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Physical characterization of Tobramycin Inhalation Powder: II. State Diagram of an Amorphous Engineered Particle Formulation Dan Miller, Trixie Tan, John Nakamura, Richard Malcolmson, Tom Tarara, and Jeff Weers Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 18 Apr 2017 Downloaded from http://pubs.acs.org on April 19, 2017

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Molecular Pharmaceutics

Physical characterization of Tobramycin Inhalation Powder: II. State Diagram of an Amorphous Engineered Particle Formulation

Authors: Dan Miller, Trixie Tan, John Nakamura, Richard Malcolmson, Tom Tarara, and Jeff Weers

Abstract:

Tobramycin Inhalation Powder (TIP) is a spray-dried engineered particle formulation used in TOBI® Podhaler®, a drug/device combination for treatment of cystic fibrosis. A TIP particle consists of two phases: amorphous, glassy tobramycin sulfate and a gel-phase phospholipid (DSPC). The objective of this work was to characterize both the amorphous and gel phases following exposure of TIP to a broad range of relative humidity and temperature. Because, in principle, changes in either particle morphology or the solid-state form of the drug could affect drug delivery or biopharmaceutical properties, understanding physical stability was critical to development and registration of this product. Studies included morphological assessments of particles, thermal analysis to measure the gel-to-liquid crystalline phase transition (Tm) of the phospholipid and the glass transition temperature (Tg) of tobramycin sulfate, enthalpy relaxation measurements to estimate structural relaxation times, and gravimetric vapor sorption to measure moisture sorption isotherms of TIP and its components. Collectively, these data enabled development of a state diagram for TIP – a map of the environmental conditions under which physical stability can be expected. This diagram shows that, at long-term storage conditions, TIP is at least 50°C below the Tg of the amorphous phase and at least 40°C below the Tm of the gel phase. Enthalpy relaxation measurements demonstrate that the characteristic structural relaxation times under these storage conditions are many orders of magnitude greater than that at Tg. These data, along with long-term physicochemical stability studies conducted during product development, demonstrate that TIP is physically stable, remaining as a mechanical solid over timescales and conditions relevant to a pharmaceutical product. This met a key design goal in the development of TIP: a room-temperature-stable formulation (three years of shelf-life) that obviates the need for refrigeration for long-term storage. This has enabled development of TOBI Podhaler – an approved inhaled drug product that meaningfully reduces the treatment burden of cystic fibrosis patients worldwide.

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Keywords: spray drying, pulmonary delivery, amorphous, structural relaxation, glass, state diagram, TOBI Podhaler


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Introduction

Tobramycin Inhalation Powder (TIP) is a key element in TOBI® Podhaler®, an integrated drug product for the treatment of cystic fibrosis (CF). This drug-device combination product relieves a CF patient’s burden of treatment, reducing the average administration time from 20 minutes (for TOBI®, a nebulized treatment) to about 6 minutes1. Beyond the time saved during dose administration, use of a device that is disposed after a week of treatment relieves the patient and caregivers from additional time spent cleaning and disinfecting nebulizer equipment. Additionally, the portability of the TOBI Podhaler device obviates the need for a power supply or refrigeration. Collectively, these benefits markedly reduce a patient’s burden of treatment. Given that preservation of lung function in persons with CF relies upon adherence to therapy, advances in existing therapies that increase patient adherence improve therapeutic outcomes2-6. TOBI Podhaler is approved in more than 70 countries worldwide. As a drug product, TOBI Podhaler integrates three key elements: an engineered powder, the primary package (a hard capsule), and an inhalation device. This work will focus on the powder. TIP is the first approved engineered dry powder manufactured using the PulmoSphere™ formulation technology. Part I of this work provided a broad overview of the powder’s physicochemical properties, demonstrating that Tobramycin Inhalation Powder comprises small, low-density, porous particles7. Raman microspectroscopy and X-ray photoelectron spectroscopy (XPS) data provide support for a two-phase model of TIP on a particle level. X-ray powder diffraction and differential scanning calorimetry (DSC) data show that the tobramycin sulfate is amorphous with a glass transition temperature above 100°C and that the main excipient, distearoylphosphatidylcholine (DSPC), is in a gel state that undergoes a gel-to-liquid-crystalline transition at approximately 80°C. Particle engineering enables control of particle architecture. By design, the main excipient (DSPC) is concentrated in a shell at the particle surface and the tobramycin sulfate drug substance is present within the core of the particle. In such core-shell particles, the hydrophobic, porous surface reduces cohesive forces and, therefore, enables high-efficiency delivery of these particles to the lungs. In addition to efficient and reproducible aerosol performance, the physicochemical stability of the drug and the particles is important. As a result of the manufacturing process, tobramycin sulfate exists as an amorphous solid in TIP. Amorphous materials pose challenges both in terms of physical and chemical stability. In addition to the ACS Paragon Plus Environment


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physicochemical stability concerns of any amorphous, solid dosage form, inhalation products carry additional development challenges. Even in the absence of recrystallization or chemical instability, changes in the size, morphology, or surface composition of particles could affect aerosol performance. Thus, amorphous drugs in inhalation products are conventionally viewed as having higher development risk than crystalline forms. This perceived risk escalates when amorphous, engineered materials are compared with most dry powders for inhalation, which are based on ordered mixtures of a micronized, crystalline drug with crystalline carrier particles, such as lactose. Thus, product development risk mitigation and regulatory concerns provide ample motivation to thoroughly understand the solid-state phase behavior of TIP. Beyond undertaking risk-mitigation exercises to assess so-called “developability” 8, health authorities expect applicants to demonstrate thorough characterization and control of the solid-state form. This motivated much of the work presented here. Because of the glassy nature of tobramycin sulfate, and the high drug loading in TIP, the physical stability of the engineered particles is a particular concern. To assess physical stability, a comprehensive study was designed in which the solid-state properties of TIP were characterized following exposure to a broad range of temperature and humidity. Of particular concern was whether recrystallization of tobramycin occurs at pharmaceutically relevant conditions and timescales, e.g., ICH storage conditions typical for Registration Stability9. Multiple physical characterization techniques were used to characterize TIP on both the bulk powder and particle levels. Particle morphology was qualitatively assessed using scanning electron microscopy (SEM). Gravimetric vapor sorption was used to measure the moisture sorption isotherm of TIP – its water content as a function of relative humidity. The solidstate morphic form of DSPC and tobramycin sulfate in TIP bulk powder were assessed using differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD), respectively. Structural relaxation times of glassy tobramycin sulfate were assessed using high-sensitivity differential scanning calorimetry.

