Amorphous Excipients Research Articles

Mechanical Characterization of Vial Strain During Freezing and Thawing Operations Using Amorphous Excipients

The purpose of this study was to investigate the mechanical stresses and strains acting on pharmaceutical glass tubing vials during freezing and thawing of model pharmaceutical formulations. Strain measurements were conducted inside of a laboratory-scale freeze-dryer using a custom wireless sensor. In both sucrose and trehalose formulations at concentrations between 5 % and 20 % w/v, the strain measurements initially increased before peaking in magnitude at temperatures close to the respective glass transition temperatures of the maximally freeze concentrated solutes, Tg’. We attribute this behavior to a shift in the mechanical properties of the frozen system from a purely elastic glass below Tg’ to a viscoelastic rubber-like material above Tg’. That is, when the interstitial region becomes mechanically compliant at temperature above Tg’. The outputs were less predictable below 5 % w/v and tended to exhibit two separate peaks in strain output, one near the equilibrium melting temperature of pure ice and the other near Tg’. The peaks merged at concentrations between 4 and 5 % w/v where the largest strain magnitude was observed. The strain on primary packaging has traditionally been applied to evaluate the risk of damage or breakage due to, for example, crystallization of excipients. However, data collected during this study suggest there may be utility in formulation design or as a process analytical technology to minimize potentially destabilizing stresses and strains in the frozen formulation.

Polymorphism of Purpurin and Low-level Detection of the Noncentrosymmetric form by Second Harmonic Generation Microscopy

Nonlinear optical imaging based on second harmonic generation (SHG) provides rapid and highly selective detection of polar crystals. Purpurin (PUR) is a natural product with multiple pharmacological activities. Two polymorphs of PUR show distinct crystal packing and structural symmetry, where form I crystallizes in a polar space group and form II crystallizes in a centrosymmetric crystal structure. The two polymorphs are monotropically related, with form I being the thermodynamically stable form, as suggested by slurry experiments, in-situ Raman spectroscopy and crystal structure prediction (CSP). The specificity of SHG to the polar crystals of form I allows rapid polymorphism detection at the limit of individual crystals. SHG is also able to detect low levels of form I in a tablet matrix dominated by amorphous excipients. This study shows that SHG microscopy can achieve the rapid and sensitive detection of noncentrosymmetric crystals in solid dosage forms, which is especially helpful for the early detection of unwanted polymorphic conversion or crystallization of amorphous drugs in formulations and final products.

Mannitol hemihydrate in lyophilized protein formulations: Impact of its dehydration during storage on sucrose crystallinity and protein stability

The high propensity of mannitol to crystallize in frozen solutions along with its high eutectic temperature enabling higher primary drying temperatures makes it a good bulking agent. In protein formulations, addition of a sugar (sucrose) that has the ability to remain amorphous throughout processing as well as storage is imperative to retain the protein in its native state. It is well known that in the presence of amorphous excipients and protein, mannitol can crystallize as a mixture of anhydrous polymorphs - α-, β- and δ-forms and a hemihydrate form [mannitol hemihydrate (MHH); C6H14O6·0.5H2O]. The conditions of formation of MHH due to processing and formulation variables are well established in the literature. However, MHH’s dehydration kinetics on storage and its impact on the stability of a protein has not been systematically evaluated. The overall objective was to identify conditions (temperature and humidity) at which MHH can dehydrate on storage and the consequences of the release of associated water on sucrose phase behavior and protein stability. In a mannitol-sucrose-protein lyophile, the purpose of this study was (i) to investigate the dehydration behavior of MHH (ii) to determine the influence of dehydration on sucrose crystallization and (iii) the effect of moisture released due to MHH dehydration on model protein (Bovine serum albumin, BSA or Human serum albumin, HSA) aggregation. MHH dehydration and sucrose crystallization was observed in cases where the relative humidity was ≥ 55% (open vials). A relative humidity of ≤ 33% RH prevented MHH dehydration while retaining sucrose amorphous. No protein aggregation was observed irrespective of presence of MHH or its dehydration.

