RESUMEN
BACKGROUND: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter. RESULTS: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ1-8PNP1-20 or LacZ1-8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression. The two most performing fusion protein systems namely short thermoinducible LacZ1-8Met-MTGase from NP668/1 and long constitutive LacZ1-8PNP1-20Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ1-8PNP1-20Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain. CONCLUSIONS: Combining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ1-8Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Proteínas Recombinantes/metabolismo , Transglutaminasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Escherichia coli/metabolismo , Fermentación , Plásmidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Streptomyces/enzimología , Streptomyces/genética , Transglutaminasas/química , Transglutaminasas/genética , Transglutaminasas/aislamiento & purificaciónRESUMEN
BACKGROUND AND OBJECTIVES: The new filgrastim formulation, BK0023, whose synthesis method is patented, was tested in a phase I clinical study that was aimed at investigating the pharmacodynamic and pharmacokinetic equivalence and the safety of BK0023 in healthy male subjects. METHODS: Single and multiple escalating doses were administered to healthy male volunteers according to a double-blind, randomised, two-way crossover design. Thirty-two subjects received subcutaneous filgrastim 2.5 µg/kg/day for 7 consecutive days in each period, 36 subjects received 5 µg/kg/day for 7 days in each period, and 22 subjects received 10 µg/kg/day for 5 days. Absolute neutrophil count (ANC) and CD34+ cell count were measured in whole blood as primary and secondary pharmacodynamic parameters. Filgrastim concentrations were measured in serum to calculate the primary pharmacokinetic parameters. RESULTS: The maximum ANC and the area under the curve of the ANC after the first dose and to the end of treatment satisfied the equivalence criterion (95 % confidence intervals within 85-115 or 85-117 % in case of log-transformation). At all three dose regimens, BK0023 was also bioequivalent to the reference product in terms of pharmacokinetic profile of serum filgrastim. The frequency of the treatment-emergent adverse events did not differ significantly between treatments, with the most frequent untoward effects being back and bone pain. CONCLUSIONS: Equivalence could be established using both the baseline-adjusted values and the original unadjusted values. The tested formulation at all three dose regimens was also bioequivalent to the reference product in terms of pharmacokinetic profile.
Asunto(s)
Filgrastim/administración & dosificación , Adulto , Química Farmacéutica , Estudios Cruzados , Método Doble Ciego , Filgrastim/farmacocinética , Filgrastim/farmacología , Humanos , Recuento de Leucocitos , Masculino , Neutrófilos/efectos de los fármacos , Proteínas Recombinantes , Equivalencia Terapéutica , Adulto JovenRESUMEN
BACKGROUND: Filgrastim or methionyl-granulocyte colony-stimulating factor (Met-G-CSF), is a recombinant therapeutic protein widely used to treat severe neutropenia caused by myelosuppressive drugs in patients with nonmyeloid malignancies. In addition to its role in the regulation of granulopoiesis, treatment with G-CSF is considered the standard approach to mobilize CD34 positive (CD34+) mononuclear cells for reconstituting hemopoietic ability for bone marrow transplantation. An intended biosimilar filgrastim (coded BK0023) was produced in GMP conditions by E.coli fermentation according to an original recombinant process and showed physico-chemical properties and purity profile similar to Neupogen®, a commercial preparation of filgrastim. The aim of the present study was to demonstrate the comparability of BK0023 to Neupogen® in terms of both in vitro biological activities and in vivo toxicology, pharmacokinetics and pharmacodynamics. METHODS: Cell proliferation and radioligand binding assays were conducted in NFS-60 cells to compare the biological activity and functional interaction with the G-CSF receptor in vitro, while preclinical in vivo studies, including pharmacokinetics and pharmacodynamics after repeated dose were performed in normal and neutropenic rats. A phase I study was carried out in healthy male volunteers treated by multiple-dose subcutaneous administration of BK0023 and Neupogen® to evaluate their pharmacodynamic effects as well as their pharmacokinetic and safety profile and to demonstrate their pharmacodynamic equivalence and pharmacokinetic bioequivalence. RESULTS: The results reported in this work demonstrate that BK0023 is comparable in terms of biological activity, efficacy and safety to Neupogen®. CONCLUSIONS: BK0023 has the same pharmacokinetic profile, efficacy and safety as the reference commercial filgrastim Neupogen® and therefore could be further developed to become a convenient option to treat neutropenia in oncological patients.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Neutropenia/tratamiento farmacológico , Animales , Línea Celular , Proliferación Celular , Estudios Cruzados , Femenino , Filgrastim , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Factor Estimulante de Colonias de Granulocitos/química , Factor Estimulante de Colonias de Granulocitos/farmacología , Recuento de Leucocitos , Masculino , Ratones , Neutrófilos/efectos de los fármacos , Conejos , Ratas , Ratas Sprague-Dawley , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Equivalencia Terapéutica , Resultado del TratamientoRESUMEN
The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values.
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Biofisica/métodos , Factor Estimulante de Colonias de Granulocitos/química , Metionina/química , Dicroismo Circular , Humanos , Luz , Modelos Estadísticos , Conformación Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Dispersión de Radiación , Espectrometría de Fluorescencia/métodos , Espectroscopía Infrarroja por Transformada de Fourier/métodos , TemperaturaRESUMEN
PEG(5 kDa)-cholane, PEG(10 kDa)-cholane and PEG(20 kDa)-cholane self-assembling polymers have been synthesised by the end-functionalisation of 5, 10 and 20 kDa linear amino-terminating monomethoxy-poly(ethylene glycol) (PEG-NH(2)) with 5ß-cholanic acid. Spectroscopic studies and isothermal titration calorimetry showed that the CMC of the PEG-cholane derivatives increased from 23.5 ± 1.8 to 60.2 ± 2.4 µM as the PEG molecular weight increased. Similarly, light scattering analysis showed that the micelle size increased from 15.8 ± 4.9 to 23.2 ± 11.1 nm with the PEG molecular weight. Gel permeation studies showed that the polymer bioconjugates associate with recombinant human granulocyte colony stimulating factor (rh-G-CSF) to form supramolecular nanocomposites according to multi-modal association profiles. The protein loadings obtained with PEG(5 kDa)-cholane, PEG(10 kDa)-cholane and PEG(20 kDa)-cholane were 7.4 ± 1.1, 2.7 ± 0.3 and 2.1 ± 0.4% (protein/polymer, w/w %), respectively. Scatchard and Klotz analyses showed that the protein/polymer affinity constant increased and that the number of PEG-cholane molecules associated to rh-G-CSF decreased as the PEG molecular weight increased. Isothermal titration calorimetry confirmed the protein/polymer multi-modal association. Circular dichroism analyses showed that the polymer association alters the secondary structure of the protein. Nevertheless, in vitro studies performed with NFS-60 cells showed that the polymer interaction does not impair the biological activity of the cytokine. In vivo studies performed by intravenous and subcutaneous administrations of rh-G-CSF to rats showed that the association with PEG(5 kDa)-cholane prolongs the body exposure of the protein. After subcutaneous administration, the protein t(max) values obtained with rh-G-CSF and 1:14 and 1:21 rh-G-CSF/PEG(5 kDa)-cholane (w/w ratio) nanocomplexes were 2, 8 and 24h, respectively. The 1:21 (w/w) rh-G-CSF/PEG(5kDa)-cholane formulation resulted in 149% relative bioavailability, and the pharmacokinetic behaviour was similar to that obtained with an equivalent protein dose of rh-G-CSF chemically conjugated with one linear 20-kDa PEG. A single administration of a 1.5mg/kg dose of a 1:21 (w/w) rh-G-CSF/PEG(5 kDa)-cholane formulation induced a high production of white blood cells for 96 h.
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Colanos/química , Preparaciones de Acción Retardada/química , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/farmacocinética , Nanocompuestos/química , Polietilenglicoles/química , Administración Intravenosa , Animales , Dicroismo Circular , Factor Estimulante de Colonias de Granulocitos/farmacología , Humanos , Inyecciones Subcutáneas , Recuento de Leucocitos , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologíaRESUMEN
We describe the preparation and characterization of a new monoPEGylated derivate of a recombinant form of filgrastim (methionyl human granulocite colony stimulating factor, rh-Met-G-CSF), BK0026, prepared by enzymatic site-specific 20kDa PEG conjugation to glutamine 135 residue by microbial transglutaminase catalyzed reaction. BK0026 was purified to a clinical grade by a single cation exchange chromatography step and characterized by using a panel of physicochemical analyses. NH(2)-terminal sequence and peptide mapping demonstrated no differences between the primary structure of BK0026 and the non-PEGylated filgrastim. The circular dichroism and fluorescence spectroscopy showed the preservation of high order protein structure. The single conjugation site on glutamine 135 was identified by endoproteinase Glu-C peptide mapping combined with mass spectrometry analysis and NH(2)-terminal sequence of the PEGylated peptides. BK0026 purity as well as product- and process-related contaminants was determined by several analytical methods, which showed that BK0026 is stable for more than 2 years when stored at 4-8°C. The advantages of enzymatic PEGylation of filgrastim are the absolute specificity of glutamine 135 conjugation combined with high PEGylation yields under very mild reaction conditions. The new site specific monoPEGylated filgrastim is a promising candidate for preclinical and clinical studies aimed at developing a long-lasting treatment of neutropenia in oncological patients under chemotherapy treatments.
