Registro sanitario de Heberprot-P®, medicamento biotecnológico innovador para el tratamiento de las úlceras del pie diabético / Sanitary registration of Heberprot-P®, an innovative biotechnological drug for the treatment of diabetic foot ulcers

Introducción: Heberprot-P® obtuvo su primer registro sanitario en Cuba en el año 2006, actualmente está aprobado en otros 26 países. Objetivo: Describir el proceso de registro sanitario en México, del medicamento biotecnológico Heberprot-P® para el tratamiento de las úlceras del pie diabético. Métodos: El proceso de registro sanitario de Heberprot-P® siguió las pautas de la reglamentación sanitaria de México sobre la base de la Ley general de salud y el Reglamento de insumos para la salud. Se revisaron además la Farmacopea de los Estados Unidos Mexicanos y las normas oficiales mexicanas en función de cumplir las exigencias para la comercialización de medicamentos en este territorio. Resultados: El proceso de registro se inició en junio de 2017 en México con acciones en función de completar los documentos e informaciones exigidas en el expediente de registro sanitario a presentarse. Entre ellos resaltan las consideraciones del Subcomité de Evaluación de Productos Biotecnológicos y el Comité de Moléculas Nuevas, la evaluación del expediente por un Tercero Autorizado y documentos emitidos por el Centro Nacional de Farmacovigilancia e Instituto Mexicano de la Propiedad Industrial. Se presentó la solicitud del registro sanitario ante Cofepris y esta se aprobó en mayo de 2018. Conclusiones: El trabajo con grupos de expertos permitió a la autoridad mexicana hacer un trabajo más expedito basado en las evidencias de las evaluaciones realizadas que son parte de la información del registro sanitario. Como resultado de este proceso, se otorgó el Registro Sanitario a Heberprot-P® en mayo de 2018 y Cofepris lo reconoció como un medicamento biotecnológico innovador(AU)

Introduction: Heberprot-P® obtained its first Sanitary Registration in Cuba in 2006, and it is currently approved in 26 other countries. Objective: Describe the sanitary registration process in Mexico of the biotechnological drug Heberprot-P® for the treatment of diabetic foot ulcers. Methods: The sanitary registration process of Heberprot-P® followed the guidelines of the sanitary regulations of Mexico on the basis of the General Health Law and the Regulation of Supplies for Health. The Pharmacopoeia of the United Mexican States and the official Mexican standards were also revised in order to comply with the requirements for the marketing of medicines in this territory. Results: The registration process began in June 2017 in Mexico with actions to complete the documents and information required in the sanitary registration file to be submitted. Among them are the considerations of the Sub-committee on the Evaluation of Biotechnological Products and the Committee on New Molecules, the evaluation of the file by an Authorized Third Party and documents issued by the National Center for Pharmacovigilance and the Mexican Institute of Industrial Property. The application for sanitary registration was submitted to Cofepris and this was approved in May 2018. Conclusions: The work with groups of experts allowed the Mexican authority to do a more expeditious work based on the evidence of the evaluations carried out that are part of the information of the sanitary registry. As a result of this process, Heberprot-P® was granted the Sanitary Registry in May 2018 and COFEPRIS recognized it as an innovative biotechnological medicine(AU)

Fructooligosaccharides production by immobilized Pichia pastoris cells expressing Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase.

Fructooligosaccharides (FOSs)-fructose-based oligosaccharides-are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.

Removal of bacterial dextran in sugarcane juice by Talaromyces minioluteus dextranase expressed constitutively in Pichia pastoris.

A gene construct encoding the mature region of Talaromyces minioluteus dextranase (EC 3.2.1.11) fused to the Saccharomyces cerevisiae SUC2 signal sequence was expressed in Pichia pastoris under the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP). The increase of the transgene dosage from one to two and four copies enhanced proportionally the extracellular yield of the recombinant enzyme (r-TmDEX) without inhibiting cell growth. The volumetric productivity of the four-copy clone in fed batch fermentation (51 h) using molasses as carbon source was 1706 U/L/h. The secreted N-glycosylated r-TmDEX was optimally active at pH 4.5-5.5 and temperature 50-60 °C. The addition of sucrose (600 g/L) as a stabilizer retained intact the r-TmDEX activity after 1-h incubation at 50-60 °C and pH 5.5. Bacterial dextran in deteriorated sugarcane juice was completely removed by applying a crude preparation of secreted r-TmDEX. The high yield of r-TmDEX in methanol-free cultures and the low cost of the fed batch fermentation make the P. pastoris pGAP-based expression system appropriate for the large scale production of dextranase and its use for dextran removal at sugar mills.

