RESUMEN

Pichia pastoris is a simple and powerful expression platform that has the ability to produce a wide variety of recombinant proteins, ranging from simple peptides to complex membrane proteins. A well-established fermentation strategy is available comprising three main phases: a batch phase, followed by a glycerol fed-batch phase that increases cell density, and finally an induction phase for product expression using methanol as the inducer. We previously used this three-phase strategy at the 15-L scale to express three different AMA1-DiCo-based malaria vaccine candidates to develop a vaccine cocktail. For two candidates, we switched to a two-phase strategy lacking the intermediate glycerol fed-batch phase. The new strategy not only provided a more convenient process flow but also achieved 1.5-fold and 2.5-fold higher space-time yields for the two candidates, respectively, and simultaneously reduced the final cell mass by a factor of 1.3, thus simplifying solid-liquid separation. This strategy also reduced the quantity of host cell proteins that remained to be separated from the two vaccine candidates (by 34% and 13%, respectively), thus reducing the effort required in the subsequent purification steps. Taken together, our new fermentation strategy increased the overall fermentation performance for the production of two different AMA1-DiCo-based vaccine candidates.

Asunto(s)

Antígenos de Protozoos/metabolismo , Biotecnología/métodos , Vacunas contra la Malaria/metabolismo , Proteínas de la Membrana/metabolismo , Pichia/metabolismo , Proteínas Protozoarias/metabolismo , Tecnología Farmacéutica/métodos , Antígenos de Protozoos/genética , Fermentación , Vacunas contra la Malaria/genética , Proteínas de la Membrana/genética , Pichia/genética , Proteínas Protozoarias/genética , Vacunas Sintéticas/genética , Vacunas Sintéticas/metabolismo

RESUMEN

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial.

Asunto(s)

Variación Antigénica , Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/inmunología , Proteínas de la Membrana/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Femenino , Fermentación , Humanos , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/efectos adversos , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Estabilidad Proteica , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Control de Calidad , Conejos , Proteínas Recombinantes

RESUMEN

The production of biopharmaceutical proteins in plants requires efficient downstream processing steps that remove impurities such as host cell proteins (HCPs) and adventitious endotoxins produced by bacteria during transient expression. We therefore strived to develop effective routines for endotoxin removal from plant extracts and the subsequent use of the extracts to generate antibodies detecting a broad set of HCPs. At first, we depleted the superabundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) for which PEG precipitation achieved the best results, preventing a dominant immune reaction against this protein. We found that a mixture of sera from rabbits immunized with pre-depleted or post-depleted extracts detected more HCPs than the individual sera used alone. We also developed a powerful endotoxin removal procedure using Polymyxin B for extracts from wild type plants or a combination of fiber-flow filtration and EndoTrap Blue for tobacco plants infiltrated with Agrobacterium tumefaciens. The antibodies we generated will be useful for quality and performance assessment in future process development and the methods we present can easily be transferred to other expression systems rendering them useful in the field of plant molecular farming.

Asunto(s)

Anticuerpos Monoclonales/metabolismo , Nicotiana/genética , Extractos Vegetales/inmunología , Polimixina B/aislamiento & purificación , Ribulosa-Bifosfato Carboxilasa/deficiencia , Agrobacterium tumefaciens/metabolismo , Animales , Especificidad de Anticuerpos , Endotoxinas , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/microbiología , Conejos , Nicotiana/inmunología , Nicotiana/microbiología

RESUMEN

The EU Sixth Framework Programme Integrated Project 'Pharma-Planta' developed an approved manufacturing process for recombinant plant-made pharmaceutical proteins (PMPs) using the human HIV-neutralizing monoclonal antibody 2G12 as a case study. In contrast to the well-established Chinese hamster ovary platform, which has been used for the production of therapeutic antibodies for nearly 30 years, only draft regulations were initially available covering the production of recombinant proteins in transgenic tobacco plants. Whereas recombinant proteins produced in animal cells are secreted into the culture medium during fermentation in bioreactors, intact plants grown under nonsterile conditions in a glasshouse environment provide various 'plant-specific' regulatory and technical challenges for the development of a process suitable for the acquisition of a manufacturing licence for clinical phase I trials. During upstream process development, several generic steps were addressed (e.g. plant transformation and screening, seed bank generation, genetic stability, host plant uniformity) as well as product-specific aspects (e.g. product quantity). This report summarizes the efforts undertaken to analyse and define the procedures for the GMP/GACP-compliant upstream production of 2G12 in transgenic tobacco plants from gene to harvest, including the design of expression constructs, plant transformation, the generation of production lines, master and working seed banks and the detailed investigation of cultivation and harvesting parameters and their impact on biomass, product yield and intra/interbatch variability. The resulting procedures were successfully translated into a prototypic manufacturing process that has been approved by the German competent authority.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Ingeniería Genética/métodos , Nicotiana/genética , Animales , Biomasa , Anticuerpos ampliamente neutralizantes , Células CHO , Cricetinae , Cricetulus , Vectores Genéticos/metabolismo , Anticuerpos Anti-VIH , Humanos , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente

RESUMEN

Radio frequency impedance spectroscopy (RFIS) is a robust method for the determination of cell biomass during fermentation. RFIS allows non-invasive in-line monitoring of the passive electrical properties of cells in suspension and can distinguish between living and dead cells based on their distinct behavior in an applied radio frequency field. We used continuous in situ RFIS to monitor batch-cultivated plant suspension cell cultures in stirred-tank bioreactors and compared the in-line data to conventional off-line measurements. RFIS-based analysis was more rapid and more accurate than conventional biomass determination, and was sensitive to changes in cell viability. The higher resolution of the in-line measurement revealed subtle changes in cell growth which were not accessible using conventional methods. Thus, RFIS is well suited for correlating such changes with intracellular states and product accumulation, providing unique opportunities for employing systems biotechnology and process analytical technology approaches to increase product yield and quality.

