Ectoine and Hydroxyectoine Stabilize Antibodies in Spray-Dried Formulations at Elevated Temperature and during a Freeze/Thaw Process.

Maintenance of protein stability during manufacture, storage, and delivery is necessary for the successful development of a drug product. Herein, the utility of two compatible solutes-ectoine and hydroxyectoine-in stabilizing a model protein labeled Fab2 has been investigated. Specifically, the performance of ectoine and hydroxyectoine in stabilizing Fab2 in a spray-dried formulation at elevated temperature and after multiple freeze/thaw cycles has been compared with the performance of a formulation containing trehalose and a formulation containing no excipient as controls. In the solid state at 90 and 37 °C and in freeze concentrate systems, ectoine and hydroxyectoine suppress protein aggregation. Like trehalose, hydroxyectoine also limits N-terminal pyroglutamate formation in Fab2 in the solid state. The extent of protein stabilization is dependent on the excipient concentration in the formulation, but at a 1:1 excipient to protein mass ratio, hydroxyectoine is better than trehalose in stabilizing Fab2. The results presented here suggest that ectoine and hydroxyectoine are effective excipients for stabilizing therapeutic antibodies.

Protein Stability and Photostability under In Vitro Vitreal Conditions - Implications for Long Acting Delivery of Protein Therapeutics for Ocular Disease.

PURPOSE: To evaluate the stability of a model Mab1 and Fab1 under in vitro vitreal conditions in the presence of various metabolites and in the presence of light at λ > 400 nM. METHODS: A full length IgG1 monoclonal antibody (Mab1) and a fab fragment (Fab1) were formulated with various metabolites typically found in the vitreous humor and subjected to visible light treatment. Both proteins were analyzed using a variety of biochemical techniques. Furthermore, Fab1 was also tested for antigen binding ability using surface plasmon resonance. RESULTS: Our data shows that some vitreal metabolites such as riboflavin and ascorbic acid affect protein stability, via formation of reactive oxygen species (ROS) and advanced glycation end products (AGE) s respectively. In contrast, metabolites such as glutathione may protect these proteins from light-induced degradation to some extent. CONCLUSIONS: Ascorbic acid and riboflavin were found to photosensitize therapeutic proteins especially when exposed to light. Ascorbic acid reacted with proteins even in the absence of light. Antioxidants such as glutathione helped limit photooxidation under ambient or blue light exposure. Since antioxidant capacity in the eye decreases with age we recommend understanding long term stability, including photooxidation and photosensitization, of new candidate proteins in the context of controlled or sustained release technologies for ocular diseases.

Hyaluronic Acid-Antibody Fragment Bioconjugates for Extended Ocular Pharmacokinetics.

Treatment of ocular diseases associated with neovascularization currently requires frequent intravitreal injections of antivascular endothelial growth factor (anti-VEGF) therapies. Reducing the required frequency of anti-VEGF injections and associated clinical visits may improve patient adherence to the prescribed treatment regimen and improve outcomes. Herein, we explore conjugation of rabbit and fragment antibodies (Fab) to the biopolymer hyaluronic acid (HA) as a half-life modifying strategy, and assess the impact on Fab biophysical properties and vitreal pharmacokinetics. HA-Fab conjugates of three distinct molecular weights and hydrodynamic radii (RH) were assessed for in vivo pharmacokinetic performance relative to unconjugated Fab after intravitreal injection in rabbits. Covalent conjugation to HA did not significantly alter the thermal stability or secondary or tertiary structure, or diminish the potency of the Fab, thereby preserving its pharmacological properties. Conjugation to HA did significantly slow the in vivo clearance of Fab from the rabbit vitreous in an RH-dependent manner. Compared to free Fab (observed vitreal half-life of 2.8 days), HA-Fab conjugates cleared with observed half-lives of 7.6, 10.2, and 18.3 days for 40 kDa, 200 kDa, and 600 kDa HA conjugates, respectively. This work elucidates a possible strategy for long-acting delivery of proteins intended for the treatment of chronic posterior ocular diseases.

