The growing impact of lyophilized cell-free protein expression systems.

Recently reported shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotherapeutics, biosensing, biocatalysis, and high throughput protein expression. Lyophilized cell-free protein expression systems not only overcome cold-storage limitations, but also enable stockpiling for on-demand synthesis and completely sterilize the protein synthesis platform. Recently reported high-yield synthesis of cytotoxic protein Onconase from lyophilized E. coli extract preparations demonstrates the utility of lyophilized cell-free protein expression and its potential for creating on-demand biotherapeutics, vaccines, biosensors, biocatalysts, and high throughput protein synthesis.

Pharmaceutical patent applications in freeze-drying.

Injectable products are often the formulation of choice for new therapeutics; however, formulation in liquids often enhances degradation through hydrolysis. Thus, freeze-drying (lyophilization) is regularly used in pharmaceutical manufacture to reduce water activity. Here we examine its contribution to 'state of the art' and look at its future potential uses. A comprehensive search of patent databases was conducted to characterize the international patent landscape and trends in the use of freeze-drying. A total of 914 disclosures related to freeze-drying, lyophilization or drying of solid systems in pressures and temperatures equivalent to those of freeze-drying were considered over the period of 1992-2014. Current applications of sublimation technology were contrasted across two periods those with patents due to expire (1992-1993) and those currently filed. The number of freeze-drying technology patents has stabilized after initial activity across the biotechnology sector in 2011 and 2012. Alongside an increasing trend for patent submissions, freeze-drying submissions have slowed since 2002 and is indicative of a level of maturity.

Effects of cooling rate in microscale and pilot scale freeze-drying - Variations in excipient polymorphs and protein secondary structure.

Microscale freeze-drying makes rapid process cycles possible for early-stage formulation development. To investigate the effects of equipment scale and cooling rate on the solid state properties and the protein's secondary structure of a sample, three binary formulations of catalase were prepared and freeze-dried with sucrose, mannitol, or (2-hydroxypropyl)-ß-cyclodextrin (HP-ß-CD). The protein's secondary structure was assessed using attenuated total reflection Fourier transform infrared spectroscopy (FTIR-ATR). The solid state properties were assessed using differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The results were interpreted with respect to the biological activity of catalase after its reconstitution. According to the results of both the protein secondary structure and the reconstituted biological activity, scale-up could be achieved with the sucrose-catalase formulation when it was prepared at a high cooling rate and with the mannitol-catalase formulation when prepared at a low cooling rate. However, differences in the polymorph composition of crystalline mannitol were noted. No cooling rate influence was found with the HP-ß-CD formulation. The results clearly indicate that the effects of the cooling rate should be closely examined during microscale formulation development and scale-up of the freeze-drying process.

New Processes for Freeze-Drying in Dual-Chamber Systems.

Dual-chamber systems can offer self-administration and home care use for lyophilized biologics. Only a few products have been launched in dual-chamber systems so far-presumably due to dual-chamber systems' complex and costly drug product manufacturing process. Within this paper, two improved processes (both based on tray filling technology) for freeze-drying pharmaceuticals in dual-chamber systems are described. Challenges with regards to heat transfer were tackled by (1) performing the freeze-drying step in a needle-down orientation in combination with an aluminum block, or (2) freeze-drying the drug product "externally" in a metal cartridge with subsequent filling of the lyophilized cake into the dual-chamber system. Metal-mediated heat transfer was shown to be efficient in both cases and batch (unit-to-unit) homogeneity with regards to sublimation rate was increased. It was difficult to influence ice crystal size using different methods when in use with an aluminum block due to its heat capacity. Using such a metal carrier implies a large heat capacity leading to relatively small ice crystals. Compared to the established process, drying times were reduced by half using the new processes. The drying time was, however, longer for syringes compared to vials due to the syringe design (long and slim). The differences in drying times were less pronounced for aggressive drying cycles. The proposed processes may help to considerably decrease investment costs into dual-chamber system fill-finish equipment. LAY ABSTRACT: Dual-chamber syringes offer self-administration and home care use for freeze-dried pharmaceuticals. Only a few products have been launched in dual-chamber syringes so far-presumably due to their complex and costly drug product manufacturing process. In this paper two improved processes for freeze-drying pharmaceuticals in dual-chamber syringes are described. The major challenge of freeze-drying is to transfer heat through a vacuum. The proposed processes cope with this challenge by (1) freeze-drying the drug product in the syringe in an orientation in which the product is closest to the heat source, or (2) freeze-drying the drug product outside the syringe in a metal tube. The latter requires filling the freeze-dried product subsequently into the dual-chamber syringe. Both processes were very efficient and promised to achieve similar freeze-drying conditions for all dual-chamber syringes within one production run. The proposed processes may help to considerably decrease investment costs into dual-chamber syringe fill-finish equipment.

Methods of amorphization and investigation of the amorphous state.