Asunto(s)

Biomasa , Espectroscopía Dieléctrica , Nicotiana/fisiología , Células Vegetales/fisiología , Reactores Biológicos , Biotecnología/métodos , Línea Celular , Espectroscopía Dieléctrica/instrumentación , Espectroscopía Dieléctrica/métodos , Fermentación , Sistemas en Línea , Plantas Modificadas Genéticamente/fisiología , Programas Informáticos

RESUMEN

Recombinant proteins can be produced in a diverse array of plant-based systems, ranging from whole plants growing in the soil to plant suspension cells growing in a fully-defined synthetic medium in a bioreactor. When the recombinant proteins are intended for medical use (plant-derived pharmaceutical proteins, PDPs) they fall under the same regulatory guidelines for manufacturing that cover drugs from all other sources, and when such proteins enter clinical development this includes the requirement for production according to good manufacturing practice (GMP). In principle, the well-characterized GMP regulations that apply to pharmaceutical proteins produced in bacteria and mammalian cells are directly transferrable to plants. In practice, the cell-specific terminology and the requirement for a contained, sterile environment mean that only plant cells in a bioreactor fully meet the original GMP criteria. Significant changes are required to adapt these regulations for proteins produced in whole-plant systems and it is only recently that the first GMP-compliant production processes using plants have been delivered.

Asunto(s)

Biotecnología/métodos , Plantas Modificadas Genéticamente/genética , Plantas Medicinales/genética , Proteínas Recombinantes/biosíntesis , Reactores Biológicos , Biotecnología/legislación & jurisprudencia , Células Vegetales/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Plantas Medicinales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/normas

RESUMEN

The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for super-transformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-gamma and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L(-1) range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MFalpha1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha.

Asunto(s)

Calnexina/genética , Calnexina/metabolismo , Dosificación de Gen , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , 6-Fitasa/genética , 6-Fitasa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Calnexina/biosíntesis , Calnexina/química , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo , Clonación Molecular , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Interferón gamma/genética , Interferón gamma/metabolismo , Datos de Secuencia Molecular , Alineación de Secuencia , Albúmina Sérica/genética , Albúmina Sérica/metabolismo , Transformación Genética

RESUMEN

Plants have been established as an useful production system for commercially relevant proteins. Plant cell cultures show certain advantages compared to field-grown plants. However, one critical drawback for the long-term use of recombinant plant cell cultures is the instability of cell cultures concerning genetic background and productivity when maintained by subculturing. The ability to store recombinant cell lines stably by cryopreservation allows to maintain an efficient and stable production system. In this work, we describe the development of a cryopreservation protocol for a transgenic BY-2 cell culture expressing human serum albumin. In 1.75-L stirred-tank bioreactors growth and production kinetics of the transgenic cell line were compared after cryopreservation to the hitherto performed maintenance by subculturing. Growth and productivity of the cryopreserved cell culture remained stable after freezing for one week. Here, we show that we developed an efficient method which allows the storage of transgenic plant cell cultures, an important requirement for industrial processes.

Asunto(s)

Criopreservación , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/metabolismo , Albúmina Sérica/metabolismo , Bancos de Tejidos , Reactores Biológicos , Técnicas de Cultivo de Célula/métodos , Línea Celular , Congelación , Humanos , Cinética , Plantas Modificadas Genéticamente/genética , Proteínas Recombinantes/metabolismo , Albúmina Sérica/genética , Nicotiana/citología , Nicotiana/genética , Nicotiana/metabolismo

RESUMEN

The use of whole plants for the synthesis of recombinant proteins has received a great deal of attention recently because of advantages in economy, scalability and safety compared with traditional microbial and mammalian production systems. However, production systems that use whole plants lack several of the intrinsic benefits of cultured cells, including the precise control over growth conditions, batch-to-batch product consistency, a high level of containment and the ability to produce recombinant proteins in compliance with good manufacturing practice. Plant cell cultures combine the merits of whole-plant systems with those of microbial and animal cell cultures, and already have an established track record for the production of valuable therapeutic secondary metabolites. Although no recombinant proteins have yet been produced commercially using plant cell cultures, there have been many proof-of-principle studies and several companies are investigating the commercial feasibility of such production systems.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Plantas/genética , Plantas/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Productos Biológicos/biosíntesis , Productos Biológicos/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Mejoramiento Genético/métodos , Transducción de Señal/fisiología

RESUMEN

The effect of major nutrients on growth and azadirachtin-related limonoids (AZRL) production in plant cell culture of Azadirachta indica (neem) was studied with the objective to increase the yield of AZRL, one of the major group of pesticidal compounds found in intact neem trees. We report the novel online monitoring of plant cell respiration activities in a new parallel shake flask measuring device. Results obtained using three standard plant cell culture media showed non-growth-associated production characteristics for AZRL. These findings were supported by the oxygen uptake rate data. Further investigations on AZRL production in a modified MS medium with different concentrations of nitrogen and phosphorus sources resulted in 0.25 mg.g(-1) dry weight of AZRL, compared to no detectable AZRL production in standard MS media. These characteristics suggest the necessity of a two-stage process for the production of AZRL in plant cell culture. Compared to the single-stage process, an almost twofold increase in the volumetric productivity of AZRL was achieved using the two-stage process.