Advancing predictions of protein stability in the solid state.

The β-relaxation associated with the sub-glass transition temperature (Tg,β) is attributed to fast, localised molecular motions which can occur below the primary glass transition temperature (Tg,α). Consistent with Tg,β being observed well-below storage temperatures, the β-relaxation associated motions have been hypothesised to influence protein stability in the solid state and could thus impact the quality of e.g. protein powders for inhalation or reconstitution and injection. Why then do distinct solid state protein formulations with similar aggregation profiles after drying and immediate reconstitution, display different profiles when reconstituted following prolonged storage? Is the value of Tg,β, associated with the β-relaxation process of the system, a reliable parameter for characterising the behaviour of proteins in the solid state? Bearing this in mind, in this work we further explore the different relaxation dynamics of glassy solid state monoclonal antibody formulations using terahertz time-domain spectroscopy and dynamical mechanical analysis. By conducting a 52 week stability study on a series of multi-component spray-dried formulations, an approach for characterising and analysing the solid state dynamics and how these relate to protein stability is outlined.EPSR

Tracking Solid State Dynamics in Spray-Dried Protein Powders at Infrared and Terahertz Frequencies

Therapeutic protein powders can be prepared by spray-drying. This process is known to result in solid particles of relatively narrow size distribution and high yield and purity [1], [2]. Additionally, the spray-drying process is rapid, semi-continuous, cost-effective, reproducible and scalable. The process transforms a liquid into dry particles by atomising the liquid feed in a hot drying gas stream [3]. One of the main advantages of spray-drying is that a wide range of formulations, including heat-sensitive materials, can be dried using this technique since the droplet surface will retain the wet-bulb temperature rather than the temperature of the hot drying gas, provided evaporation is taking place at the droplet surface. By the time the evaporation at the droplet/particle surface stops, the drying gas will already have cooled down, thus limiting the heat exposure of the formulation components to the relatively high inlet gas temperatures, and, in combination with the short process duration, making spray-drying a feasible process for heat-sensitive materials, including proteins [1], [2], [3]. While spray-drying is a well established process for small molecules, the additional challenge of ensuring protein stability of the dried product during storage currently limits its use for biopharmaceutical products [2], [4]. A major concern during the spray-drying process is the entire or partial unfolding of proteins due to their high susceptibility to migrate to the air-liquid interfaces where the surface energies can cause the protein to expose hydrophobic regions, resulting in facilitated protein-protein interactions and ultimately aggregation [5]. In order to prevent such undesired aggregation non-ionic surfactants, for example polysorbate, are often used to prevent accumulation of protein at the air-liquid interface, as these small and more mobile surfactants will preferentially position themselves at the interfaces [6]. To put more generally, the excipients of a formulation are vital in providing stability to the protein by maintaining its native conformation during the spray-drying process.T.A.S. and J.A.Z. acknowledge funding from AstraZeneca UK Limited/MedImmune Limited and the UK Engineering and Physical Sciences Research Council (EP/N022769/1). T.A.S. would like to thank the AJA-Karten Trust and the AIA-Kenneth Lindsay Trust for their financial support