Temperature-dependence of riboflavin phosphorescence in cryosolvents

The molecular mobility of amorphous excipients is important for the stability of biomaterials during preservation, facilitating matrix formulation and product design. Phosphorescence spectroscopy is a sensitive optical method to study molecular mobility. However, there is a need to expand the pool of probes available for analysis since molecules differ in sensitivity. This research explored the feasibility and limitations of using riboflavin as a phosphorescent probe for monitoring matrix molecular mobility. Phosphorescence decays of riboflavin in four amorphous cryosolvents (aqueous solutions of glycerol, ethanol, sucrose, and dextran) were collected at 77 K to capture its natural phosphorescence lifetime (estimated at 170 ms). Decays were also collected during ballistic heating to assess the sensitivity of riboflavin towards changes in matrix molecular mobility. Riboflavin exhibited good sensitivity towards matrix secondary relaxations in the glass, indicating that riboflavin has excellent potential as an edible phosphorescent probe for molecular mobility in food and pharmaceutical products.

Be Aggressive! Amorphous Excipients Enabling Single-Step Freeze-Drying of Monoclonal Antibody Formulations.

Short freeze-drying cycles for biopharmaceuticals are desirable. Formulations containing an amorphous disaccharide, such as sucrose, are prone to collapse upon aggressive primary drying at higher shelf temperature. We used 2-hydroxypropyl-betacyclodextrin (HPBCD) in combination with sucrose and polyvinylpyrrolidone (PVP) to develop an aggressive lyophilization cycle for low concentration monoclonal antibody (mAb) formulations. Glass transition temperature and collapse temperature of the formulations were determined, and increasingly aggressive cycle parameters were applied. Using a shelf temperature of +30 °C during primary drying, the concept of combining sublimation and desorption of water in a single drying step was investigated. Cake appearance was evaluated visually and by micro-computed tomography. Lyophilisates were further analyzed for reconstitution time, specific surface area, residual moisture, and glass transition temperature. We demonstrated the applicability of single-step freeze-drying, shortening the total cycle time by 50% and providing elegant lyophilisates for pure HPBCD and HPBCD/sucrose formulations. HPBCD/PVP/sucrose showed minor dents, while good mAb stability at 10 mg/mL was obtained for HPBCD/sucrose and HPBCD/PVP/sucrose when stored at 40 °C for 3 months. We conclude that HPBCD-based formulations in combination with sucrose are highly attractive, enabling aggressive, single-step freeze-drying of low concentration mAb formulations, while maintaining elegant lyophilisates and ensuring protein stability at the same time.

Application of a mixture DOE for the prediction of formulation critical temperatures during lyophilisation process optimisation.

During lyophilisation cycle design, primary drying parameters (chamber pressure and shelf temperature) are adjusted to maximize the sublimation rate and prevent cake collapse, by maintaining the product continuously below its critical temperatures. The objective of this study was to employ mixture design of experiments to generate empirical models capable of predicting glass transition of the maximally freeze concentrated solution (Tg') and collapse temperature (Tc) of amorphous protein (BSA and IgG1) formulations. Additionally, the models developed aid the design of high concentration protein formulations with maximised critical temperatures to obtain shorter and more cost-effective lyophilisation cycles. Formulations contain sucrose as cryo/lyo-protectant and arginine/arginine-HCl as multifunctional excipient (e.g. solubility enhancer, viscosity and aggregation suppressor). The impact of formulation components at varied ratios on critical temperatures was evaluated; the amorphous excipients decrease critical temperatures, on the contrary, the protein increases critical temperatures. The robustness of the empirical models generated with BSA formulations was verified with BSA and IgG1 formulations. The models showed greater accuracy in predicting Tg' than the Fox-Flory equation. For the first time, empirical models are reported to predict both critical temperatures. Finally, unconventional collapse events observed for formulations with and without arginine/arginine-HCl at different protein concentrations are also discussed.

