A Brief Reminder of Systems of Production and Chromatography-Based Recovery of Recombinant Protein Biopharmaceuticals.

Recombinant proteins are produced for various applications in laboratory and industrial settings. Among them, therapeutic applications have evolved into a mature field in recent years, affecting the face of contemporary medical treatment. This, in turn, has stimulated an ever-greater need for innovative technologies for the description, expression, and purification of recombinant protein biopharmaceuticals. Therefore, many biopharmaceuticals are synthesized in heterologous systems to obtain satisfactory yields that cannot be provided by natural sources. As more than 35 years has passed since the first recombinant biopharmaceutical (human insulin) successfully completed clinical trials in humans, we provide a brief review of the available prokaryotic and eukaryotic expression systems, listing the advantages and disadvantages of their use. Some examples of therapeutic proteins expressed in heterologous hosts are also provided. Moreover, technologies for the universal extraction of protein molecules are mentioned here, as is the methodology of their purification.

Development and validation of a HPTLC method for analysis of Sunitinib malate

ABSTRACT A simple high performance thin layer chromatography (HPTLC) has been developed and validated for determination of sunitinib malate and possible impurities. The samples were applied in forms of bands on an aluminum TLC plate pre-coated with silica gel and were separated using dichloromethane: methanol: toluene: ammonia solution as the mobile phase. Sunitinib malate was thoroughly separated from impurities including E-isomer, sunitinib N-oxide and impurity B with a retention factor (RF) of 0.35±0.02. Quantitative analysis of sunitinib was carried out using a mobile phase consisting of dichloromethane:methanol:ammonia solution, RF value was 0.53±0.02 for Z isomer. Detection was performed densitometrically in absorbance mode at 430 nm. This method was found to produce sharp, symmetrical, and well resolved peaks. Linear relationship with the coefficients of determination > 0.99 was achieved over the concentration range of 27.34 to 437.5 ng/spot. This method provides robust, replicable and accurate results with acceptable sensitivity.

Chromatographic analysis and antiproliferative potential of aqueous extracts of Punica granatum fruit peels using the Allium cepa test

Punica granatum L., locally known as romanzeira, is native to Asia but found throughout Brazil. P. granatum is used for treating inflammatory, infectious and respiratory diseases. The aim of this study was to evaluate the chromatography and genotoxicity of an aqueous extract of P. granatum (pomegranate) fruit peel using the Allium cepa L. test. The experiment set-up entailed 7 treatments: T1-distilled water, T2-tea 5 g.L-1, T3-tea 10 g.L-1, T4-glyphosate at 9.6%, T5-glyphosate with subsequent recovery in distilled water, T6-glyphosate with subsequent recovery in tea 5 g.L-1 and T7-glyphosate with subsequent recovery in tea 10 g.L-1. The rootlets were collected and fixed in ethanol:acetic acid (3:1) for 24 hours, then stored in 70% ethanol under refrigeration. Analysis was performed using high performance liquid chromatography for the quantification of the extracted phenolic compounds. Gallic acid, catechin, caffeic acid, and rutin were abundant in the extracts of P. granatum. The extracts were found to exhibit antiproliferative potential but not antimutagenic or genotoxic activity.

Punica granatum L., conhecida como romanzeira, é originária da Ásia e encontra-se distribuída por todo Brasil. É usada para o tratamento de doenças inflamatórias, infecciosas e respiratórias. Em decorrência da grande utilização de recursos fitoterápicos, é necessário esclarecer à população sobre a grande quantidade de substâncias existentes nas plantas e sobre os benefícios e prejuízos de tais substâncias à saúde. O presente trabalho objetivou realizar a análise cromatográfica e o estudo da genotoxicidade dos extratos aquosos das cascas dos frutos de P. granatum através do teste de Allium cepa L. Para a montagem do experimento, foram utilizados 7 tratamentos: T1-água destilada, T2-chá 5 g.L-1, T3-chá 10 g.L-1, T4-glifosato a 9,6%, T5-glifosato para recuperação em água destilada, T6-glifosato para recuperação em chá 5 g.L-1 e T7-glifosato para recuperação em chá 10 g.L-1. As radículas foram coletadas e fixadas em etanol:ácido acético (3:1) por 24 horas, e armazenadas em álcool 70%, sob refrigeração. Realizou-se análise por cromatografia líquida de alta eficiência para quantificação dos compostos fenólicos. Nos extratos de P. granatum foram observados em maior quantidade: ácido gálico, catequina, ácido cafeico e rutina. Além disso, os extratos demonstraram potencial antiproliferativo, sem apresentar atividade antimutagênica e genotóxica.

Preparative purification of recombinant proteins: current status and future trends.

Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications.