Materials and Methods Materials


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To understand the solid-state phase behavior of TIP, it was necessary to also characterize the phase behavior of its individual components. Thus, a vehicle formulation, PulmoSphere Placebo Inhalation Powder (hereafter be referred to as “placebo”), was manufactured using a process and scale of manufacture similar to that used for TIP. To characterize the phase behavior of amorphous tobramycin sulfate in the absence of excipients, a powder was prepared by spray drying an aqueous tobramycin sulfate feedstock. The composition of TIP and placebo, and a description of the manufacturing process and materials used were provided in the previous publication7. To assess the response of materials to various relative humidity environments, bulk, unpackaged powders (TIP, placebo, and tobramycin sulfate) were stored for at least five days in the headspace of saturated salt solutions (at either 25°C or 40°C) in vacuum desiccators or in stability chambers. All salt solutions were prepared using HPLC-grade water and ACS-grade reagents, and all were purchased from Sigma and used without further purification (see Table 1).

Table 1: Saturated salt solutions used to control relative humidity at either 25°C or 40°C 10.

% RH

Temperature (°C)

Salt solution (or alternative approach)

0 %RH

25°C

(phosphorus pentoxide)

11.3 %RH

25°C

lithium chloride

17.6 %RH

25°C

lithium iodide

22.5 %RH

25°C

potassium acetate

32.8 %RH

25°C

magnesium chloride

43.2 %RH

25°C

potassium carbonate



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60 %RH

25°C

(stability chamber)

75.3 %RH

25°C

sodium chloride

11.2 %RH

40°C

lithium chloride

22.7 %RH

40°C

potassium fluoride

31.6 %RH

40°C

magnesium chloride

48.4 %RH

40°C

magnesium nitrate

75 %RH

40°C

(stability chamber)

Methods Methods for X-ray powder diffraction (XRPD), modulated differential scanning calorimetry, and scanning electron microscopy were provided in the previous publication7. Additional methods used in this work are provided below.

Dynamic Vapor Sorption Moisture sorption isotherms at 25°C were measured using a DVS-1000 dynamic vapor sorption (DVS) instrument. This instrument gravimetrically measures uptake and loss of water vapor by a material. The moisture sorption isotherm of a given material is the relationship between its water content and the relative humidity (RH) at a given temperature. The DVS system is equipped with a recording microbalance with a resolution of ±0.1 µg and a daily drift of approximately ±1 µg. In the first step of the experimental run, the sample was dried at 25°C and 0%RH for about 2000 minutes to bring the sample to a constant mass. Then, the instrument was programmed from 0 to 2%RH, to 5% RH, and then RH was increased in steps of 5% RH to 90% RH and decreased in steps of 10%RH from 90% to 0% RH (including an extra step at 5%RH). An equilibration criterion of dm/dt =0.005%/min ACS Paragon Plus Environment

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was chosen for the system to achieve at each RH step before automatically proceeding to the next RH step. A sample mass of about 10 mg was used in this study.

Thermogravimetric Analysis The weight loss upon drying of each powder was measured using either a TGA-2950 or Q5000IR instrument made by TA Instruments. To increase the sample mass and sensitivity, a disc of powder was prepared using a 4 mm diameter custom stainless steel press. Gentle pressure was applied to produce a 6 to 14 mg disc that fits inside the sample pan. A previous study showed that the pressures employed do not influence the mass loss (thermogravimetric analysis) or the phase transition temperatures and enthalpies (differential scanning calorimetry) of these materials. The TGA was equilibrated at 30°C and then heated to 110°C at 10°C/min. The temperature was held at 110°C for at least 90 minutes. The % weight loss was calculated between the initial sample mass and the sample mass at 60 minutes.

Karl Fischer Titrimetry The water content of TIP powders was measured using coulometric Karl Fischer titrimetry. The Karl Fischer system used consists of a Metrohm model 831 Karl Fischer Coulometer, a model 800 Dosino, and a model 774 Oven Sample Processor. To analyze a sample, the autosampler inserts the sample vial into the oven at 130°C. Dry nitrogen (60 ± 5 mL/min) flows over the heated sample to carry the evaporated water to the titration cell where it reacts with dry Karl Fischer reagent. Titration ends when the drift falls below 5 µg/min. System performance was checked by measuring the water content of standard samples of potassium citrate monohydrate (Hydranal-Water Standard KF Oven, Cat. No. 34748) before and after measurements of the samples of unknown water content. For each sample, about 20 to 50 mg of TIP powder was weighed into a glass vial, which was crimped and then placed on the autosampler and measured within 12 hours. Samples were prepared in triplicate. To measure the water contributed from the KF system and the environment, three empty ‘blank’ vials were prepared at the same RH and temperature conditions as used for preparation of the powder samples. The average blank water content was subtracted from the water titrated from each sample. ACS Paragon Plus Environment