Investigation of nanocarriers and excipients for preparation of nanoembedded microparticles

Colloidal drug delivery systems often face physical and chemical instability as well as challenges with directed delivery. In order to overcome these challenges the colloidal formulations can be processed into microparticulate form (nanoembedded microparticles (NEMs)). In this study, different polymer nanocarriers (poly(lactide-co-glycolide), poly(styrene), chitosan and dendrimers) were used for preparing NEMs by spray-drying. Further, distinct matrix excipients were investigated including sugars (i.e., trehalose, sucrose, mannitol) and polymers (poly(vinyl pyrrolidone) and poly(ethylene glycol)), and the characteristics and performance of NEMs were studied in detail. It was found that with increasing hydrophilicity of the polymer nanocarriers, an increasing amount of excipient was necessary to stabilize the nanoparticles. NEMs containing polyplexes and nanogels required a matrix-to-nanoparticle (M:N) ratio of 50:1 and 10:1, respectively, whereas NEMs with poly(styrene) and poly(lactide-co-glycolide) only required an M:N ratio of 1:1 and 1:4, respectively. Investigation of different excipients demonstrated that water soluble sugars and polymers can be used to prepare NEMs and that spray-dried amorphous excipients (trehalose, sucrose, poly(vinyl pyrrolidone)) are superior to spray-dried crystalline excipients (mannitol, poly(ethylene glycol)) for stabilizing NEMs. It is therefore important to select an appropriate excipient for stabilization of a given nanoparticle system and identify a suitable level of this excipient to keep the nanoparticles viable.

Impact of vacuum-induced surface freezing on inter- and intra-vial heterogeneity

This paper aimed to study the impact of freezing on both within-batch (inter-vial) and within-product (intra-vial) heterogeneity. This analysis has been carried out using two freezing protocols, the conventional shelf-ramped method and the Vacuum Induced Surface Freezing, and placebo formulations containing both crystallizing (mannitol) and amorphous (lactose and sucrose) excipients. The freezing conditions (i.e., the temperature of freezing, the temperature and time of the equilibration phase, and the filling volume) were found to have a dramatic impact on both the within-batch and the within-product homogeneity. Overall, we observed that the control of freezing can effectively minimize the variability in product characteristics, and moisture content, within the same batch. In addition to more uniform production, the control of freezing was found to be fundamental to achieve a more uniform product than that produced by the shelf-ramped freezing method. The influence of the freezing protocol on the crystallization process of mannitol was also investigated, showing that the temperature of freezing plays a key role in the formation of the mannitol polymorphs.

Free Surface Electrospinning of Microemulsions Containing Vitamin E

In research and development of pharmaceutical products, an estimated 90% of active pharmaceutical ingredients (APIs) are insoluble or partially soluble in water. Due to poor solubility, these APIs exhibit poor bioavailability in solid dosage forms. To improve the release rate of APIs, free surface electrospinning of a microemulsion was considered as a means of producing submicron size domains of API dispersed in an amorphous excipient. Microemulsions containing a poor solubility API, vitamin E, and an excipient, Polyvinylpyrrolidone, are electrospun to produce a highly porous material with a high surface area, which promotes rapid drug release. The materials were characterized by scanning electron microscopy and high performance liquid chromatography to determine the morphology of the fibers and the bioavailability of the final material. The final product contained significant amounts of Vitamin E encapsulated within the excipient, and release rates were significantly improved over commercial products. In addition to improving the bioavailability of APIs, this technique may be utilized to streamline the downstream processing of pharmaceuticals, resulting in lower operating cost and improved uniformity over current batch manufacturing techniques.

Cryopellets based on amorphous organic calcium salts: Production, characterization and their usage in coagulation diagnostics

Abstract This work deals with the production and characterization of powders of amorphous organic calcium salts (excipients) as a possible recalcification source for citrated whole blood samples. Rapidly dissolving cryopellets were also prepared from these excipients. The amorphous excipients show high oversaturation which allows the manufacture of cryopellets containing a high amount of organic calcium salt. The characterization of the cryopellets revealed very rapid dissolution and breaking forces sufficient to withstand substantial mechanical stresses. Of the various individual and compounded excipients examined, calcium lactogluconate and calcium lactobionate are the best alternatives to CaCl2 for recalcification of citrated whole blood. Their influence on coagulation was minimal and both substances showed a low hygroscopic tendency during dynamic vapor sorption. Both excipients are non-hygroscopic under ambient conditions up to a relative humidity of p/p0 = 60%.

Thermodynamics of Water–Solid Interactions in Crystalline and Amorphous Pharmaceutical Materials

Pharmaceutical materials, crystalline and amorphous, sorb water from the atmosphere, which affects critical factors in the development of drugs, such as the selection of drug substance crystal form, compatibility with excipients, dosage form selection, packaging, and product shelf-life. It is common practice to quantify the amount of water that a material sorbs at a given relative humidity (RH), but the results alone provide minimal to no physicochemical insight into water-solid interactions, without which pharmaceutical scientists cannot develop an understanding of their materials, so as to anticipate and circumvent potential problems. This research was conducted to advance the science of pharmaceutical materials by examining the thermodynamics of solids with sorbed water. The compounds studied include nonhygroscopic drugs, a channel hydrate drug, a stoichiometric hydrate excipient, and an amorphous excipient. The water sorption isotherms were measured over a range of temperature to extract the partial molar enthalpy and entropy of sorbed water as well as the same quantities for some of the solids. It was found that water-solid interactions spanned a range of energy and entropy as a function of RH, which was unique to the solid, and which could be valuable in identifying batch-to-batch differences and effects of processing in material performance.

Investigation on formulation and preparation of adenovirus encoding human endostatin lyophilized powders

A recombinant adenovirus encoding human endostatin gene, E10A, has finished phase II trials for head and neck cancer. However, the rigid storage temperature (−80°C) and the toxicity of glycerol in the E10A liquid preparation limited its clinical application. In this study, lyophilization was applied to develop a stable E10A lyophilized powder without glycerol that is able to maintain biological activity at 4°C and suitable for intravenous administration. The E10A lyophilized formulations composed of nontoxic and already clinically used excipients were characterized in terms of the pH change during freezing, the eutectic melting temperature (Teu) and the collapse temperature (Tc). Freeze thawing tests were carried out to examine the protective effect of various excipients during freezing. Mannitol and its combinations with sucrose or inulin showed effective protection of E10A. The E10A lyophilized powders were analyzed by particle size measurement, residual humidity quantification, infectivity assay and gene expression level. An optimized formulation (formulation I1) yielded a good recovery of 76% of the starting infectivity after lyophilization and 89% of the original infectivity after storage at 4°C for 180 days. Also the gene expression capability of E10A in formulation I1 was maintained after lyophilization. In addition, it was found that the matrix of amorphous excipients, mannitol combinations with sucrose or inulin, was indispensible in protecting E10A against the stress of freezing and dehydration. Hereby, the E10A lyophilized powder with eliminated glycerol toxicity and improved stability could enhance the applicability of E10A for cancer gene therapy through intravenous administration.

The effect of water on the solid state characteristics of pharmaceutical excipients: Molecular mechanisms, measurement techniques, and quality aspects of final dosage form.

In this paper we give an overview about the interaction of water molecules with pharmaceutical excipients. Most of these excipients are amorphous or partially amorphous polymers and their characteristics are very sensitive to the water content. In the course of the manufacturing processes water sorption is possible, therefore in some cases it is important to strictly control the residual moisture content of a dosage form. There are several mechanisms of water sorption, like water is able to bind to polar groups of hygroscopic excipients and could also exist in the capillary system of amorphous excipients. Several techniques are available to characterise the states of water inside the materials and the effects of residual water on polymers. For this purpose water sorption measurements, differential scanning calorimetry and the Fourier-transform infrared spectroscopy are reviewed. The importance of water content and storage conditions of pharmaceuticals on the properties of the final dosage forms are also demonstrated with practical examples.

Perichromism: A Novel, Rapid, Spectroscopic Technique to Distinguish between Amorphous and Crystalline Material

