EVALUACIÓN DE ACTIVOS INTANGIBLES EN LA INDUSTRIA BIOFARMACÉUTICA CUBANA: UN PROYECTO DE NEUROPROTECCIÓN COMO CASO DE ESTUDIO

The Center for Genetic Engineering and Biotechnology has developed a project, based on the co-administration of Epidermal Growth Factor (EGF) and Growth Hormone Releasing Peptide 6 (GHRP6) for its application in the indication of ischemic stroke. The article aims to develop a prospective business proposal for the introduction of the project in the international market. The four stages deployed for project negotiation are presented: characterization of the product and its application, definition of the target market, determination of the value of the project and proposal of the business model. The negotiation proposal can be generalized to many other innovative Research and Development (R&D) projects in the Cuban Biopharmaceutical industry.El Centro de Ingeniería Genética y Biotecnología ha desarrollado un proyecto, basado en la coadministración del Factor de Crecimiento Epidérmico (EGF) y del Péptido 6 liberador de hormona de crecimiento (GHRP6) para su aplicación en la indicación de ictus isquémico. El artículo tiene como objetivo desarrollar una propuesta prospectiva de negocio para la introducción del proyecto en el mercado internacional. Se presentan las cuatro etapas desplegadas para la negociación: caracterización del producto y su aplicación, definición del mercado objetivo, determinación del valor del proyecto y propuesta del modelo del negocio. La propuesta de negociación puede ser generalizada a otros muchos proyectos novedosos de Investigación y Desarrollo (I+D) de la industria Biofarmacéutica Cubana

Conformational and thermal stability improvements for the large-scale production of yeast-derived rabbit hemorrhagic disease virus-like particles as multipurpose vaccine.

Recombinant virus-like particles (VLP) antigenically similar to rabbit hemorrhagic disease virus (RHDV) were recently expressed at high levels inside Pichia pastoris cells. Based on the potential of RHDV VLP as platform for diverse vaccination purposes we undertook the design, development and scale-up of a production process. Conformational and stability issues were addressed to improve process control and optimization. Analyses on the structure, morphology and antigenicity of these multimers were carried out at different pH values during cell disruption and purification by size-exclusion chromatography. Process steps and environmental stresses in which aggregation or conformational instability can be detected were included. These analyses revealed higher stability and recoveries of properly assembled high-purity capsids at acidic and neutral pH in phosphate buffer. The use of stabilizers during long-term storage in solution showed that sucrose, sorbitol, trehalose and glycerol acted as useful aggregation-reducing agents. The VLP emulsified in an oil-based adjuvant were subjected to accelerated thermal stress treatments. None to slight variations were detected in the stability of formulations and in the structure of recovered capsids. A comprehensive analysis on scale-up strategies was accomplished and a nine steps large-scale production process was established. VLP produced after chromatographic separation protected rabbits against a lethal challenge. The minimum protective dose was identified. Stabilized particles were ultimately assayed as carriers of a foreign viral epitope from another pathogen affecting a larger animal species. For that purpose, a linear protective B-cell epitope from Classical Swine Fever Virus (CSFV) E2 envelope protein was chemically coupled to RHDV VLP. Conjugates were able to present the E2 peptide fragment for immune recognition and significantly enhanced the peptide-specific antibody response in vaccinated pigs. Overall these results allowed establishing improved conditions regarding conformational stability and recovery of these multimers for their production at large-scale and potential use on different animal species or humans

Evaluation of methodologies to determine the effect of specific active immunotherapy on VEGF levels in phase I clinical trial patients with advanced solid tumors

Two phase I clinical trials were conducted to evaluate, among other parameters, the humoral response elicited by a vascular endothelial growth factor (VEGF)-based therapeutic vaccine in cancer patients with advanced solid tumors. VEGF reduction was studied using an indirect methodology named as “Platelet VEGF”. This methodology is based on the estimation of VEGF within platelets by subtracting the plasma VEGF level from the serum level and dividing this by the platelet count, and then this latter expression is additionally corrected by the hematocrit. However, there is broad debate, whether serum or plasma VEGF or platelet-derived VEGF measurements is the most appropriate strategy to study the changes that occur on ligand bioavailability when patients are submitted to a VEGF-based immunotherapy.The current research is a retrospective study evaluating the changes on VEGF levels in serum and plasma as well as platelet-derived measurements. Changes in VEGF levels were related with the humoral response seen in cancer patients after an active immunotherapy with a VEGF-based vaccine. The present study indicates that “Platelet VEGF” is the most reliable methodology to investigate the effect of VEGF-based immunotherapies on ligand bioavailability. “Platelet VEGF” was associated with those groups of individuals that exhibited the best specific humoral response and the variation of “Platelet VEGF” showed the strongest negative correlation with VEGF-specific IgG antibody levels. This methodology will be very useful for the investigation of this VEGF-based vaccine in phase II clinical trials and could be applied to immunotherapies directed to other growth factors that are actively sequestered by platelets

Analysis of the RHDV VLP purified under different conditions.

