Physicochemical and biological characterization of 1E10 Anti-Idiotype vaccine

<p>Abstract</p> <p>Background</p> <p>1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)₃, in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained <it>in vivo </it>from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the <it>in vivo </it>to a bioreactor-based method.</p> <p>Results</p> <p>Here, we present a comprehensive molecular and immunological characterization of 1E10 produced by the two different production processes in order to determine the impact of the manufacturing process in vaccine performance. We observed differences in glycosylation pattern, charge heterogeneity and structural stability between <it>in vivo</it>-produced 1E10 and bioreactor-obtained 1E10. Interestingly, these modifications had no significant impact on the immune responses elicited in two different animal models.</p> <p>Conclusions</p> <p>Changes in 1E10 primary structure like glycosylation; asparagine deamidation and oxidation affected 1E10 structural stability but did not affect the immune response elicited in mice and chickens when compared to 1E10 produced in mice.</p

N-Glycosylation of Plant Recombinant Pharmaceuticals

International audienceN-glycosylation is a maturation event necessary for the correct function, efficiency, and stability of a high number of biopharmaceuticals. This chapter presented here proposes various methods to determine whether, how, and where a plant pharmaceutical is N-glycosylated. These methods rely on blot detection with glycan-specific probes, specific deglycosylation of glycoproteins followed by mass spectrometry, N-glycan profile analysis, and glycopeptide identification by LC-MS

Resultados del tratamiento laparoscópico de la colecistitis aguda Laparoscopy treatment of acute cholecystitis: Results

INTRODUCCIÓN. Desde la introducción de la colecistectomía laparoscópica, numerosos autores han documentado las ventajas de esta técnica sobre la colecistectomía abierta, y se considera el abordaje estándar para el tratamiento de la colelitiasis no complicada. Sin embargo, existía cierta resistencia por parte de los cirujanos cuando se trataba de la colecistitis aguda. El objetivo de nuestro estudio fue presentar los resultados obtenidos con el tratamiento laparoscópico precoz de la colecistitis aguda en nuestro servicio. MÉTODOS. De un total de 142 pacientes ingresados con el diagnóstico de colecistitis aguda, 49 fueron intervenidos mediante colecistectomía laparoscópica de urgencia durante el período comprendido entre febrero del 2003 y febrero del 2007. Se establecieron 5 criterios para la selección de los pacientes: comienzo de los síntomas antes de 72 h, diagnóstico clínico y ultrasonográfico de colecistitis aguda, vía biliar principal no dilatada, ausencia de imagen sugestiva de litiasis coledociana, ausencia de cirugía del abdomen superior y pacientes sin contraindicación para la cirugía laparoscópica. RESULTADOS. El tiempo quirúrgico promedio fue de 65 min. Hubo una lesión de la vía biliar principal que se detectó durante la cirugía (herida de colédoco), 2 infecciones de la herida, 2 pacientes precisaron de conversión: uno por lesión de la vía biliar y otro por sangrado del lecho vesicular. La estadía hospitalaria promedio fue de 2,5 días. No hubo reintervenciones ni mortalidad en esta serie. CONCLUSIONES. Se concluyó que el método laparoscópico en la colecistitis aguda, cuando se indica precozmente, es seguro y factible. Recomendamos que sea la primera opción terapéutica en estos pacientes.INTRODUCTION: From introduction of laparoscopic cholecystectomy, many authors have verified advantages of this technique on open cholecystectomy, and it is consider the standard approach for treatment of non-complicated cholelitiasis. However, there was some resistance by surgeons when to be about the acute cholecystitis. Aim of present paper was to present results achieved with early laparoscopic treatment of acute cholecystitis in our service. METHODS: From a total of 142 patients admitted diagnosed with acute cholecystitis, 49 of them were operated on by means emergent laparoscopic cholecystectomy from February 2003 to February 2007. Five criteria was established for patients selection: start of symptoms before 72 hours, clinical and ultrasound diagnosis of acute cholecystitis, non-dilated main biliary route, lack of suggestive image of choledochal lithiasis, lack of high abdomen surgery, and patients with contraindications for laparoscopic surgery. RESULTS: Average surgical time was of 65 minutes. There was a lesion of main biliary route detected at surgery (choledochal wound), two wound infections, and two patients needed conversion: one by biliary route lesion and another by vesicular roof bleeding. Average hospital stay was or 2, 5 days. There were neither re-interventions nor mortality in this series. CONCLUSIONS: We conclude that laparoscopic method in case of acute cholecystitis, when it is early prescribed, is safe and feasible. We suggested that it be the first therapeutical option in these patients

Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants

International audiencePlants synthesize N-glycans containing the antigenic sugars α(1,3)-fucose and β(1,2)-xylose. Therefore it is important to monitor these N-glycans in monoclonal antibodies produced in plants (plantibodies). We evaluated several techniques to characterize the N-glycosylation of a plantibody produced in tobacco plants with and without the KDEL tetrapeptide endoplasmic reticulum retention signal which should inhibit or drastically reduce the addition of α(1,3)-fucose and β(1,2)-xylose. Ammonium hydroxide/carbonate-based chemical deglycosylation and PNGase A enzymatic release were investigated giving similar 2-aminobenzamide-labeled N-glycan HPLC profiles. The chemical release does not generate peptides which is convenient for MS analysis of unlabeled pool but its main drawback is that it induces degradation of α1,3-fucosylated N-glycan reducing terminal sugar. Three analytical methods for N-glycan characterization were evaluated: (i) MALDI-MS of glycopeptides from tryptic digestion; (ii) negative-ion ESI-MS/MS of released N-glycans; (iii) normal-phase HPLC of fluorescently labeled glycans in combination with exoglycosidase sequencing. The MS methods identified the major glycans, but the HPLC method was best for identification and relative quantitation of N-glycans. Negative-mode ESI-MS/MS permitted also the correct identification of the linkage position of the fucose residue linked to the inner core N-acteylglucosamine (GlcNAc) in complex N-glycans

A propósito de un caso de liposarcoma retroperitoneal Apropos of a case of retroperitoneal liposarcoma

El liposarcoma retroperitoneal es un tumor maligno de origen mesenquimatoso, y el más frecuente de los sarcomas de partes blandas de localización retroperitoneal. Su crecimiento es lento e insidioso, lo que le permite adquirir grandes dimensiones permaneciendo asintomático. Se detecta en el examen físico o mediantes exámenes complementarios. Se presenta el caso de una paciente con una gran masa irregular hacia la mitad derecha y superior del abdomen y se muestran los resultados de los estudios complementarios que sugieren la presencia de un tumor retroperitoneal. Se encontró un tumor bien encapsulado que se extirpó totalmente. El estudio histológico confirmó un liposarcoma retroperitoneal bien diferenciado. La paciente ha tenido una evolución satisfactoria durante 2 años de seguimientoThe retroperitoneal liposarcoma is a malignant tumor of mesenchymatous origin, and it is the most frequent of the sarcomas of soft tissues of retroperitoneal localization. Its growth is slow and insidious, which allows it to acquire large dimensions in an asymptomatic way. It is detected in the physical examination or by complementary tests. The case of a patient with a large irregular mass on the upper right half of the abdomen is presented. The results of the complementary studies suggesting the presence of a retroperitoneal tumor are showed. A well encapsulated tumor that was totally removed was found. The histological study confirmed a well differentiated retroperitoneal liposarcoma. The patient has had a satisfactory evolution during 2 years of follow-u

Contribution for analitical methodology for quality control of antigens of Cuban vaccine VA-MENGOC BC®and for a new antityphoidic vaccine