The amorphous form of pharmaceutical materials represents the most energetic solid state of a material. It provides advantages in terms of dissolution rate and bioavailability. This review presents the methods of solid- -state amorphization described in literature (supercooling of liquids, milling, lyophilization, spray drying, dehydration of crystalline hydrates), with the emphasis on milling. Furthermore, we describe how amorphous state of pharmaceuticals differ depending on the method of preparation and how these differences can be screened by a variety of spectroscopic (X-ray powder diffraction, solid state nuclear magnetic resonance, atomic pairwise distribution, infrared spectroscopy, terahertz spectroscopy) and calorimetry methods.

A procedure to optimize scale-up for the primary drying phase of lyophilization.

This article describes a procedure to facilitate scale-up for the primary drying phase of lyophilization using a combination of empirical testing and numerical modeling. Freeze dry microscopy is used to determine the temperature at which lyophile collapse occurs. A laboratory scale freeze-dryer equipped with manometric temperature measurement is utilized to characterize the formulation-dependent mass transfer resistance of the lyophile and develop an optimized laboratory scale primary drying phase of the freeze-drying cycle. Characterization of heat transfer at both lab and pilot scales has been ascertained from data collected during a lyophilization cycle involving surrogate material. Using the empirically derived mass transfer resistance and heat transfer data, a semi-empirical computational heat and mass transfer model originally developed by Mascarenhas et al. (Mascarenhas et al., 1997, Comput Methods Appl Mech Eng 148: 105-124) is demonstrated to provide predictive primary drying data at both the laboratory and pilot scale. Excellent agreement in both the sublimation interface temperature profiles and the time for completion of primary drying is obtained between the experimental cycles and the numerical model at both the laboratory and pilot scales. Further, the computational model predicts the optimum operational settings of the pilot scale lyophilizer, thus the procedure discussed here offers the potential to both reduce the time necessary to develop commercial freeze-drying cycles by eliminating experimentation and to minimize consumption of valuable pharmacologically active materials during process development.

Oral lyophilisate and food immunotherapy: from research to clinical practice

Thanks to its excellent safety profile, sublingual immunotherapy has served as a basis for launching two important lines of research in current allergology: sublingual immunotherapy with pharmaceutical registry (oral lyophilizates or tablets), and sublingual immunotherapy with food. At present, clinical trials are being conducted which use rapid dissolution oral lyophilizates. The results of the clinical trials carried out in large patient groups and based on a double-blind methodological design have allowed pharmaceutical registry of this form of treatment, with the therapeutic indications of rhinitis and allergy to grasses. Phleum lyophilizate indicated for the treatment of rhinoconjunctivitis will be marketed in Spain in the coming months. In parallel to development of the sublingual route, advances in our knowledge of pollen allergy and its relationship to plant food allergies have facilitated the conducting of studies involving sublingual immunotherapy for allergy to kiwi fruit, hazel nut and peach – thus giving rise to promising future perspectives for affected patients

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Pharmacovigilance study of a regional intravenous immunoglobulin (II): evaluation and comparison of an improved pharmaceutical form / Estudio de farmacovigilancia de una inmunoglobulina intravenosa regional (II): evaluación y comparación de una forma farmacológica mejorada

Introduction: Intravenous immunoglobulin (IVIG) therapy is an effective treatment in patients with different diseases. This product must comply with all the regulatory requirements established by the World Health Organization and the European Pharmacopoeia for clinical tolerance, therapeutic efficacy, and viral safety. Although IVIG are effective and safe products, in some patients they may cause adverse reactions. Objectives: The aim of this study was to assess the clinical tolerance of two pharmaceutical forms (lyophilized and liquid) of Imunoglobulina G Endovenosa UNC® (IVIG UNC®), a regional IVIG preparation, and to compare the reported data. Patients and methods: The pharmacovigilance reports of 149 infusions in 51 patients treated with lyophilized IVIG UNC® and of 157 infusions in 53 patients treated with liquid IVIG UNC® were statistically evaluated. Clinical tolerance was evaluated through the adverse reactions reported. Results: Lyophilized IVIG UNC®: adverse reactions were reported in 6.7 % of the total number of infusions. Of these reactions, 4.0 % were mild, 2.0 % were moderate, and 0.7 % severe. Liquid IVIG UNC®: adverse reactions were reported in 3.2 % of the total number of infusions; of these, 1.3 % were mild, 1.9 % were moderate, and 0.0 % were severe. Statistical analysis showed no association between tolerance and the pharmaceutical form used (p > 0.05) and indicated similar tolerance for both preparations. Conclusions: Based on the results obtained, the excellent clinical tolerance of both pharmaceutical forms of IVIG UNC® can be confirmed