Excipient screening for spray drying of monoclonal antibodies

INTRODUCTION & GOALS Spray drying (SD) was selected for converting monoclonal antibody (mAb) solutions into powders for reconstitution, which could increase mAb (storage) stability. The technique is able to yield readily dispersible powders, but addition of excipients is required to stabilise the mAbs during drying and subsequent storage 1, 2. Therefore, a large scale excipient screening was conducted to assess the impact of sugars, surfactant and amino acids (AAs) on mAb stability. METHODS mAB formulations were spray dried using a Büchi B-290 Mini Spray dryer, equipped with a two-fluid nozzle (0.7 mm internal diameter). Feed solutions contained the model mAb at a concentration of 50 mg/mL. For analysis, spray dried mAb powders were reconstituted to 100 mg/mL solutions. Moisture content was analysed using a Metrohm Titrino 831 Coulometer. Aggregation was assessed using a size exclusion chromatography (Tososh TSKgel G3000SWxl column) combined with multi-angle light scattering analysis (Wyatt miniDAWN TREOS), dynamic light scattering (Wyatt Möbius) and image analysis (Occhio ipac). RESULTS & DISCUSSION Addition of a non-ionic surfactant (polysorbate 20) to the formulation maintained the model mAb’s physical integrity during the SD process. Formulations containing a single AA, a combination of two AAs or their respective salts, were unable to adequately stabilise the mAb during 4 weeks of storage at 40°C, although basic AAs were found to stabilise the mAb to a greater extent than other tested AAs. Stability was further improved by combining these AAs with a disaccharide. CONCLUSION Formulations containing a basic AA, a disaccharide and a surfactant were found to have superior mAb stabilising properties compared to other tested formulations. However, further formulation optimisation is deemed necessary, as well as investigating interactions between excipients and identifying process parameters impacting mAb stability. REFERENCES 1. C. J. Roberts, Protein aggregation and its impact on product quality, Curr. Opin. Biotechnol.. 30 (2014) 211 – 217. 2. A. Ajmera, R. Scherließ, Stabilisation of proteins via mixtures of amino acids during spray drying, Int. J. Pharm. 463 (1) (2014) 98 – 107.status: publishe

Excipient screening for spray drying of monoclonal antibodies

PURPOSE Spray drying (SD) was selected for converting monoclonal antibody (mAb) solutions into powders for reconstitution, which could increase mAb (storage) stability. The technique is able to yield readily dispersible powders, but addition of excipients is required to stabilise the mAbs during drying and subsequent storage 1, 2. Therefore, a large scale excipient screening was conducted to assess the impact of sugars, surfactant and amino acids (AAs) on mAb stability. METHODS mAB formulations were spray dried using a Büchi B-290 Mini Spray dryer, equipped with a two-fluid nozzle (0.7 mm internal diameter). Feed solutions contained the model mAb at a concentration of 50 mg/mL. For analysis, spray dried mAb powders were reconstituted to 100 mg/mL solutions. Moisture content was analysed using a Metrohm Titrino 831 Coulometer. Aggregation was assessed using a size exclusion chromatography (Tososh TSKgel G3000SWxl column) combined with multi-angle light scattering analysis (Wyatt miniDAWN TREOS), dynamic light scattering (Wyatt Möbius) and image analysis (Occhio ipac). RESULTS Addition of a non-ionic surfactant (polysorbate 20) to the formulation maintained the model mAb's physical integrity during the SD process. Formulations containing a single AA, a combination of two AAs or their respective salts, were unable to adequately stabilise the mAb during 4 weeks of storage at 40°C, although basic AAs were found to stabilise the mAb to a greater extent than other tested AAs. Stability was further improved by combining these AAs with a disaccharide, where the combination of L-lysineHCl, trehalose and polysorbate 20 was found to stabilise the model mAb to a greater extent than the other formulations. CONCLUSION Formulations containing a basic AA, a disaccharide and a surfactant were found to have superior mAb stabilising properties compared to other tested formulations. However, further formulation optimisation is deemed necessary, as well as investigating interactions between excipients and identifying process parameters impacting mAb stability. REFERENCES 1. C. J. Roberts, Protein aggregation and its impact on product quality, Curr. Opin. Biotechnol.. 30 (2014) 211 - 217. 2. A. Ajmera, R. Scherließ, Stabilisation of proteins via mixtures of amino acids during spray drying, Int. J. Pharm. 463 (1) (2014) 98 - 107.status: publishe

Formulating monoclonal antibodies as powders for reconstitution at high concentration using spray drying: Models and pitfalls

In anticipation of non-invasive routes capable of delivering adequately high, systemic monoclonal antibody (mAb) concentrations, subcutaneous (SC) injection is arguably the most patient friendly alternative administration route available for this drug class. However, due to the limited volume that can be administered through this route and mAbs' relatively low therapeutic activity, solutions for subcutaneous injection often need to be highly concentrated, making them inherently more prone to potentially detrimental protein (self-) interaction, which is why mAb formulations for SC injection and other highly concentrated mAb solutions are often dried to increase their stability. In this work we investigated spray drying (SD) as a drying technique for formulating mAbs as powders for reconstitution, assessing the influence of SD process parameters, as well as excipients present in the feed solution on both mAb stability and relevant powder characteristics for reconstitution using a model mAb. By employing a design of experiments approach, we were able to provide statistically substantiated evidence for the reconstitution time reducing and stability improving properties of l-arginineHCl, l-histidineHCl, l-lysineHCl and polysorbate 20 when combined with a disaccharide in SD mAb powders for reconstitution. Additionally, the study yielded several statistical models describing process parameter influences on relevant powder and mAb stability characteristics.status: publishe