Engineered thermostable ß-fructosidase from Thermotoga maritima with enhanced fructooligosaccharides synthesis.

The thermostable ß-fructosidase (BfrA) from the bacterium Thermotoga maritima converts sucrose into glucose, fructose, and low levels of short-chain fructooligosaccharides (FOS) at high substrate concentration (1.75 M) and elevated temperatures (60-70 °C). In this research, FOS produced by BfrA were characterized by HPAE-PAD analysis as a mixture of 1-kestotriose, 6G-kestotriose (neokestose), and to a major extent 6-kestotriose. In order to increase the FOS yield, three BfrA mutants (W14Y, W14Y-N16S and W14Y-W256Y), designed from sequence divergence between hydrolases and transferases, were constructed and constitutively expressed in the non-saccharolytic yeast Pichia pastoris. The secreted recombinant glycoproteins were purified and characterized. The three mutants synthesized 6-kestotriose as the major component of a FOS mixture that includes minor amounts of tetra- and pentasaccharides. In all cases, sucrose hydrolysis was the predominant reaction. All mutants reached a similar overall FOS yield, with the average value 37.6% (w/w) being 3-fold higher than that of the wild-type enzyme (12.6%, w/w). None of the mutations altered the enzyme thermophilicity and thermostability. The single mutant W14Y, with specific activity of 841 U mg-1, represents an attractive candidate for the continuous production of FOS-containing invert syrup at pasteurization temperatures.

Fructooligosaccharides production by Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase constitutively expressed to high levels in Pichia pastoris.

The non-saccharolytic yeast Pichia pastoris was engineered to express constitutively the mature region of sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Tall fescue (Schedonorus arundinaceus). The increase of the transgene dosage from one to nine copies enhanced 7.9-fold the recombinant enzyme (Sa1-SSTrec) yield without causing cell toxicity. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (38%) and extracellular release (62%) of Sa1-SSTrec to an overall activity of 102.1 U/ml when biomass reached (106 g/l, dry weight) in fed-batch fermentation using cane sugar for cell growth. The volumetric productivity of the nine-copy clone PGFT6x-308 at the end of fermentation (72 h) was 1422.2 U/l/h. Sa1-SSTrec purified from the culture supernatant was a monomeric glycoprotein optimally active at pH 5.0-6.0 and 45-50 °C. The removal of N-linked oligosaccharides by Endo Hf treatment decreased the enzyme stability but had no effect on the substrate and product specificities. Sa1-SSTrec converted sucrose (600 g/l) into 1-kestose (GF2) and nystose (GF3) in a ratio 9:1 with their sum representing 55-60% (w/w) of the total carbohydrates in the reaction mixture. Variations in the sucrose (100-800 g/l) or enzyme (1.5-15 units per gram of substrate) concentrations kept unaltered the product profile. Sa1-SSTrec is an attractive candidate enzyme for the industrial production of short-chain fructooligosaccharides, most particularly 1-kestose.

Scaling-up batch conditions for efficient sucrose hydrolysis catalyzed by an immobilized recombinant Pichia pastoris cells in a stirrer tank reactor

Background: Invert sugar is used greatly in food and pharmaceutical industries. This paper describes scaling-up batch conditions for sucrose inversion catalyzed by the recombinant Pichia pastoris BfrA4X whole cells expressing Thermotoga maritima invertase entrapped in calcium alginate beads. For the first time, we describe the application of a kinetic model to predict the fractional conversion expected during sucrose hydrolysis reaction in both, a model and a prototype bioreactor with 0.5- and 5-L working volume, respectively. Results: Different scaled-up criteria used to operate the 0.5-L bioreactor were analyzed to explore the invert sugar large scale production. After model inversion studies, a 5-L scaled-up reaction system was performed in a 7-L stirred reactor. Both scaled-up criteria, immobilized biocatalyst dosage and stirring speed, were analyzed in each type of bioreactors and the collected data were used to ensure an efficient scale-up of this biocatalyst. Conclusions: To date, there is not enough information to describe the large-scale production of invert sugar using different scaled-up criteria such as dose of immobilized biocatalyst and stirring speed effect on mass transfer. The present study results constitute a valuable tool to successfully carry out this type of high-scale operation for industrial purposes.