Asunto(s)
Portadores de Fármacos/química , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/química , Polietilenglicoles/química , Tecnología Farmacéutica/métodos , Transglutaminasas/química , Secuencia de Aminoácidos , Animales , Catálisis , Línea Celular Tumoral , Fenómenos Químicos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Filgrastim , Glutamina/química , Factor Estimulante de Colonias de Granulocitos/farmacocinética , Masculino , Ratones , Datos de Secuencia Molecular , Mapeo Peptídico , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Espectrometría de Fluorescencia , Espectrometría de Masa por Ionización de Electrospray , Streptomyces/enzimologíaRESUMEN
Human glucagon-like peptide-1 (GLP-1) is a physiological gastrointestinal peptide with glucose-dependent insulinotropic effects which is therefore considered an interesting antidiabetic agent. However, after in vivo administration, exogenous GLP-1 does not exert its physiological action due to the combination of rapid proteolytic degradation by ubiquitous dipeptidyldipeptidase IV (DPP IV) enzyme and renal clearance resulting in an extremely short circulating half-life. In this work we describe the conjugation of GLP-1-(7-36)-amide derivatives with polyethylene glycol (PEG) by enzymatic site-specific transglutamination reaction as an approach to reduce both the proteolysis and the renal clearance rates. The compound GLP-1-(7-36)-amide-Q(23)-PEG 20 kDa monopegylated on the single glutamine residue naturally present in position 23 maintained the ability to activate the GLP-1 receptor expressed in the rat ß-cell line RIN-m5F with nanomolar potency along with an increased in vitro resistance to DDP IV and a circulating half-life of about 12 h after subcutaneous administration in rats. These properties enabled GLP-(7-36)-amide-Q(23)-PEG 20 kDa to exert a glucose-stabilizing effect for a period as long as 8 h, as demonstrated by a single subcutaneous injection to diabetic mice concomitantly challenged with an oral glucose load. The results reported in this work indicate that GLP-(7-36)-amide-Q(23)-PEG 20 kDa could be a lead compound for the development of long-lasting anti-diabetic agents useful in the treatment of type 2 diabetes affected patients.
RESUMEN
The protein delivery properties of polymer supramolecular assemblies were investigated by using recombinant human growth hormone (rh-GH) and two polyhydroxyethylaspartamide (PHEA) derivatives: (a) PHEA-C16 obtained by PHEA random grafting with hexadecylalkylamine; (b) PHEA-PEG 5000-C16 obtained by PHEA random co-grafting with hexadecylalkylamine and 5 kDa poly(ethylene glycol). The two polymers possessed similar self-assembling properties: critical micelle concentration (CMC) and particle size. The protein loading (protein/polymer, w/w, %) was 12.1+/-1.3% and 8.5+/-0.4% with PHEA-C16 and PHEA-PEG 5000-C16, respectively. The rh-GH/polymer association constant calculated by Scatchard analysis was 1.87 x 10(5)M(-1) with PHEA-C(16) and 0.27 x 10(5)M(-1) with PHEA-PEG 5000-C16. The Klotz analysis showed that 5 PHEA-C16 and 9 PHEA-PEG 5000-C16 polymer chains associated with one protein molecule. The protein dissociation from the PHEA-C16 and PHEA-PEG 5000-C16 supramolecular complexes was complete in about 350-450 and 450-550 h, respectively. With both polymers, the protein release was faster as the protein/polymer ratio increased. Pharmacokinetic studies were performed by subcutaneous administration to rats of protein/polymer solutions at different w/w ratios (1:75 and 1:150). Both polymer formulations slowed the protein absorption. The protein bioavailability increased as the protein/polymer complex stability decreased and the protein/polymer w/w ratio increased indicating that efficient protein delivery can be achieved by proper polymer choice and formulation composition.