Spray Drying of Monoclonal Antibodies for Subcutaneous Injection: Models and Pitfalls

INTRODUCTION Even though considerable research has been devoted to the drying of monoclonal antibodies (mAbs) and other pharmaceutical proteins, the majority of this research has focused on lyophilisation or spray drying (SD) for inhalation. Powders for reconstitution and subcutaneous (SC) injection need to comply with a significantly different set of product characteristics than those for inhalation. Therefore, following a formulation screening, the impact of spray drying process parameters on both powder characteristics and short- and long-term stability was determined for a model mAb formulation, using a design of experiments (DoE) approach that combined statistical tools with a set of orthogonal analytical techniques. MATERIALS AND METHODS The mAb formulation was spray dried using a ProCepT Micro-Spray with inlet air flow rate, inlet air temperature, nozzle air flow rate, feed rate, feed concentration and nozzle diameter as factors which were varied at three levels according to a D-optimal design. The following responses were measured and evaluated for this study: Yield, angle of repose, residual water content (Karl Fischer titration), reconstitution time, particle size distribution (laser diffraction), particle morphology (SEM), solid state characterisation (XRPD and DSC), aggregation (SEC, DLS, OD600). RESULTS Descriptive statistical models were obtained for several responses, suggesting the inlet air flow rate to be one of the overall most influential process parameters. Large variations in particle morphology were observed with SEM, revealing that particle size distribution models, although statistically significant, were not representative for any relevant powder property. Additionally, the location of powder collection, i.e. collector/cyclone versus transport tube, had a noticeable influence on the particle size distribution of the spray dried powders. CONCLUSIONS Descriptive models were generated for a model mAb, which could prove to be useful tools in improving our understanding of process parameter influences on the mAb stability and particle characteristics of powders for reconstitution. The presented data also emphasises the importance of using orthogonal techniques and keeping the physicochemical meaning of responses in mind when designing a (bio)pharmaceutical drying process.status: publishe

Advancing Predictions of Protein Stability in the Solid State

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Tracking solid state dynamics in spray-dried protein powders at infrared and terahertz frequencies

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Feasibility of electrospraying fully aqueous bovine serum albumin solutions

Electrospraying or electrohydrodynamic atomisation, i.e. the formation of tiny droplets from a jet of conductive liquid under the influence of an electric field, has been gaining in popularity as a particle engineering technique in recent years. In addition to general benefits for particle engineering, e.g. the ability to generate nanometre sized particles with a very narrow size distribution, electrospraying also possesses a number of characteristics, like its applicability at ambient conditions, which could make it especially interesting for formulating therapeutic proteins. However, as fully aqueous solutions of proteins tend to have relatively high electrical conductivities and surface tensions, obtaining a stable Taylor cone-jet mode for these solutions is inherently challenging. This is why in the majority of studies reporting the successful electrospraying of proteins, either emulsions, aqueous suspensions or a mixture of water and one or more organic solvents were used instead of fully aqueous solutions. Therefore, an ab initio electrospraying formulation development study was conducted, using only fully aqueous feed solutions containing protein stabilising excipients commonly used in spray- and freeze-drying of therapeutic proteins. The study included bovine serum albumin (BSA) as a model protein and consisted out of two parts: (1) a one parameter at a time screening study, designed to improve the understanding of how various formulation components influence relevant physicochemical properties and the electrospraying process and (2) two subsequent mixture design of experiments (DoE) studies, designed to aid in the statistical description and prediction of the influence of different protein-excipient combinations on the electrospraying process. Additionally, the influence of physicochemical properties relevant to the electrospraying process, i.e. the volumetric mass density, electrical conductivity, kinematic viscosity and surface tension, was assessed for all feed solutions included in the study.status: publishe