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High-Sensitivity Differential Scanning Calorimetry (HS-DSC) Isothermal enthalpy relaxation experiments were conducted using a Setaram Micro DSC III (Setaram Inc., Caluire, France). Approximately 700 mg of spray-dried tobramycin sulfate was sealed into a 0.85 cm3 stainless steel ampoule. To reduce the effect of moisture redistribution between the powder and the headspace in the ampoule during heating, the headspace volume was minimized by tamping the powder. Thus, each experiment was conducted at nearly constant water content. The thermal history of the sample was controlled by heating at 1°C/minute to about 20°C above the calorimetric Tg and then cooling at the same rate to an annealing temperature (below Tg) at which enthalpy relaxation was monitored for six hours. To reduce the effects of moisture redistribution and the presence of temperature gradients across the sample during establishment of thermal equilibrium, the first 15 minutes of data were ignored in the analysis of isothermal enthalpy relaxation. Also, because relaxation rate decreases with time, most of the meaningful heat flow signal was within the first few hours of the six-hour aging period. To avoid overfitting the noise in the baseline at long times, only the data up to four hours were fit. To determine structural relaxation parameters, the Levenberg-Marquardt algorithm was used to perform a non-linear least squares minimization (Microcal OriginPro 2016, Northampton, MA) of each set of relaxation data using the differentiated form of the modified stretched exponential (MSE) equation11,12,13 . The detailed methodology for determination of structural relaxation parameters is provided elsewhere14.

Results and Discussion

Influence of long-term storage on particle architecture of TIP The solid-state phase composition of the TIP core/shell particle is a consequence of the formulation and process technology used in its manufacture. Under the spray-drying process conditions employed, an atomized droplet becomes a solid particle over a timescale on the order of 10 milliseconds 15. Due to its molecular size and conformational flexibility, tobramycin (or tobramycin sulfate) cannot recrystallize over the short timescale of spray drying. Instead, it becomes an amorphous, glassy solid upon particle formation. Using ACS Paragon Plus Environment

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multiple orthogonal physical characterization techniques (DSC, XRPD, ESCA, and Raman), we showed in previous work7 that a TIP particle consists of two phases: a core of amorphous, glassy tobramycin sulfate with a glass transition temperature (Tg) of about 100°C and a shell that predominantly comprises a gel-phase (Lβ’) phospholipid (DSPC) with a gel-to-liquid-crystal transition temperature of about 80ºC. Note that this ‘chain-melting’ transition is due to an increase in the degree of disorder of the two acyl chains of DSPC; it occurs without impairing the long-range order of the phospholipid. At temperatures well below Tm, the chains are in an all-trans configuration (designated as Lβ’). Above Tm, the chains become disordered (Lα), with introduction of a number of gauche conformers. In contrast to crystalline solids, the molecules in a glassy material do not reside in fixed positions in a three-dimensional lattice. Because of this, glasses have long-range molecular mobility and, under some conditions, can undergo viscous flow over practical time scales. Because this could affect the particle size and morphology, it was of interest to qualitatively assess particle morphology over timescales relevant to long-term product storage. As part of stability testing during product development, blister packages containing TIP in hydroxypropyl methylcellulose (HPMC) capsules (TOBI Podhaler Capsules) were exposed to 25°C/60%RH and 30°C/75%RH for 36 months. SEM images of tobramycin inhalation powder show that both exposed and unexposed powders consist of spherical, porous particles (Figure 1). Based on a qualitative assessment of these images, there is no change in particle morphology over long-term stability testing at these storage conditions; all powders consist of spherical, porous particles. The physical stability of the particles was also quantitatively demonstrated via assessments of product performance during long-term stability studies. The aerodynamic particle size distribution and the delivered dose uniformity – aerosol properties relevant to delivery of a therapeutic dose – remain unchanged over a three-year shelf life (data not shown). Collectively, these data demonstrate that core/shell particles in the filled, blister-packaged capsules are physically stable, remaining as mechanical solids over timescales and conditions relevant to pharmaceutical products.



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Figure 1. Scanning electron microscopy images of TIP at time t=0 and after storage of packaged product at 25°C/60%RH or 30°C/75%RH for 36 months. The scale bar in the images represents 5 µm.

Moisture sorption isotherm The moisture sorption isotherm of a material provides the relationship between water content and relative humidity (RH) at a given temperature. This is important in inhaled product development because, while water content is often measured and reported on a certificate of analysis, relative humidity is usually the governing variable for moisture uptake during manufacture, storage, and product use. To determine how RH affects water content, the moisture sorption isotherms of TIP, spray-dried tobramycin sulfate, and placebo were measured using gravimetric (or dynamic) vapor sorption (DVS). Figure 2 shows the moisture sorption isotherms (25°C) of these materials. The isotherms of the two PulmoSphere powders were mathematically adjusted using KF titrimetry data. This approach is necessary because phospholipid-containing powders cannot be completely dried under the mild drying conditions typically employed during DVS measurements (in the first step of a typical DVS experiment, the powder is dried at 0%RH at 25°C until a constant mass is attained). Thus, the water associated with the phospholipid head groups was determined using KF titrimetry, which measures the total water liberated by vaporization at ACS Paragon Plus Environment

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high temperatures (>100°C). This approach was validated by confirming that the water content given by the adjusted moisture sorption isotherm agrees with that measured on an independent sample using KF titrimetry (i.e., a sample that was not used to adjust the moisture sorption isotherm).

Figure 2. Moisture sorption isotherms of TIP, tobramycin sulfate, and placebo at 25°C. The calculated isotherm is based on Equation 1.

The spray-drying process parameters employed to manufacture these engineered particles typically result in a powder with an equilibrium RH between about 5 and 10%RH. Based on the moisture sorption isotherm, this would correspond to water contents between 4.8% and 5.7% w/w. This is consistent with the measured (KF) initial water contents of TIP. The calculated (ideal) moisture sorption isotherm of TIP is also shown in Figure 2 (i.e., the dotted curve). Here, the ideal isotherm of TIP is calculated based on a linear superposition of the isotherms of its two principal components, tobramycin sulfate, and placebo. The weighting factors are based on the composition of the TIP formulation, as given by:

0.85 0.15 ACS Paragon Plus Environment

Equation 1


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where

= calculated water content (g/100 g) in TIP at RHi = measured water content (g/100 g) in tobramycin sulfate at RHi = measured water content (g/100 g) in placebo at RHi

The calculated or ‘ideal’ isotherm of TIP lies between those of tobramycin sulfate and placebo and is closer to that of tobramycin sulfate, which would be expected based on the high drug loading of tobramycin sulfate in TIP.