There are currently no techniques that allow facile on-line determination between amorphous and crystalline material. Herein, a new colorimetric method, perichromism, has been developed to distinguish between amorphous and crystalline material, based on diffuse reflection ultraviolet–visible (UV-visible) spectroscopy. A probe molecule, phenol red, at 0.1% w/w concentration was added to sucrose and dissolved in water. The solution was freeze-dried, which resulted in an amorphous excipient with the probe molecule incorporated in an intimate mixture. The samples were then stored at a selected controlled temperature and relative humidity to allow control of crystallization of the sucrose. The amorphous or crystalline nature of each sample, both with and without the addition of the phenol red probe molecule, was monitored through X-ray diffraction, differential scanning calorimetry, and Raman spectroscopy. It was found that sucrose remained amorphous when stored in an atmosphere with a relative humidity below 32%; above this level, crystallization occurred. It was found that the presence of the probe molecule did not alter the extent of crystallinity of sucrose. Analysis of the samples containing the phenol red probe by diffuse reflection spectroscopy revealed that spectra could be classified into two groups depending on the λmax. Furthermore, correlation of the diffuse reflection spectra with X-ray diffraction and differential scanning calorimetry data showed that sucrose was amorphous in one group and crystalline in the other group, thereby revealing that diffuse reflection spectroscopy can, when phenol red is used as a probe molecule, quickly and easily determine whether sucrose is crystalline or amorphous.

Excipient crystallinity and its protein-structure-stabilizing effect during freeze-drying.

The relationship between mannitol crystallization during freeze-drying and its effects on stabilizing protein structures was studied using lysozyme, bovine serum albumin, ovalbumin, beta-lactoglobulin and lactate dehydrogenase as model proteins. FT-IR analysis of the protein secondary structure indicated perturbation of both alpha-helix and beta-sheet regions in freeze-drying without cosolutes, whereas the proteins retained most of their native structure in co-lyophilization with sucrose. Mannitol protected the protein structure to different degrees depending on the crystallinity. The combination of mannitol with potassium phosphate buffer reduced the mannitol crystallinity and the structural changes occurring during freeze-drying, whereas mannitol by itself showed little stabilizing effect. Heat-treatment of the frozen solutions at -10 degrees C resulted in a higher mannitol crystallinity and a smaller stabilizing effect in freeze-drying. The secondary structure perturbation was mostly reversible in rehydrated solutions. The varied structure-stabilizing effects of mannitol paralleled its effects on maintaining lower concentrations of enzyme activity during freeze-drying. These results confirm the contribution of molecular interactions between amorphous excipients and proteins (e.g. hydrogen bonding) to structure stabilization during freeze-drying.

Implications of Global and Local Mobility in Amorphous Excipients as Determined by DSC and TM DSC

The paper explores the use of differential scanning calorimetry (DSC) and temperature modulated differential scanning calorimetry (TM DSC) to study α- and β- processes in amorphous sucrose and trehalose. The real part of the complex heat capacity is evaluated at the frequencies, f, from 5 to 20mHz. β-relaxations were studied by annealing glassy samples at different temperatures and subsequently heating at different rates in a differential scanning calorimeter.

Investigation of nanocapsules stabilization by amorphous excipients during freeze-drying and storage

Freeze-drying was recently applied to improve the long-term storage stability of nanoparticles. Nanocapsules have a thin polymeric envelope that may not withstand the stresses of such process. So, cryoprotectants and lyoprotectants are usually added to the formulation to protect these vectors during freezing and desiccation steps. The aim of this paper was to investigate the importance of the vitrification of cryoprotectants on the stabilization of nanocapsules during freezing, desiccation, and storage steps. Furthermore, the effect of stabilizer crystallization on the conservation of nanocapsules properties was studied. Finally, the effect of temperature storage and relative humidity on the stability of nanocapsules was tested through an accelerated stability study. Results indicate that nanocapsules stabilization during the different steps of freeze-drying requires their dispersion within a vitrified matrix of amorphous excipient to protect them against the stress of freezing and dehydration. The crystallization of this stabilizer during the freezing, the desiccation or the storage steps can destabilize these fragile particles. Electron spectroscopy for chemical analysis revealed the adsorption of nanocapsules at the interface ice/liquid during the freezing step. Such adsorption must be avoided in the case of freeze-drying of immuno-nanoparticles to preserve the native structure of proteins attached to their surface.

