additionally be complicated by the simultaneous presence of interfacial, hydrodynamic and mechanical stresses, whose contributions may be difficult to deconvolute. As a consequence, it remains difficult to identify the key chemical and physical determinants and define appropriate analytical methods to monitor and predict protein instability at these interfaces. In this review, we first discuss the main mechanisms of surface-induced protein aggregation. We then review the types of contact materials identified as potentially harmful or detected as potential triggers of proteinaceous particle formation in formulations and discuss proposed mitigation strategies. Finally, we present current methods to probe surface-induced instabilities, which represent a starting point towards assays that can be implemented in early-stage screening and formulation development of biologics.     

Promiscuous beta-strand interactions and the conformational diseases

Conformational change plays an important role in the life of all proteins, starting from when they fold, through their function and often their fate. For an increasing number of proteins inappropriate conformational change leads to a chain of events, which culminate in the deposition of proteinacious aggregates and disease. In this review we consider the current literature on a number of proteins which form part of the Conformational Disease family. We describe here two types of aggregate that can be formed, Type I aggregates are typified by the Serpin superfamily and consist of non-fibrillar polymeric species. Type II aggregates are of the classical fibrillar form formed by a diverse range of proteins. Through biochemical and biophysical analysis of the aggregation reaction of members of these two classes we show that they form these aggregates through highly similar pathways. Essentially, the whole process can be summed up in two key stages. Firstly, the existence of conditions which increase the conformational flexibility of the protein, enabling it to adopt a partially folded state. Secondly, the propensity of this intermediate conformer to form intermolecular linkages leads to multimeric forms, a step often mediated via hydrophobic or beta -strand interactions. Our understanding of these structural changes has facilitated the rationale design of specific aggregation inhibitors. We will discuss the successes and pitfalls of such approaches to demonstrate how similar approaches may be applied to any misfolding protein.     

IgG1 Aggregation and Particle Formation Induced by Silicone–water Interfaces on Siliconized Borosilicate Glass Beads: A Model for Siliconized Primary Containers

Understanding and mitigating particle formation in prefilled syringes are critical for ensuring stability of therapeutic proteins. In the current study, siliconized beads were used as a model for the silicone–water interface to evaluate subvisible particle formation and aggregation of a monoclonal antibody (IgG1). Agitation with siliconized beads greatly accelerated the formation of protein aggregates and particles, an effect that was enhanced at pH 7.4 relative to pH 5 and in the presence of 0.5 M sucrose or 150 mM NaCl. Aggregation and particle formation were minimal in samples agitated without siliconized beads or in quiescent samples with siliconized beads. At pH 5, 0.01% (w/v) polysorbate 20 substantially inhibited aggregation during agitation with siliconized beads, but had minimal protective effect at pH 7.4. Transient exposure of IgG₁ formulations to the silicone–water interface by flowing formulations through a column packed with siliconized beads led to the formation of subvisible particles, with increased levels observed at pH 7.4 compared to pH 5. Agitation of protein formulations in the presence of siliconized glass beads provides a model for baked-on silicone oil–water interface in prefilled syringes and a means by which to evaluate particle formation and aggregation during formulation screening. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:852–865, 2013