Although the measured isotherm of TIP closely matches the ideal TIP isotherm for RH>60%, there is a modest deviation at lower RH values. Using a propagation of uncertainty analysis, this difference was found to be statistically meaningful. This deviation is a measure of the molecular interaction between tobramycin sulfate and DSPC in TIP. A related approach was used by Costantino et al. 16 to assess molecular interactions between sugars and proteins in lyophilized mixtures. We hypothesize that, at low-to-moderate RH values, tobramycin interacts with the phospholipid headgroup of DSPC via hydrogen bonding. This results in less water uptake for TIP than would be expected based upon a linear superposition of the isotherms of its two principal components. These interactions are disrupted at much higher water contents (RH>60%), leading to good agreement between the calculated and measured isotherms. This is consistent with the molecular interactions observed in chemically analogous systems, such as phospholipids stabilized by amorphous disaccharides17, 18. Note that calorimetric measurements of the main transition, Tm, support the existence of a molecular interaction between tobramycin and DSPC. The thermogram of TIP shows an endotherm with an extrapolated onset temperature at about 80 °C, whereas the thermogram of placebo (at the same water activity) shows an endotherm at about 89°C7. Thus, tobramycin sulfate depresses Tm to some extent.

Effect of water Like most spray-dried materials, TIP contains residual water. And, because all packaging materials have a non-zero water vapor permeability, the water content of TIP tends to slowly ACS Paragon Plus Environment

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increase over its shelf life. Thus, it is important to understand how residual water affects the transition temperatures relevant to the amorphous tobramycin sulfate phase (Tg) and the gelphase phospholipid (Tm). Moisture uptake increases long-range molecular mobility and depresses Tg. This phenomenon, known as plasticization, is common to amorphous organic materials. Knowledge of the glass transition temperature is important because it is used as a scaling parameter to study multiple phenomena relevant to physical stability. Examples include recrystallization, which can occur at temperatures both above and below Tg, and viscoelastic flow, which begins to occur on practical timescales as temperature is raised somewhat above Tg (or, conversely, when moisture uptake depresses Tg to values below the storage temperature). Water content also affects material properties of phospholipids. The gel-to-liquid-crystal phase transition depends on the extent of hydration, most strongly for phospholipids of low water content19, 20. Thus, knowledge of the relationships between water content and Tg and Tm provides a useful means to understand how environmental conditions (temperature and relative humidity) influence material properties during product manufacture, storage, and use. Results from studies on the effect of water content and temperature also provide some of the fundamental information required for regulatory submissions. Byrn et al. 21 and ICH Q6A22 published a series of decision trees to provide guidance to the drug developer on an approach to demonstrate understanding and control of morphic form. Given that the physical properties of amorphous particles could differ from those of their crystalline counterparts and that the dissolution behavior of such particles in the epithelial lining fluid of the lungs is difficult to measure, the usual outcome of an exercise using these decision trees during inhaled drug product development is threefold: to thoroughly characterize the amorphous form, to demonstrate that it can be reproducibly manufactured, and to demonstrate that it does not change over the course of long-term stability on several representative product batches. Because of the two-phase nature of TIP and the high amorphous content (all of the tobramycin sulfate, which comprises 85% of the formulation, is amorphous), the physical stability of TIP particles across a broad range of temperature and humidity is a concern. To assess physical stability of both the amorphous and gel phases, a comprehensive study was conducted to characterize the solid-state properties of TIP bulk powders following exposure to environments of different relative humidity (at either 25°C or 40°C). To enable interpretation of the results of TIP, experiments were also done on its principal components – spray-dried tobramycin sulfate (prepared at the same H2SO4/ tobramycin free base mole ACS Paragon Plus Environment


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ratio as used in TIP) and PulmoSphere Placebo. After exposure for at least five days using saturated salt solutions in vacuum desiccators or in stability chambers, the water content of each RH-equilibrated powder was then measured using KF titrimetry. Because the KF method requires flowable powders for preparation of samples, this technique could not be used for samples that visibly underwent viscoelastic collapse (i.e., those that flowed during storage at temperatures sufficiently above Tg). In cases of samples with extreme water content, thermogravimetric analysis (TGA) was used to measure the water content, as estimated by the loss on drying. Recrystallization is a concern for the development of any glassy, amorphous drug formulation. This makes it important to understand at what temperatures and RH conditions, if any, would any of the components of TIP begin to recrystallize or change morphic form. XRPD was used to assess the solid-state form of the drug in TIP following processing and during long-term storage. Because the X-ray powder diffraction pattern of TIP is characteristic of a formulation that contains an amorphous drug (see Miller et al. 7 and accompanying discussion), the absence of any sharp diffraction peaks attributable to tobramycin would confirm that the solid-state form of the drug remains unchanged during processing or storage. This study can be thought of as the physical stability analogue of stressed degradation studies commonly used to assess the types of possible chemical degradants and the conditions under which they occur. Stressed (chemical) degradation studies usually involve extreme conditions that are far beyond those expected during routine product manufacturing, storage, and use. Likewise, in this work, an extreme range of conditions was deliberately surveyed to explore a broad range of ‘phase space’. Figure 3 shows a comparison of XRPD patterns of TIP after exposure to various RH values (ranging from 0 to 60%RH) at 25°C. Following direct exposure of the powder to any of the RH and temperature conditions investigated, there was no change in the solid-state morphic form of the drug. These results help establish knowledge of the solid-state landscape of the drug product and, in part, address regulatory expectations to demonstrate that the polymorphic form does not change.