Complete sucrose hydrolysis by heat-killed recombinant Pichia pastoris cells entrapped in calcium alginate.

BACKGROUND: An ideal immobilized biocatalyst for the industrial-scale production of invert sugar should stably operate at elevated temperatures (60-70°C) and high sucrose concentrations (above 60%, w/v). Commercial invertase from the yeast Saccharomyces cerevisiae is thermolabile and suffers from substrate inhibition. Thermotoga maritima ß-fructosidase (BfrA) is the most thermoactive and thermostable sucrose-hydrolysing enzyme so far identified and allows complete inversion of the substrate in highly concentrated solutions. RESULTS: In this study, heat-killed Pichia pastoris cells bearing N-glycosylated BfrA in the periplasmic space were entrapped in calcium alginate beads. The immobilized recombinant yeast showed maximal sucrose hydrolysis at pH 5-7 and 90°C. BfrA was 65% active at 60°C and had no activity loss after incubation without the substrate at this temperature for 15 h. Complete inversion of cane sugar (2.04 M) at 60°C was achieved in batchwise and continuous operation with respective productivities of 4.37 and 0.88 gram of substrate hydrolysed per gram of dry beads per hour. The half-life values of the biocatalyst were 14 and 20 days when operated at 60°C in the stirred tank and the fixed-bed column, respectively. The reaction with non-viable cells prevented the occurrence of sucrose fermentation and the formation of by-products. Six-month storage of the biocatalyst in 1.46 M sucrose (pH 5.5) at 4°C caused no reduction of the invertase activity. CONCLUSIONS: The features of the novel thermostable biocatalyst developed in this study are more attractive than those of immobilized S. cerevisiae cells for application in the enzymatic manufacture of inverted sugar syrup in batch and fixed-bed reactors.

Un thermostable exo-b-fructosidase inmovilizado por diseño racional / A thermostable exo-b-fructosidase immobilised through rational design

Thermotoga maritima exo-b-fructosidase (BfrA) secreted by a recombinant Pichia pastoris strain was optimall y immobilised on Glyoxyl–Sepharose CL 4B using the Rational Design of Immobilised Derivatives(RDID) strategy. Covalent attachment of the N-glycosylated BfrA on to the activated support at pH 10allowed total recovery of the loaded enzyme and its activity. The immobilisation process caused no variationin the catalytic properties of the enzyme and allowed further enhancement of the thermal stability.Complete inversion of cane sugar (2.04 M) in a batch stirred tank reactor at 60 C was achieved with aproductivity of 22.2 g of substrate hydrolysed/gram of biocatalyst/hour. Half-life of the immobilisedenzyme of 5 days at 60 C was determined in a continuously operated fixed-bed column reactor. Ourresults promote the applicability of the BfrA-immobilised biocatalyst for the complete hydrolysis of concentratedsucrose solutions under industrial conditions, especially at a high reaction temperature(AU)

A thermostable exo-ß-fructosidase immobilised through rational design.

Thermotoga maritima exo-ß-fructosidase (BfrA) secreted by a recombinant Pichia pastoris strain was optimally immobilised on Glyoxyl-Sepharose CL 4B using the Rational Design of Immobilised Derivatives (RDID) strategy. Covalent attachment of the N-glycosylated BfrA onto the activated support at pH 10 allowed total recovery of the loaded enzyme and its activity. The immobilisation process caused no variation in the catalytic properties of the enzyme and allowed further enhancement of the thermal stability. Complete inversion of cane sugar (2.04 M) in a batch stirred tank reactor at 60 °C was achieved with a productivity of 22.2 g of substrate hydrolysed/gram of biocatalyst/hour. Half-life of the immobilised enzyme of 5 days at 60 °C was determined in a continuously operated fixed-bed column reactor. Our results promote the applicability of the BfrA-immobilised biocatalyst for the complete hydrolysis of concentrated sucrose solutions under industrial conditions, especially at a high reaction temperature.

Constitutive high-level expression of a codon-optimized ß-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris.

Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable ß-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115 g/l, dry weight) and overall invertase activity (241 U/ml) were reached at 72 h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44 %) and extracellular release (56 %) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60 % (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70 °C, with average productivity of 4.4 g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup.