RESUMEN
Since 1983, several recombinant antibodies have been expressed in important agronomic plant species. However, to date no evaluation has been published about prolonged antibody stability within plant tissues under cryo-preservation conditions. This current report presents an approach to the KDEL-plantibody HB-01 (PHB-01) stability in frozen tobacco leaves by presenting scientific evidence about the stability of a plantibody to a prolonged low temperature exposure in this biological source. Results clearly show that the PHB-01 amount is maintained during the storage of tobacco leaves at -20 degrees C for 90 days. The PHB-01 recovery was not affected by any irreversible physical and/or chemical change produced in tobacco leaves after this cryo-preservation time. The amount of total soluble proteins in the clarified extract decreased in proportion with the storage time and the PHB-01 molecules isolated from frozen leaf extracts were highly pure, >95%, according to an SDS-PAGE assessment under reducing conditions. Low temperature exposure of tobacco leaves did not reveal visible changes in frozen leaves, which is essential for the further extractability of proteins. The PHB-01 is stable in tobacco leaves at -20 degrees C during 90 days, which offers the possibility to overcome problems associated with detrimental climate conditions and optimize purification capabilities.
Asunto(s)
Anticuerpos contra la Hepatitis B/biosíntesis , Antígenos de Superficie de la Hepatitis B/inmunología , Nicotiana/metabolismo , Hojas de la Planta/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Criopreservación , Electroforesis en Gel de Poliacrilamida , Congelación , Anticuerpos contra la Hepatitis B/genética , Anticuerpos contra la Hepatitis B/inmunología , Vacunas contra Hepatitis B/biosíntesis , Vacunas contra Hepatitis B/inmunología , Ratones , Hojas de la Planta/genética , Plantas Modificadas Genéticamente , Ingeniería de Proteínas/métodos , Estabilidad Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Reproducibilidad de los Resultados , Tecnología Farmacéutica/métodos , Factores de Tiempo , Nicotiana/genéticaRESUMEN
Antibodies have been one of the proteins widely expressed in tobacco plants for pharmaceutical purposes, which demand contaminant free preparations. Rubisco constitutes 40-60% of tobacco leaf soluble proteins; therefore it is the major potential protein contaminant of plantibodies, while mycotoxins are toxic compounds that could be introduced during the biomass production and post-harvest stages with important consequences to human health. The objective of this paper was to investigate whether Rubisco and mycotoxins are present in Plantibody HB-01 preparations used in the immunopurification of the hepatitis B surface antigen. Rubisco was purified from Nicotiana tabacum yielding 154 microg of protein per gram of leaves and purity over 95%. Among mouse monoclonal antibodies generated against this enzyme, the CBSS.Rub-2 was selected for its immunodetection. It recognizes a conserved sequential epitope of Rubisco large subunit with an affinity constant of 0.13 x 10(8)M(-1). Rubisco quantification limit was 1 microg spreading to the measurement of this contaminant less than 4% of plantibodies samples. Additionally, according to a Reverse Phase-HPLC used to measure the level of adventitiously introduced contaminants, it can be concluded that aflatoxins B1, B2, G1 and G2 were undetected in the purified Plantibody HB-01 samples.
Asunto(s)
Aflatoxinas/análisis , Anticuerpos contra la Hepatitis B/aislamiento & purificación , Planticuerpos/aislamiento & purificación , Ribulosa-Bifosfato Carboxilasa/análisis , Aflatoxinas/química , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Western Blotting , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/estadística & datos numéricos , Contaminación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Anticuerpos contra la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/inmunología , Humanos , Ratones , Planticuerpos/genética , Plantas Modificadas Genéticamente , Nicotiana/genética , Nicotiana/inmunologíaRESUMEN
This paper evaluates the immunopurification behavior of a plantibody HBsAg specific plantibody coupled to Sepharose CL-4B at different ligand densities. Results show no significant differences in the adsorption and elution capacities, and rHBsAg recovery of immunosorbents at 3.43, 4.45, and 5.31 mg/mL of ligand densities compared to its mouse-derived mAb counterpart consistently used in the rHBsAg purification process. Therefore, plantibody ligand densities higher than 3.43 mg/mL do not improve the immunopurification behavior of this immunosorbent, but increase the antibody consumption and the Hepatitis B vaccine cost. Immunosorbent of 2.23 mg/mL of ligand density demonstrated a poor performance. The IgG leached detectable level never exceeded the approved limit (3 ng IgG/microg rHBsAg). Values close to this limit were only observed at the ligand density of 5.31 and 2.27 mg/mL. In the case of the ligand density of 2.23 mg/mL the IgG leached value was high (2.90 ng IgG/microg rHBsAg) due to a low level of eluted antigen. In conclusion, it supports feasibility of using this plantibody at 3.43 mg/mL of ligand density for large-scale immunopurification of rHBsAg for human use, avoiding the biosafety and ethical concerns of the massive use of animals for this purpose.
Asunto(s)
Cromatografía de Afinidad/métodos , Antígenos de Superficie de la Hepatitis B/aislamiento & purificación , Planticuerpos/inmunología , Sefarosa/química , Secuencia de Aminoácidos , Animales , Reacciones Antígeno-Anticuerpo , Western Blotting , Cromatografía en Gel/métodos , Electroforesis en Gel de Poliacrilamida , Técnicas para Inmunoenzimas , Ligandos , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
An enzyme-linked immunosorbent assay to quantify the mAb CB.Hep-1 during downstream purification process was standardized and validated. This assay is characterized by a short time of incubation at high temperature, allowing the detection of this antibody with high specificity and sensitivity. Detection of antigen-antibody reaction was achieved using a horseradish peroxidase conjugated anti-mouse IgG whose enzyme activity was revealed with o-phenylenediamine substrate. The immunoassay is linear in a range between 3.12 and 50 ng/mL, with a recovery of 98.55-107.62%. According to results, it is possible to estimate the mAb CB.Hep-1 concentration with high precision and reproducibility. The intra- and interassay coefficient of variation ranged from 0.25 to 8.64% and 1.84 to 9.43%, respectively. Significant differences were not observed in the plant-derived antibody quantification by HRP-ELISA and PhoA-ELISA (n=18), demonstrating that plant endogenous peroxidases do not produce interferences in the quantification of this molecule. Therefore, both antibodies can be tested with the same immunoassay with high precision, specificity and accuracy during their respective purification processes without interference of the buffers and sample characteristics.
Asunto(s)
Anticuerpos Monoclonales/análisis , Antígenos de Superficie de la Hepatitis B/inmunología , Planticuerpos/análisis , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática/métodos , Ratones , Ratones Endogámicos BALB C , Pichia/inmunología , Planticuerpos/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100-200kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice.
Asunto(s)
Anticuerpos/química , Antígenos de Superficie de la Hepatitis B/aislamiento & purificación , Plantas/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos/metabolismo , Western Blotting , Línea Celular Tumoral , Cromatografía , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Vacunas contra Hepatitis B/química , Humanos , Inmunoglobulina G/metabolismo , Ligandos , Ratones , Ratones Endogámicos BALB C , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes/química , Nicotiana/química , Nicotiana/inmunología , Vacunas Sintéticas/químicaRESUMEN
The application of bioengineering to plants for production of biological products for human and animal use has expanded in recent years. The reasons for this expansion are several and include advances in the technology for novel production systems and the need for very large quantities of therapeutic proteins. The process of growing pharmaceutical proteins in plants, extracting, and purifying is a hard task considering the lack of available information concerning these topics. In this work, a recombinant murine monoclonal antibody specific for the hepatitis B surface antigen, expressed in stably transformed transgenic Nicotiana tabacum plants, was purified by means of a recombinant protein A Streamline chromatography as the main purification step. The antibody expression level varied with the age of the plants and the number of harvests from 40 to 15microg/ml and the maximum process yield was about 25mg of plantibody/kg of biomass. Protein A Streamline chromatography was successfully used in the purification process yielding a recovery of about 60% and a plantibody SDS-PAGE purity of over 90% but unexpectedly, previous clarification steps could not be totally avoided. The amino acid sequence recognized by this affinity purified plantibody was similar to its murine counterpart verifying the potentiality of plants to replace animals or bioreactors for large-scale production of this monoclonal antibody.