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Figure 3. XRPD patterns of TIP after exposure to relative humidity values as high as 60%RH (25°C).

Here, XRPD was used to qualitatively assess the solid-state morphic form of tobramycin sulfate in TIP. The suitability of this approach as a means to control the solid-state form depends on the sensitivity of the method for detection of small amounts of crystalline material in the amorphous matrix. This necessitates evaluation of the limit of detection (LOD) – the minimum concentration of crystalline tobramycin that could be detected. The LOD of this method was assessed using both empirical and statistical approaches. Using an empirical approach, TIP was spiked with increasing quantities of crystalline tobramycin free base and the LOD was determined from visual inspection of the measured powder patterns. For this work, a finely ground sample of crystalline tobramycin free base was used because there is no commercially available crystalline tobramycin sulfate, nor could a highly crystalline sample be prepared in our laboratories. Furthermore, based on Ostwald’s rule of stages23,24, it is hypothesized that the free base form of tobramycin would be more likely to recrystallize first (given its lower solubility). Figure 4 shows a comparison of the X-ray ACS Paragon Plus Environment


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powder diffraction patterns of the spiked, physical mixtures. From visual inspection of the most intense diffraction peaks (found between 17°2θ and 19°2θ), one can see that peaks indicative of crystalline material are visible at concentrations at and above 4% crystallinity. Thus, the estimated limit of detection (LOD) of crystalline tobramycin in TIP is about 4% crystallinity by this XRPD method. A comparable estimate of LOD was determined using a statistical approach in which the LOD was estimated based on repeated measurements of the baseline noise in the range between 17°2θ and 19°2θ in the powder pattern of TIP.

Figure 4. X-ray powder diffraction patterns of physical mixtures of TIP and crystalline tobramycin free base. Compositions range from 2% to 17% crystallinity. The XRPD patterns are vertically offset for comparison.

It is noteworthy that tobramycin sulfate could not be recrystallized even when samples were stored near or well above their glass transition temperature. Furthermore, when packaged in ACS Paragon Plus Environment

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a tight container (glass vial), spray-dried tobramycin sulfate could not be recrystallized following storage for 10 years in our laboratories at ambient temperature. Several hypotheses can explain these observations. First, tobramycin sulfate in TIP is not a salt form according to the usual definition. Tobramycin sulfate in this formulation could be considered to be co-amorphous – a recently coined term that describes a single, homogeneous phase of low molecular weight components25. In this sense, tobramycin and its counterion comprise an amorphous mixture. Not only does the location of the sulfate counterion vary in the solidstate, but the sulfate:tobramycin mole ratio (approximately 5:3) of this ‘in situ’ salt form is non-stoichiometric. This is empirically supported by the lack of commercially available crystalline tobramycin sulfate as well as our own inability to prepare a highly crystalline tobramycin sulfate sample in our laboratories. Second, excipients are also known to inhibit crystallization26, 27. Given the molecular interaction between tobramycin sulfate and DSPC (Figure 2), TIP would be expected to be even more resistant to recrystallization than tobramycin sulfate (i.e., formulated without DSPC). Third, surface enrichment of DSPC on the surfaces of TIP particles could impede crystallization of tobramycin sulfate. There is a growing body of evidence regarding the greater mobility of molecules at surfaces relative to that in the bulk28, 29. At a given temperature below Tg, surface diffusion in molecular glasses can be several orders of magnitude greater than bulk diffusion. In TIP, DSPC comprises more than 90% of the particle surface, a six-fold enrichment over that in the bulk7. Thus, the reduced surface concentration of tobramycin sulfate in TIP could make it less prone to crystallize. Although X-ray powder diffraction results indicate that the drug (in either TIP or tobramycin sulfate) remains amorphous following exposure to any of the RH and temperature conditions investigated, exposure of unpackaged bulk powder to some conditions resulted in viscoelastic collapse of the samples, as assessed visually. Collapse, as manifested by a change in sample shape, occurred for both tobramycin sulfate and TIP during storage at RH values at and above 60%RH at 25°C and 48%RH at 40°C. This is indicative of viscous flow of the samples. This is expected to occur given that, under such extreme conditions, the glass transition temperature of the unpackaged drug decreases below the storage temperature. For example, equilibration at 60%RH depresses Tg to 15°C (see later discussion of Tg results), conditions sufficient to induce viscous flow. Because of the change in sample shape, the powder patterns of those samples appear qualitatively different from those of the other samples (see, for example, the X-ray powder pattern of TIP at 60%RH in Figure 3). And, because viscous flow would be expected to also occur following exposure to higher RH ACS Paragon Plus Environment


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values at these temperatures, unpackaged samples stored at more aggressive storage conditions were not tested by XRPD. Note that, from visual inspection, these samples were optically clear, pale yellow liquids devoid of crystalline matter. Studies of phase behavior rely on methods such as XRPD and spectroscopic techniques (e.g., Raman, FTIR) to identify structural changes. These provide a useful, empirical means to understand the environmental conditions under which phase changes occur on pharmaceutically relevant timescales. In addition to such studies, thermal analysis (e.g., DSC) provides quantitative information on Tg and thermodynamic phase transition temperatures. Such data are useful because temperature is a key environmental variable during spray drying, packaging, storage, and product use. Figure 5 and Figure 6 show the DSC (total heat flow) thermograms of spray-dried tobramycin sulfate and TIP bulk powders, respectively, following exposure to several relative humidity values at 25°C. These thermograms show that the Tg of spray-dried tobramycin sulfate decreases with increased relative humidity, as would be expected based on the plasticizing effect of water on amorphous organic materials. Similarly, plasticization also depresses the glass transition temperature of the amorphous tobramycin sulfate phase within the TIP formulation (Figure 6). In many cases, the glass transition event is accompanied by a so-called ‘overshoot’ – see, for example, the thermogram of the sample equilibrated at 43.2%RH at 25°C (Figure 6). This overshoot is due to enthalpy recovery of the glass upon reheating. In turn, this recovery is a consequence of structural relaxation of the glass toward the equilibrium, supercooled liquid during storage (and, to some extent, during reheating). The samples that were stored above their Tg values (60%RH and 75%RH at 25°C, 75%RH at 40°C) exhibit no enthalpy recovery because these samples were equilibrium, supercooled liquids at their storage conditions. This provides indirect evidence that these samples underwent viscous flow. When it occurs, enthalpy recovery does not always occur at Tg. At lower relative humidity values, enthalpy recovery occurs at progressively lower temperatures relative to the Tg (see thermograms at 11.3, 22.5, and 32.8%RH in Figure 5). The shift in the enthalpy recovery event and the depression of Tg introduce complexity into the thermograms of TIP as RH is increased. Also, note that desiccation (by storage at 0%RH) leads to changes in the shape of the DSPC main transition, as well as makes the Tg event undetectable (see Figure 6). Efforts were made to measure the Tg values of desiccated samples, including extending the scanning range to higher temperatures and using of extremely fast heating rates. Neither approach was ACS Paragon Plus Environment

