Design of the lyophilization process of a L-leucyl-L-alanine dipeptide formulation based on its thermal properties

Arién Sánchez-González1, Gerardo R Hernández-González2 1 Centro de InmunoEnsayo Calle 134 y Avenida 25. Cubanacán, Playa, La Habana, Cuba 2 Centro de Ingeniería Genética y Biotecnología Ave. 31 e/158 y 190, Cubanacán, Playa, CP 11600, La Habana, Cuba  ABSTRACT The system for phenylketonuria diagnostic in newborns (UMTEST® PKU) is produced at the Center for Immunoassay (Havana, Cuba). L-leucyl-L-alanine is one of the kit components and requires lyophilization due to its limited aqueous solution stability. Results from Diffferential Thermal Analysis/Impedance (DTA/ Zsinφ) and lyomicroscopy determinations, identify that during the freezing step product temperature must be below –30 ºC, while throughout the primary drying process product temperature at sublimation front cannot exceed –18.9 ºC. The use of a procedure based on the critical variables determinations, allow the lyophilization of three industrial batches that finally fulfilled the quality specifications and achieve a reduction of 32 % in the cycle total time. Keywords: lyophilization, L-leucyl-L-alanine, sublimation, electrothermal analysis, lyomicroscopy RESEARCH Design of the lyophilization process of a L-leucyl-L-alanine dipeptide formulation based on its thermal properties Biotecnología Aplicada 2014;32:1221-5 RESUMEN Diseño del ciclo de liofilización de una formulación del dipéptido L-leucil-L-alanina basado en sus propiedades térmicas. El sistema para la detección de la fenilcetonuria (UMTEST® PKU) en recién nacidos, se produce en el Centro de Inmunoensayo (CIE, La Habana, Cuba). La L-leucil-L-alanina, uno de los componentes del diagnosticador, requiere ser liofilizado debido a su limitada estabilidad en solución acuosa. La utilización de las técnicas de Análisis Térmico Diferencial/Impedancia (ATD/Zsinφ) y la liomicroscopía permitieron definir que en la liofilización de este reactivo, la congelación requiere ser realizada a temperaturas inferiores a –30 °C, mientras que en el secado primario la temperatura del frente de sublimación no puede sobrepasar el valor de temperatura de colapso (Tc) de –18.9 ºC. La liofilización de tres lotes a escala industrial, ejecutada con el procedimiento diseñado según los resultados de las determinaciones previamente descritas, garantizó la obtención de un producto acorde a las especificaciones de calidad, con una reducción del 32 % en el tiempo total del ciclo. Palabras clave: liofilización, L-leucil-L-alanina, análisis térmico diferencia, impedancia, liomicroscopía Introduction Phenylketonuria (PKU) is an inborn metabolic error manifested as an autosomic-inherited recessive monogenic disorder [1]. From the biochemical point of view, it leads to the accumulation of phenylalanine (Phe) in blood and other organic fluids and tissues, also slightly decreasing tyrosine levels [2]. PKU patients without control treatment could suffer from post-natal brain damage, further developing irreversible cognitive impairment, skin modifications and absence or difficulty of speech [3]. For these reasons, neonatal screening programs for the massive detection and treatment of hyperphenyalaninemias started as early as in 1961, with the Guthrie’s Bacterial Inhibition test [4]. That assay was used in Cuba from 1983 to 2000, with an incidence of 1:50 000 inhabitants been detected [5]. A pilot program started that year for PKU screening using the UMTEST® PKU kit, which consists on a fluorescent ultramicroassay for the quantitative detection of Phe in dry blood on filter paper, by using the ultramicroanalytical system (SUMA®) developed at the Center of Immunoassays (CIE), Cuba [6]. The screening compound, L-Leucyl-L-alanine, requires to be lyophilized to guarantee its two-year validity. Lyophilization is a freeze-drying operation commonly used for the long-term stabilization of substances unstable in water and sensitive to high temperatures. It occurs in three main steps: freezing, sublimation or primary drying, and desorption or secondary drying [7]. During freezing, most of the water in the product is turned into ice, practically achieving the complete immobilization of the system. The microstructure formed during this step determines mostly the final quality of the material and lyophilizate structure also. Ice is further eliminated by sublimation during primary drying [8]. This step requires the decrease of the chamber pressure below the ice vapor pressure (which is temperature-dependent) in the sublimation front [9]. The water remaining in the structure at the end of the sublimation step (5-20 %) is eliminated during the secondary drying step. This last is applied at temperatures higher enough to guarantee water desorption from the structure, but without compromising the integrity of the product [10, 11]. One of the major challenges during lyophilization resides on shortening the process’ duration without compromising the quality of the final product [12-16]. The appropriate characterization of the formulation of interest is a key step prior to freeze-drying cycle design [17, 18]. The knowledge on the temperature of glass transition (Tg’) or the eutectic temperature (Teu) determines the freezing temperature value of the product 1. Kaye CI; the Committee on Genetics. Newborn Screening Fact Sheets. Pediatrics 2006;118:e934-63. 2. Arneson W, Brickell J. Clinical Chemistry: A Laboratory Perspective. Philadelphia: F. A. Davis Co.; 2007. 3. Moyle JJ, Fox AM, Arthur M, Bynevelt M, Burnett JR. Meta-analysis of neuropsychological symptoms of adolescents and adults with PKU. Neuropsychol Rev. 2007;17(2):91-101. 4. Mei JV, Alexander JR, Adam BW, Hannon WH. Use of filter paper for the collection and analysis of human whole blood specimens. J Nutr. 2001;131(5):1631S-6S. 5. Martínez L, Robaina Z, García S, Gutiérrez E. The Cuban program for neonatal screening of phenylketonuria. Twenty years of experience: the clinical and social attention. Rev Cubana Genet Comunit. 2008;2:45-51. 6. Gonzalez EC, Marrero N, Perez PL, Frometa A, Zulueta O, Herrera D, et al. An enzyme immunoassay for determining 17alpha-hydroxyprogesterone in dried blood spots on filter paper using an ultramicroanalytical system. Clin Chim Acta. 2008;394(1-2):63-6. Arién Sánchez-González et al. Lyophilization process of a L-leucyl-L-alanine required to achieve the complete solidification of the matrix. At the same time, the collapse temperature (TC) for each formulation determines the operational parameters during primary drying [19], and the proper characterization of the formulation facilitates the execution of a safe and efficient process [20]. There are several analytical tools available to accomplish such characterization. However, all of them have advantages and limitations, their combination being essential to accomplish reliable lyophilization rates [21]. Differential scanning calorimetry (DSC) and Differential thermal analysis (DTA) take advantage of any physical change occurring in the structure of the lyophilized compou (...truncated)