Excipient selection can significantly affect solid-state phase transformation in formulation during wet granulation

Phase transformations in formulations can lead to instability in physicochemical, biopharmaceutical, and processing properties of products. The influences of formulation design on the optimal dosage forms should be specified. The aim here was to investigate whether excipients with different water sorption behavior affect hydrate formation of nitrofurantoin in wet masses. Nitrofurantoin anhydrate was used as a hydrate-forming model drug, and 4 excipients with different water-absorbing potential (amorphous low-substituted hydroxypropylcellulose, modified maize starch, partially amorphous silicified microcrystalline cellulose, and crystalline alpha-lactose monohydrate) were granulated with varying amounts of purified water. Off-line evaluation of wet masses containing nitrofurantoin anhydrate and excipient (1:1) was performed using an X-ray powder diffractometer (XRPD) and near-infrared spectroscopy, and drying phase was evaluated by variable temperature XRPD. Only amorphous excipient in the formulation retarded hydrate formation of an active pharmaceutical ingredient (API) at high water contents. Hygroscopic partially crystalline excipient hindered hydrate formation of API at low water contents. Crystalline excipient was unable to control hydrate formation of API. The character of excipient affects the stability of formulation. Thus, correct selection of excipients for the formulation can control processing-induced phase transitions and improve the storage stability of the final dosage form.

Determination of the viscosity of an amorphous drug using thermomechanical analysis (TMA).

To evaluate thermomechanical analysis (TMA) as a technique for determining the viscosity of amorphous pharmaceutical materials. This property of amorphous drugs and excipients is related to their average rate of molecular mobility and thus to their physical and chemical stability. Indomethacin was selected as a model amorphous drug whose viscosity has previously been reported in the literature. A Seiko TMA 120C thermomechanical analyzer was utilized in isothermal penetration mode to determine the viscosity of the amorphous drug over the maximum possible range of temperatures. Using a cylindrical penetration geometry it was possible to accurately determine the viscosity of amorphous indomethacin samples by TMA over the temperature range from 35 to 75 degrees C. The results were consistent with those reported in the literature using a controlled strain rheometer over the range 44-75 degrees C. The limiting lower experimental temperature for the TMA technique was extended to significantly below the calorimetric glass transition temperature (Tg approximately 42 degrees C), thus allowing a direct experimental determination of the viscosity at Tg to be made. Thermomechanical analysis can be used to accurately determine the viscosity of amorphous pharmaceutical materials at temperatures near and above their calorimetric glass transition temperatures.

Effects of Formulation and Process Variables on the Aggregation of Freeze-Dried Interleukin-6 (IL-6) After Lyophilization and on Storage

ABSTRACTThis study assessed the impact of residual moisture, Tg, and excipient physical state of different formulations on the “in-process” and shelf-life stability of freeze-dried interleukin-6 (IL-6). The effect of an annealing procedure was also evaluated. Characterization of the lyophilizates was done by Karl Fischer titration, differential scanning calorimetry (DSC), and x-ray measurements. Analysis of protein stability was carried out by size exclusion chromatography (SEC), sodium dodecyl sulfate-polyacry-lamidegel electrophoresis (SDS-PAGE), and turbidity measurements. During freeze-drying, the most effective protection against aggregation was provided by completely amorphous formulations consisting of trehalose or sucrose either alone or in combination with glycine or mannitol. Other amorphous formulations like those of sucrose with lysine–HCl or dextran could not provide comparable stabilization. In lyophilizates containing a crystallized excipient such as glycine or mannitol, IL-6 suffered destabilization, which was less pronounced if an additional amorphous excipient was present. For the completely amorphous formulations, aggregation was prevented during a 9-month storage at 25 and 40°C as long as the storage temperature did not exceed theT value of the lyophilizate, otherwise severe damage occurred. Formulations containing amorphous dextran or lysine–HCl could not effectively stabilize IL-6 even when stored below Tg Annealing helped to improve cake robustness and appearance, but for lyophilizates containing an excipient crystallized by annealing an increase of IL-6 aggregation was observed despite a storage below Tg. Thus, the amorphous state of the excipients and a high Tg can be considered necessary conditions for preventing aggregation of freeze-dried IL-6. Whether the conditions are also sufficient depends on the choice of excipients. De-stabilization can occur with some excipients despite their amorphous state as well as in the presence of crystallized excipients despite a storage below Tg Compared to sucrose, trehalose is a more favorable excipient for protein lyophilization because it exhibits a higher Tgpossesses better stabilizing properties, and can reduce protein aggregation which may have been caused by annealing.