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able to clearly discern a Tg of these samples. It is suspected that thermal degradation of the sample at high temperatures precluded measurement of Tg.

Figure 5. DSC thermograms of spray-dried tobramycin sulfate after exposure to several relative humidities at 25°C. (DSC thermograms are offset for clarity)



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Figure 6. DSC thermograms of TIP bulk powder after exposure to several relative humidity values at 25°C. (DSC thermograms are offset for clarity) Figure 7 shows the Tg values of both TIP bulk powder and tobramycin sulfate as a function of water content. The Tg depression behavior, as given by the slopes of the data sets, appears to be similar for both spray-dried tobramycin sulfate and TIP. This would be expected based on the similar composition of the amorphous phase of both of these materials. It is somewhat surprising, however, that the absolute Tg values of both materials at any given water content are so similar given that the water content in the amorphous phase would be expected to differ. That is, because some of the water in TIP will partition into the DSPC phase, it would not be expected to plasticize the (amorphous) tobramycin sulfate phase. Based on this argument, the water content in the amorphous phase of TIP would be lower than the total water content. Thus, at any non-zero water content, the Tg of the amorphous phase in TIP would be expected to be higher than that of tobramycin sulfate. However, given the large difference in the hygroscopicities of tobramycin sulfate and placebo and that DSPC is a minor component of TIP, the expected difference in Tg based on such an analysis would be modest. For example, when compared at 10% w/w water content, the Tg of TIP would be expected to only be about 5°C greater than that of tobramycin sulfate. The Tg vs. water content data of tobramycin sulfate can be fit to the Gordon-Taylor equation30, which has often been used to model plasticization of organic glasses by water31:

Equation 2

Here, w1 and w2 are the mass fractions of tobramycin sulfate and water, respectively, and Tg1 and Tg2 are the glass transition temperatures of tobramycin sulfate and water, respectively. The glass transition temperature of water is not without controversy. While data from the earliest work supports the use of 136K 32, more recent work by Angell and coworkers argues that the glass transition temperature of water is closer to 165K 33, 34. Most recently, in a broad-based analysis of data on glass-forming aqueous mixtures, and water in confined spaces and in bulk, Capaccioli and Ngai concluded that the glass transition temperature of water must be near the generally accepted value of 136K35. ACS Paragon Plus Environment

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The Tg depression data of tobramycin sulfate were fit to the Gordon-Taylor equation using Tg2=136 K, and both Tg1 and k as adjustable parameters. The best-fit values are Tg1=529 K (256 ± 6°C) and k=7.9 ± 0.3 [adjusted R2=0.996]. This k value is greater than those of monosaccharide glasses (ribose, 3.02; glucose, 4.52) and somewhat greater than those of disaccharide glasses (sucrose, 5.42; lactose, 6.56)31. Given that k increases with molecular weight, the k value of tobramycin sulfate follows the trend of amorphous sugar glasses. The best-fit value for the glass transition temperature of tobramycin sulfate, 256°C, provides some support for the experimental difficulty in measuring it. Thermal degradation of an aminoglycoside during heating to such a high temperature would not be unexpected. Note that a high glass transition temperature was a key consideration in selection of the sulfate counterion, as this provides greater physical stability. When compared at the same relative humidity (11.3%RH), the Tg of tobramycin sulfate is nearly 40°C greater than that of the free base (105°C vs. 67°C)7.

Figure 7. Glass transition temperature (Tg) as a function of water content of TIP and tobramycin sulfate (TS).



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Typically, Tg values of amorphous organic materials at low to moderate water contents will decrease by approximately 10 to 15°C for every 1 wt% increase in water content31. Thus, for a range of water contents between 4.8% and 5.7% w/w (typical of TIP), the Tg values would be expected to vary by about 10°C to 15°C. This is consistent with the variation in Tg values of TIP, as determined by differentiation of the Gordon-Taylor equation at the above range of water contents using the best-fit values for tobramycin sulfate (dTg/dw1=17°C/wt% H2O). This well-established relationship between water content and Tg provided the rationale to justify that there is no need to routinely measure Tg of TIP as part of quality control testing; measurement of residual water content is sufficient to control Tg. In addition to the plasticization of the amorphous phase, the water content in the gel-phase phospholipid decreases Tm, as shown in Figure 8. The effect of water on the gel-to-liquidcrystalline transition temperature of phospholipids is well known; several studies have been published on the phase diagram of the DPPC-water system36,19,37. Because Tg has a much stronger dependence on water content than does Tm, these two thermal events cross over one another at about 75°C and 9% w/w H2O. While this effect introduces complexity into the thermograms of TIP as RH is increased, such complexity does not occur in thermograms of samples with (lower) water contents relevant to manufacture and long-term storage of TIP.