Se requería incorporar nuevos elementos al conocimiento acerca de la caracterización y control de la calidad de la vacuna antimeningocócica cubana VA-MENGOC BC® para demostrar su seguridad y elevar su competitividad en el mercado. Se combinaron métodos como la cromatografía de tamizaje molecular, microscopía electrónica, SDS-PAGE, Western Blot, tipificación del lipopolisacárido, Western Blot específico (DNAsa y RNAsa); para la evaluación de la morfología vesicular, identidad proteica y pureza de las vesículas de membrana externa de Neisseria meningitidis serogrupo B. Se incorporaron métodos para la evaluación de la viscosidad y de la identidad mediante ensayos inmunoquímicos y resonancia magnético nuclear, para los polisacáridos C de N. meningitidis y Vi de Salmonella typhi. Los resultados permitieron establecer referencias para la caracterización y control de estos antígenos, en términos de integridad, identidad, pureza y estabilidad. La aplicación de estos métodos permitieron demostrar la presencia de estructuras con comportamientos físico- químicos y patrones inmunoquímicos específicos, similares a los obtenidos por instituciones internacionales de control, lo que permitió conformar la metodología analítica para el control de la nueva vacuna antitifoidea. La combinación de estos métodos resultó en una metodología analítica armónica y validable.New elements to the knowledge were necessary for the characterization and quality control of cuban meningococcal vaccine VA-MENGOC BC® in order to demonstrate security and increasing market competitiveness. Matched methods as gel chromatography, electronic microscopy, SDS-PAGE, Western Blot, lipopolysaccharide classification, specific Western Blot (DNAsa and RNAsa contaminant enzymes), for evaluating of vesicular morphology, protein identity and purity of outer membrane vesicle of N. meningitidis were carried out. We included some methods to evaluate the viscosity and identity for immunochemical assays and nuclear magnetic resonance for meningococcal C polysaccharide and Vi polysaccharide from Salmonella typhi. Results established some references to characterization and control of that antigens according to the identity, integrity, purity and stability parameters. Application of the methods allowed demonstrating the physical-chemical and specific immunochemical behavior like the obtained by international control institutes. All results allowed to complete the analytical methodology for the control of a new antityphoid vaccine. Combination of methods resulted in an harmonic and validated methodology.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Contribution for analitical methodology for quality control of antigens of Cuban vaccine VA-MENGOC BC®and for a new antityphoidic vaccine

Se requería incorporar nuevos elementos al conocimiento acerca de la caracterización y control de la calidad de la vacuna antimeningocócica cubana VA-MENGOC BC® para demostrar su seguridad y elevar su competitividad en el mercado. Se combinaron métodos como la cromatografía de tamizaje molecular, microscopía electrónica, SDS-PAGE, Western Blot, tipificación del lipopolisacárido, Western Blot específico (DNAsa y RNAsa); para la evaluación de la morfología vesicular, identidad proteica y pureza de las vesículas de membrana externa de Neisseria meningitidis serogrupo B. Se incorporaron métodos para la evaluación de la viscosidad y de la identidad mediante ensayos inmunoquímicos y resonancia magnético nuclear, para los polisacáridos C de N. meningitidis y Vi de Salmonella typhi. Los resultados permitieron establecer referencias para la caracterización y control de estos antígenos, en términos de integridad, identidad, pureza y estabilidad. La aplicación de estos métodos permitieron demostrar la presencia de estructuras con comportamientos físico- químicos y patrones inmunoquímicos específicos, similares a los obtenidos por instituciones internacionales de control, lo que permitió conformar la metodología analítica para el control de la nueva vacuna antitifoidea. La combinación de estos métodos resultó en una metodología analítica armónica y validable.New elements to the knowledge were necessary for the characterization and quality control of cuban meningococcal vaccine VA-MENGOC BC® in order to demonstrate security and increasing market competitiveness. Matched methods as gel chromatography, electronic microscopy, SDS-PAGE, Western Blot, lipopolysaccharide classification, specific Western Blot (DNAsa and RNAsa contaminant enzymes), for evaluating of vesicular morphology, protein identity and purity of outer membrane vesicle of N. meningitidis were carried out. We included some methods to evaluate the viscosity and identity for immunochemical assays and nuclear magnetic resonance for meningococcal C polysaccharide and Vi polysaccharide from Salmonella typhi. Results established some references to characterization and control of that antigens according to the identity, integrity, purity and stability parameters. Application of the methods allowed demonstrating the physical-chemical and specific immunochemical behavior like the obtained by international control institutes. All results allowed to complete the analytical methodology for the control of a new antityphoid vaccine. Combination of methods resulted in an harmonic and validated methodology.Colegio de Farmacéuticos de la Provincia de Buenos Aire