Un nuevo método de cromatografía de líquidos de alto rendimiento y de fase inversa para la determinación de flutamida en liposomas, plasma de sangre de rata y formas de dosificación en comprimidos / A new reverse-phase high performance liquid chromatographic method for determination of flutamide in liposomes, rat blood plasma and tablet dosage forms

La flutamida es un antiandrógeno ampliamente utilizado en el tratamiento del cáncer de próstata que afecta al nivel de antígeno específico de la próstata (PSA) en sangre, lo que requiere que sea estable en la sangre durante el suficiente tiempo. Se ha desarrollado una nueva forma de dosificación farmacéutica, los liposomas, para mejorar la eficacia de este fármaco. El objeto de este estudio es desarrollar un método HPLC UV para la determinación de la FLT en los liposomas, en el plasma de sangre de rata y en distintas formas de dosificación en comprimidos comercializadas. Los pasos de preparación de muestras requieren menos tiempo. El estándar interno (IS) para el procedimiento de ensayo fue la 6-mercaptopurina. Las muestras se inyectaron en la columna de la fase inversa (Thermosil ® C18 ). La fase móvil, metanol: agua (80:20, v/v), se llevo a cabo con una velocidad de flujo de 1 ml/min. durante 8 min. La FLT se detectó mediante un detector de UV con una longitud de onda de 295 nm. Los tiempos de retención del IS y la FLT fueron de 3,03 y 4,02 min. respectivamente. El método fue lineal en el rango de concentración de 20-1000 ng/ml y 50-1000 µg/ml en la fase móvil y en el plasma de sangre de rata respectivamente. Se validó la exactitud, precisión, robustez y recuperación del método. El límite de detección fue de 0,099 µg/ml y de 0,106 µg/ml en la fase móvil y en el plasma de sangre de rata respectivamente. El método demostró ser muy reproducible y parece ser adecuado para el control rutinario de fármaco terapéutico. Se podría utilizar sin ninguna interferencia por parte de lípidos, excipientes de comprimidos y sustancias endógenas de las muestras de plasma

Flutamide is an anti-androgen widely used in treatment of prostate cancer by affecting Prostate Specific Antigen (PSA) level in blood, which requires its stability in blood for enough time. A new pharmaceutical dosage form, liposomes, has been developed for improving efficacy of this drug. The aim of this study is to develop HPLC UV method for the determination of FLT in liposomes, rat blood plasma and different marketed tablet dosage forms. The method involved less time-consuming sample preparation steps. The internal standard (IS) for the assay procedure was 6-Mercaptopurine. The samples were injected on to reverse-phase (Thermosil ® C18 ) column. The mobile phase, Methanol: Water (80:20, v/v)was run at a flow rate of 1 ml/min for 8 min. The FLT was detected by UV detector at 295 nmwavelength. The retention times for IS and FLT were 3.03 and 4.02 min, respectively. The method was linear over the concentration range 20-1000 ng/ml and 50-1000 µg/ml in mobile phase and rat blood plasma, respectively. The method was validated for accuracy, precision, robustness and recovery. The limit of detection was 0.099µg/ml and 0.106µg/ml in mobile phase and rat blood plasma, respectively. The method was shown to be highly reproducible and it seems to be adequate for routine therapeutic drug monitoring. It could be used without any interference from lipids, tablet excipients and endogenous substances from the plasma samples

CD34+ cell selection in chronic phase chronic myeloid leukaemia: a comparison of laboratory grade columns.

CD34 positive (CD34+) cell selection is increasingly used for a number of important applications including gene therapy studies, ex vivo expansion and purging. However there are no data regarding the use of different technologies for CD34+ cell selection in chronic myeloid leukaemia (CML). We therefore compared the performance of three laboratory grade CD34+ selection columns (MiniMACS, Cellpro Ceprate LC and Baxter Isolex 50), using CML chronic phase peripheral blood (PB) and bone marrow (BM). With different CML samples the CD34+ purity from the three columns was equivalent, but comparing five paired samples the Ceprate purity was greater than MiniMACS, at 92.5 and 80.9%, respectively, P = 0.04. Combining results from paired and unpaired CML samples, MiniMACS (n = 7) gave a higher CD34+ yield than Ceprate LC (n = 8) or Isolex 50 (n = 4) with a mean of 51.1%, 24.3% and 13.2% respectively, (P = 0.04 and 0.01). Cell losses with all columns were similar. Attempts to improve the yield from the Ceprate LC columns by modifying the method were unsuccessful. Following MiniMACS and Ceprate LC separation the clonogenic potentials of CD34+ cells in the pre- and positive cell fractions were the same. The proportion of CD34+ 38- or CD34+ DR- cells was unchanged following column separation. These data suggest that the MiniMACS column may be the best column for CD34+ cell selection in CML but these results must be confirmed using large scale clinical columns once the MiniMACS column is licensed. It is possible that variations in CD34+ cell yields between the different columns reflect differences in antibody binding affinity to CML cells, or differences in column technologies.