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Figure 8. Effect of water on the amorphous and gel phases of TIP. Water plasticizes the amorphous phase and depresses Tg, as well as reduces the gel-toliquid crystal phase transition temperature (Tm) of DSPC.

Molecular mobility of a glassy material can be quantitated using the concept of structural relaxation time. A glass is a non-equilibrium state and, over time, it will relax toward the (hypothetical) equilibrium supercooled liquid. As it relaxes via collective molecular motions, its volume and enthalpy will decrease. These enthalpic changes enable calorimetric measurement of molecular mobility. Generally, the calorimetric Tg is associated with a structural relaxation time on the order of 100 seconds38. As temperature is reduced below Tg, structural relaxation times markedly increase. In this work, these times were determined by fitting isothermal enthalpy relaxation data to the differentiated form of the MSE equation; Sadrzadeh et al. 14 employed a similar approach to determine structural relaxation times of a spray-dried insulin formulation. Figure 9a shows the temperature dependence of the characteristic relaxation function, τ Dβ , of amorphous tobramycin sulfate samples equilibrated at each of three RH values, 11.3, 17.6, and 32.8%RH (corresponding to 6.7, 7.7, and 10.3 %



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w/w H2O, respectively). As expected, plasticization of the amorphous matrix with water (i.e., increasing RH) reduces the structural relaxation time at a given temperature. When plotted as a function of the annealing temperature relative to the glass transition temperature (Tg/T), the structural relaxation times collapse into a narrower band (Figure 9b). This is an example of time-temperature superposition. This scaling relationship is useful in that it enables prediction of structural relaxation times based on Tg/T data. For example, if the Tg at a given water content is known (see Figure 7), Tg/T can be calculated at any temperature below Tg. Then, the principle of time-temperature superposition can be used to estimate the structural relaxation times at that given combination of water content and temperature. This approach was used to plot the family of iso-relaxation time contours shown in Figure 10.

Figure 9. (a) Temperature dependence of the stretched structural relaxation time, τ Dβ , of amorphous tobramycin sulfate samples of different water content. (b) time-temperature superposition of stretched structural relaxation times when expressed on a Tg/T scale.


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A single sample was used for structural relaxation measurements at each water content. A question remains, therefore, on whether repeated heating cycles led to degradation of the sample, ultimately influencing the measured relaxation times. To assess the influence of the sample’s thermal history on the results, enthalpy relaxation at the same isothermal annealing temperature (43°C) was measured at the beginning, middle, and end of a series of isothermal periods. The stretched structural relaxation times ( τ Dβ ) of the samples equilibrated at 11.3 and 17.6%RH each spanned a modest range (mean ± standard deviation are 336±44 hours and 209±4 hours, respectively). Thus, the thermal history of the sample had only a minor effect on the structural relaxation time.

State diagram Development of any solid dosage form requires knowledge of the solid-state phase behavior in the context of the manufacturing, storage, and use conditions. Being a two-phase formulation, development of TIP requires consideration of the phase behavior of both amorphous tobramycin sulfate and the gel-phase phospholipid. Because amorphous materials are inherently less stable than crystalline forms, it becomes especially important to define conditions under which one could expect long-term physicochemical stability. In the previous sections, we have used multiple, complementary analytical techniques to characterize the structural changes in TIP as a function of both RH (or water content) and temperature. This section will focus on integrating those results to put them into the context of development of a stable, engineered particle formulation for inhalation. Collectively, the observations and measurements in this manuscript can be used to construct a state diagram, as shown in Figure 10. Lechuga-Ballesteros et al. 39 have constructed state diagrams for other materials, including excipients (amorphous sucrose) and amorphous solid dispersions (aspartame or Asn-hexapeptide stabilized in a PVP matrix). Here, the term ‘state diagram’ is preferred over the more familiar term ‘phase diagram’ because amorphous materials are non-equilibrium systems and Tg is not a thermodynamic parameter (in contrast to Tm). The state diagram provides useful information regarding the solid-state behavior of TIP as a function of water content and temperature – two key variables during spray drying, packaging, long-term ACS Paragon Plus Environment


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storage, and even product use. Note that the diagram could also be expressed in terms of RH instead of water content. Although RH is an environmental variable, perhaps making it a more natural variable for use in a state diagram, water content is used here because it is an absolute quantity (i.e., it is not a function of temperature). In addition to the Tg and Tm data, two key features are inscribed on the state diagram. First, there is an area that denotes the environment of the particles during spray-drying (the temperature and the water content of particles at the outlet of the drying chamber). Second, there is a box that delineates the range of water content and temperature that could be experienced by TIP during long-term storage. The diagram is supplemented with structural relaxation time data; each short, dashed line below the Tg curve represents a given relaxation time. These contours were determined using time-temperature superposition of the data in Figure 9.

Figure 10. State diagram of TIP, with ‘phase’ regions delineated by Tg and Tm curves (TS=Tobramycin sulfate, DSPC= distearoylphosphatidylcholine). The short, dashed curves represent iso-relaxation time (τ Dβ ) contours of amorphous tobramycin sulfate.