Trehalose Limits Fragment Antibody Aggregation and Influences Charge Variant Formation in Spray-Dried Formulations at Elevated Temperatures.

The preparation of PLGA rods for sustained release applications via a hot-melt extrusion process employs heat and mechanical shear. Understanding protein stability and degradation mechanisms at high temperature in the solid state is therefore important for the preparation of protein-loaded PLGA rods. The stability of a model protein, labeled Fab2, has been investigated in solid-state formulations containing trehalose at elevated temperatures. Spray-dried formulations containing varying levels of trehalose were exposed to temperatures ranging from 90 to 120 °C. Measurement of aggregation and chemical degradation rates suggests that trehalose limits Fab2 degradation in a concentration-dependent manner, but the effect tends to saturate when the mass ratio of trehalose to protein is around 1 in the solid formulation. The Fab2 secondary structure and spray-dried particle morphology were studied using circular dichroism and scanning electron microscopy techniques, respectively. On the basis of temperature and trehalose-dependent aggregation kinetics as well as changes in spray-dried particle morphology, a mechanism is proposed for the trehalose stabilization of proteins in solid state at elevated temperatures. The results reported here suggest that when fragment antibodies in the solid state are formulated with trehalose as excipient, a high temperature process such as hot-melt extrusion can be successfully accomplished with minimal degradation.

In Situ Characterization of the Microstructural Evolution of Biopharmaceutical Solid-State Formulations with Implications for Protein Stability.

Lyophilized and spray-dried biopharmaceutical formulations are used to provide long-term stability for storage and transport, but questions remain about the molecular structure in these solid formulations and how this structure may be responsible for protein stability. Small-angle neutron scattering with a humidity control environment is used to characterize protein-scale microstructural changes in such solid-state formulations as they are humidified and dried in situ. The findings indicate that irreversible protein aggregates of stressed formulations do not form within the solid-state but do emerge upon reconstitution of the formulation. After plasticization of the solid-state matrix by exposure to humidity, the formation of reversibly self-associating aggregates can be detected in situ. The characterization of the protein-scale microstructure in these solid-state formulations facilitates further efforts to understand the underlying mechanisms that promote long-term protein stability.

Bio-Orthogonal Cross-Linking Chemistry Enables In Situ Protein Encapsulation and Provides Sustained Release from Hyaluronic Acid Based Hydrogels.

Chemically cross-linked hydrogels are promising systems for protein delivery applications, but their utility may be limited due to the possibility of protein reaction with hydrogel precursors. Herein, a catalyst-free inverse-demand Diels-Alder reaction between tetrazine and norbornene groups was used to demonstrate the bio-orthogonal nature of cross-linking chemistry that is chemically inert to proteins. Tetrazine-modified hyaluronic acid and norbornene-modified polyethylene glycol were used as hydrogel precursors for in situ encapsulation of a model protein, Fab1. Measurement of gelation kinetics demonstrates that network formation and gel stiffness are temperature-dependent but independent of Fab1 concentration. In vitro release testing shows that Fab1 is completely released from the hydrogel matrix over a period of several weeks. Analytical characterization suggests that Fab1 is released without any physical or chemical modifications and retains its antigen binding capacity. Thus, the bio-orthogonal and catalyst-free aqueous phase chemistry enables efficient in situ protein encapsulation in a single step and provides sustained protein release.

Characterization of Protein-Excipient Microheterogeneity in Biopharmaceutical Solid-State Formulations by Confocal Fluorescence Microscopy.

Protein-stabilizer microheterogeneity is believed to influence long-term protein stability in solid-state biopharmaceutical formulations and its characterization is therefore essential for the rational design of stable formulations. However, the spatial distribution of the protein and the stabilizer in a solid-state formulation is, in general, difficult to characterize because of the lack of a functional, simple, and reliable characterization technique. We demonstrate the use of confocal fluorescence microscopy with fluorescently labeled monoclonal antibodies (mAbs) and antibody fragments (Fabs) to directly visualize three-dimensional particle morphologies and protein distributions in dried biopharmaceutical formulations, without restrictions on processing conditions or the need for extensive data analysis. While industrially relevant lyophilization procedures of a model IgG1 mAb generally lead to uniform protein-excipient distribution, the method shows that specific spray-drying conditions lead to distinct protein-excipient segregation. Therefore, this method can enable more definitive optimization of formulation conditions than has previously been possible.