Introducción: La administración de inmunoglobulina endovenosa (IGEV) es una terapéutica eficaz en pacientes con diversas patologías. Este medicamento debe cumplir con todos los requerimientos reguladores establecidos por la Organización Mundial de la Salud (OMS) y la Farmacopea Europea (FE) en cuanto a tolerancia clínica, eficacia terapéutica y seguridad viral. Aunque las IGEV son productos eficaces y seguros, en algunos pacientes pueden provocar reacciones adversas. Objetivos: El objetivo de este trabajo fue evaluar la tolerancia clínica de dos formas farmacéuticas (liofilizada y líquida) de Inmunoglobulina G Endovenosa UNC® (IGEV UNC®), una IGEV regional, y comparar los datos reportados. Pacientes y métodos: Se evaluaron estadísticamente los Registros de Farmacovigilancia de 149 infusiones administradas a 51 pacientes tratados con IGEV UNC® liofilizada y 157 infusiones administradas a 53 pacientes con IGEV UNC® líquida. La tolerancia clínica se evaluó considerando las reacciones adversas reportadas. Resultados: IGEV UNC® liofilizada: Se informaron reacciones adversas en 6,7% de las infusiones administradas, de las cuales 4,0% fueron leves, 2,0% moderadas y 0,7% graves. IGEV UNC® líquida: Se informaron reacciones adversas en 3,2% de las infusiones administradas, de las cuales 1,3% fueron leves, 1,9% moderadas y 0,0% graves. El análisis estadístico no mostró asociación entre tolerancia y forma farmacéutica empleada (p>0,05) indicando una tolerancia similar para ambas preparaciones. Conclusiones: Los resultados obtenidos permiten confirmar la excelente tolerancia clínica de ambas formas farmacéuticas de IGEV UNC®

Freeze-drying of proteins: some emerging concerns.

Freeze-drying (lyophilization) removes water from a frozen sample by sublimation and desorption. It can be viewed as a three-step process consisting of freezing, primary drying and secondary drying. While cryoprotectants can protect the protein from denaturation during early stages, lyoprotectants are needed to prevent protein inactivation during drying. The structural changes as a result of freeze-drying have been investigated, especially by FTIR (Fourier-transform IR) spectroscopy. In general, drying results in a decrease of alpha-helix and random structure and an increase in beta-sheet structure. In the case of basic fibroblast growth factor and gamma-interferon, enhanced FTIR showed large conformational changes and aggregation during freeze-drying, which could be prevented by using sucrose as a lyoprotectant. It is now well established that structural changes during freeze-drying are responsible for low activity of freeze-dried powders in nearly anhydrous media. Strategies such as salt activation can give 'activated' enzyme powders, e.g. salt-activated thermolysin-catalysed regioselective acylation of taxol to give a more soluble derivative for therapeutic use. In the presence of moisture, freeze-dried proteins can undergo disulphide interchange and other reactions which lead to inactivation. Such molecular changes during storage have been described for human insulin, tetanus toxoid and interleukin-2. Some successful preventive strategies in these cases have also been mentioned as illustrations. Finally, it is emphasized that freeze-drying is not an innocuous process and needs to be understood and used carefully.

Comparação da resistência à compressão de ossos bovinos congelados e liofilizados / Compression resistance of deep frozen and freeze dried bovine bone

Efetuou-se o estudo de ossos bovinos congelados e liofilizados visando verificar a existência ou não de diferenças na resistência à compressão resultantes da utilização destes diferentes processos de estocagem de osso utilizados atualmente em bancos de enxertos ósseos. Foram comprimidos em uma máquina de ensaios mecânicos universal 78 corpos de prova cilíndricos retirados de côndilos femorais bovinos. Os corpos de prova foram divididos em três grupos de 26 peças. O grupo I foi composto por cilindros de osso liofilizado e reidratado durante uma hora e meia antes do ensaio: o trupo II foi composto por cilindros de osso congelado e posteriormente descongelado durante uma hora; e o grupo III foi composto por cilindros de osso liofilizado e reidratado no momento do ensaio. Os resultados mostraram que não diferença estatisticamente significativa nem na resistência à compressão entre os grupos de estudo (Força máxima média de compressão e respectivo desvio padrão - GI 216 ± 147 N; GII 324 ± 245 N; GIII 284 ± 216 N), nem na razão de deformação (Razão de deformação média e respectivo desvio padrão - GI 422 ± 363 N/mm; GII 686 ± 500 N/mm; GIII 559 ± 481 N/mm).

Basic aspects and future trends in the freeze-drying of pharmaceuticals.

Freeze-drying, as a unit operation in chemical engineering, is a complex multistage process which has to be carefully adjusted to each individual case. Its essential feature, drying from the frozen state, makes it particularly attractive for the stabilization of labile hydrated organic compounds of biological origin. However, it offers equal opportunities for the preparation of three-dimensional porous matrices for diversified physical and chemical applications. As such it is both an advanced technology for the long term preservation of unstable material and a refined operation for the development of new structures. Since freeze-drying, also called lyophilization, has been known for almost a century, and has given rise to numerous publications, this paper will not attempt to give a comprehensive review of its evolution but will focus on some of its basic requirements and potential trends.