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Note that the state diagram covers a broader range of temperature and water content (or RH) than would be experienced by TIP during long-term storage at typical conditions (25°C to 40°C). This design space was selected to encompass these conditions as well as short-term conditions, such as those experienced by TIP during spray drying and packaging. The state diagram contains information regarding environmental conditions (temperature and water content) as well as timescales. All are important to describe a non-equilibrium system such as a glass. For example, under the conditions incurred during spray drying, TIP would have a Tg much greater than 100°C and Tm of about 80°C. Thus, during spray drying, the processing temperatures would be below both the Tg and Tm of TIP. Furthermore, the structural relaxation time under these conditions is several-fold longer than the duration of spray drying, minimizing structural relaxation of the amorphous tobramycin sulfate within the particles. In contrast to the timescale of spray drying (hours), long-term storage requires stability over multiple years. One must also consider that the water content of the product could rise due to moisture uptake during storage, especially under hot, humid conditions such as those found in Climatic Zone IV (30°C/65%RH or 30°C/75%RH, depending on the region)40 or accelerated storage conditions (40°C/75%RH). By design, the materials (aluminum foil-foil blister) and process used for packaging TOBI Podhaler capsules control the water content of the powder within a narrow range, below 10% w/w over several years of storage. Based on the state diagram, the storage temperature (30°C) would be well below Tg (and Tm), even for a water content as high as 10% w/w. Under these conditions, the stretched relaxation time, τ Dβ , is on the order of 80 (with τ expressed in units of hours), with a mean stretching parameter (β) of approximately 0.3. Using this stretching parameter, a τ Dβ value of 80 would correspond to a τ D value that is exceptionally longer than the long-term storage times. The physical stability of TIP particles is well supported by stability studies. Over storage for as long as three years, TIP has remained amorphous; crystalline drug has never been detected. Beyond solid-state form, particle morphology has remained stable over this period, as determined by SEM and aerosol performance measurements.



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The physical and chemical stability of TIP was a key consideration in formulation design. Heretofore, the discussion has focused on physical stability with respect to minimizing the molecular mobility that could lead to recrystallization and viscoelastic flow (related to Tg) and phospholipid phase changes (related to Tm). It is of interest to explore the relationship between the solution-state and solid-state chemistry of tobramycin. In dilute aqueous solution at neutral pH, Brandl and Gu show that the predominant degradation mechanism for tobramycin is auto-oxidation41. In their study, degradation was investigated at 60, 80, and 100°C. The kinetic data followed an Arrhenius relationship which, when extrapolated to ambient temperature, resulted in degradation rates on the order of 7%/year. In contrast, the measured degradation rates of solid-state, glassy tobramycin in TIP are much lower. Long-term stability studies conducted during development as part of TIP product registration show no significant chemical degradation of tobramycin, with the sum of degradation products being 0.3% over four years at 25°C. This nearly 100-fold difference in the degradation rates in solution and in the solid state suggests that the molecular rearrangements involved in solid-state degradation of tobramycin are diffusion controlled. The observed solid-state chemical stability of TIP under long-term storage conditions would be consistent with a diffusion-controlled reaction with markedly slowed kinetics, as supported by the long structural relaxation times in this region of the state diagram. Note, however, that it is also possible that the solution and solidstate degradation processes proceed via different mechanisms. While development of a state diagram is retrospective in nature, the physicochemical stability of TIP is by design. The counterion (sulfate) and the phospholipid (DSPC) were selected to provide Tg and Tm values, respectively, that are well above the temperatures used for storage of TIP (25ºC and 40ºC). Also by design, the packaging materials and process conditions selected for packaging TOBI Podhaler capsules control the water content of the powder over a narrow range over several years of storage. Based on the state diagram, even for a water content as high as 10% w/w, pharmaceutically relevant storage temperatures would be well below Tg (and Tm) of TIP. Thus, the TIP state diagram illustrates that the molecular mobility of amorphous tobramycin sulfate is low and the gel-phase phospholipid is maintained, favoring stability under long-term storage conditions.


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Conclusions

Physicochemical stability is critical to development and registration of dry powder inhalation products. Changes in particle size, morphology, or surface composition could affect aerosol performance and drug delivery to the lungs. Likewise, changes in solid-state form could influence biopharmaceutical properties. This was an especially important consideration during development of TOBI® Podhaler® because the drug exists as an amorphous solid in TIP. Besides concerns regarding physical stability, amorphous materials also tend to have lower chemical stability. Thus, demonstration of thorough characterization and control of the solid-state form and product performance was not only important during product development, but also a regulatory expectation. Because of the glassy nature of the tobramycin sulfate and the high drug loading in TIP, the physical stability of the engineered particles was of particular concern, as would be the case for any amorphous drug product. Besides plasticization of the drug by water, the gel-to-liquid-crystal phase transition of the main excipient (DSPC) depends on the extent of hydration. Thus, to assess physical stability of both the amorphous and gel phases, a comprehensive study was designed in which the solidstate properties of TIP were characterized following exposure to a broad range of relative humidity and temperature. Short-term studies, such as the comprehensive RH/T study described in this work, provided a useful means to survey ‘phase space’ and to develop a state diagram for TIP. This diagram helps establish the environmental (RH and temperature) conditions under which physical stability can be expected. For storage of TIP of typical water content under ICH storage conditions for Registration Stability (either 25°C/60% RH or 40°C/75% RH) the storage temperature is about 50°C to 70°C lower than the Tg of the amorphous ‘phase’ and about 40°C to 60°C lower than the Tm of the gel phase. Thus, storage under such conditions reduces long-range molecular mobility, resulting in improved physical and chemical stability. Enthalpy relaxation measurements demonstrate that the structural relaxation times under these storage conditions are many orders of magnitude greater than at Tg. This is further supported by long-term stability studies conducted during product development. After exposure of packaged drug product to either 25°C/60%RH or 30°C/75%RH for at least three years, there was no change in solid-state form. The extent of chemical degradation of tobramycin ACS Paragon Plus Environment


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was negligible. SEM images showed no change in particle morphology, and aerosol properties relevant to delivery of a therapeutic dose remained unchanged. This demonstrates that the glassy particles are physicochemically stable, remaining as mechanical solids over timescales and conditions relevant to pharmaceutical products. Overall, rational formulation design has enabled a room-temperature stable product – the first porous engineered particle approved for inhaled delivery. As part of a thorough characterization effort during development, it was demonstrated that TIP can be reproducibly manufactured and that it does not change over the course of longterm stability. This met a key design goal in the development of TIP: a roomtemperature-stable formulation (three year shelf-life) that obviates the need for refrigeration for long-term storage. Most importantly, particle engineering has enabled development of TOBI® Podhaler® - an approved inhaled drug product that meaningfully reduces the treatment burden to cystic fibrosis patients worldwide.


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