Hydroxypropyl methyl cellulose derivatives stabilize fragment antibody against aggregation in spray dried formulations at elevated temperature and resist pH changes.

The ability to deliver stable and active dried protein therapeutics from biopharmaceutical drug delivery systems is critical for solid dosage formulation development. Spray dried formulations with carefully selected excipients provide a unique opportunity in amorphous phase stabilization of the therapeutic proteins. Herein, we discuss the role of hydroxypropyl methylcellulose acetate succinate (HPMCAS) derivatives as polymeric excipients for stabilizing a model fragment antibody (Fab2) during high temperature processing and in possible low pH environments of a drug delivery platform. The effects of high temperature processing and microenvironmental pH sensitivity are of particular interest to us due to their adverse impact on stability of molecules that demonstrate temperature and pH dependent inactivation within drug delivery devices. It appears in solid state at 90 °C and 37 °C and within low pH micro-environment HPMCAS protects protein against aggregation. The high temperature performance of HPMCAS is comparable to that of a disaccharide excipient like trehalose in spray dried protein powder. Simultaneously, inside a poly(lactic-co-glycolic acid) (PLGA) based delivery system HPMCAS provides protection to a pH sensitive protein against acidic degradation products from aqueous hydrolysis of PLGA.

Design of PLGA-Based Drug Delivery Systems Using a Physically-Based Sustained Release Model.

An extensive data set has been developed and used to further the progress of a model-informed design of controlled drug release. An improved drug-release model with mechanistic modeling of hydrolytic polymer degradation is used and validated by comparing model predictions to in vitro experiments. Combining parameter estimates from the literature with model fits to the data set, this study can aid in achieving a priori design of controlled drug release from a model PLGA release system. A systematic series of model release systems were formulated with FITC-labeled dextran, as a surrogate for biopharmaceuticals, in PLGA rods over a broad range of compositions. While general comparisons between the model and experiments were favorable, important discrepancies were identified for several formulations with significant first-phase drug release. Supported by cross-sectional fluorescence microscopy images of the FITC-dextran distribution within the rods, this first-phase release was attributed to a combination of two main factors: (1) percolation of the drug particles and (2) swelling of and pore formation in the rods due to water uptake. These observations indicate the importance of careful selection of the PLGA polymer grade when designing drug release systems but also reflect a need for better understanding of phenomena such as pore formation. Adapting model parameters, without modifying the physical processes included in the model, enabled accurate fitting of the experimental data for all formulations, highlighting the applicability of the model.

Long-Term Stability of Anti-Vascular Endothelial Growth Factor (a-VEGF) Biologics Under Physiologically Relevant Conditions and Its Impact on the Development of Long-Acting Delivery Systems.

The port delivery system with ranibizumab (PDS) is an investigational long-acting drug delivery system for the continuous release of ranibizumab, an anti-VEGF biologic, in the vitreous humor. The efficacy of the PDS implant relies on the maintenance of long-term drug stability under physiological conditions. Herein, the long-term stability of three anti-VEGF biologics - ranibizumab, bevacizumab and aflibercept - was investigated in phosphate buffered saline (PBS) at 37 °C for several months. Comparison of stability profiles shows that bevacizumab and aflibercept are increasingly prone to aggregation whereas ranibizumab undergoes minimal aggregation. Ranibizumab also shows the smallest loss in antigen binding capacity after long-term incubation in PBS. Even though the aggregated forms of bevacizumab and aflibercept bind to VEGF, the consequences of aggregation on immunogenicity, implant function and efficacy are unknown. These results highlight the importance of maintaining long-term drug stability under physiologically relevant conditions which is necessary for achieving efficacy with an in vivo continuous drug delivery device such as the PDS implant.