<p>(<b>A</b>) Chromatographic profile of RHDV VLP after disruption and purification conducted at different pH values. Purification was performed by sec-HPLC using a TSK G-3000 preparative column. In all cases multimeric VP1 was detected in the first peak and quantified by sandwich ELISA using monoclonal antibodies. Retention times are indicated. (<b>B</b>) Electron micrographs of negatively stained samples containing VP1 multimers obtained after separation in sec-HPLC. According to measurements by ELISA, the highest recoveries were obtained at pH 4 and 7. The morphology and size of the VLP was analyzed by TEM using 2% uranyl acetate for negative stain. The bars indicate 200 nm. Magnifications: x30 000 and x40 000. (<b>C</b>) SDS-PAGE (lanes 1, 2) and Western blot analysis (lanes 3, 4) of purified VLP. Lane 1 corresponds to disruption supernatant from <i>P. pastoris</i> PVP12 strain. Lanes 2 and 3 were loaded with purified RHDV VLP. A minor band migrating close to 60 kDa was also detected in lane 3. For comparison, lane 4 shows glycosylated VP1 expressed associated to the yeast membranous system in PVP11 strain, in which various protein bands were recognized by the hyperimmune serum. The molecular weight of approximately 60 kDa is shown in the left and indicated by an arrow in the right. (<b>D</b>) Analysis with conformation dependent monoclonal antibodies of epitopes located on the VLP obtained after cell disruption, purification, and rounds of stressing steps at pH values of 4 and 7. RHDV VLP and native RHDV were detected using mAbs 1H8, 6F9, 5B2, and 6G2. Standard deviation bars of measurements performed are indicated.</p

Hemagglutination inhibition titers detected in sera of rabbits immunized with the recombinant VLP and challenged with RHDV.

a<p>The HI titer was expressed as the maximum dilution capable to completely inhibit the agglutination of human type O erythrocytes by RHDV. Each titer value corresponds to an individual animal. In the assay, a hyperimmune serum against RHDV (with an HI titer of 1/2048) was used as positive control. All animals were immunized at days 0 and 21. n.d.: hemagglutination inhibition titers not detected.</p>b<p>The result is also expressed as the mean ± standard deviation of inhibition values obtained in cELISA from individual animals. Different letters within a row indicate statistical significant differences for <i>p</i><0.05, according to the Kruskal-Wallis and Dunn’s tests.</p

Recovery of VLP obtained after buffer variations and with the use of stabilizers during storage.

a<p>Various excipients were evaluated at different concentrations during VLP in storage at 4°C for a time period of 24 weeks after purification. The initial concentration of VLP in all samples was 500 µg mL⁻¹. The experiments were conducted using duplicated lots. The percent of recovery was calculated as the percent of aggregation inhibition in the presence of additive = aggregation inhibition in the presence of additive * 100/concentration of aggregated proteins in the absence of additive where:</p><p>aggregation inhibition in the presence of additive = concentration of aggregated proteins in the absence of additive - concentration of aggregated proteins in the presence of additive.</p><p>Similar results in terms of recovery were obtained at pH 4 or 7 with the additives assayed. For simplicity, only recoveries at pH 7.0 are shown.</p><p>As a control lot, VLP were kept in identical storage conditions without stabilizers in phosphate buffer pH 7.0, 300 mM NaCl. In such case, the recovery of VLP accounted for a 48.5%.</p

Analysis of the recombinant VLP extracted from vaccine formulations subjected to an accelerated stress treatment experiment.

<p>(<b>A</b>) Analysis in size-exclusion HPLC. The temperature at which the emulsion was kept for one week and the retention times corresponding to elution of multimeric VP1 are indicated in each chromatogram. The inner figures show Western blot analyses and detection of VP1 in the main peaks detected, which were designated as 1 and 2 according to their appearance in the chromatogram. An anti-RHDV hyperimmune serum was used. The 60 kDa band is indicated. (<b>B</b>) Electron micrographs of negatively stained samples containing the extracted VLP. The VLP structure was analyzed by TEM using 2% uranyl acetate. The bar indicates 200 nm. Magnification: x40 000. (<b>C</b>) Detection of RHDV VLP epitopes with the conformational-sensitive monoclonal antibodies 1H8, 6F9, 6H6, 5B2, and 6G2 after thermal stresses. Standard deviation bars are indicated.</p

Recovery of properly assembled RHDV VLP during the evaluation of different pH in the disruption and purification processes.

<p>Purification was performed by size exclusion chromatography using a preparative TSK G3000 column. Alternatively, an additional step known to induce instability and protein aggregation (rounds of freezing-thawing) was included. Quantification of recovered VLP was accomplished by sandwich ELISA with the use of mAbs.</p><p>VLP directly detected after cell disruption and clarification were expressed in the first raw as µg per mL of disruption supernatant. Starting from those values, percents of recovery are shown for the next two steps. All measurements were done in duplicate starting from two independent bioreactions. Protein amounts and percents of recovery refer only to properly assembled VLP.</p

Evaluation of the apparent viscosity in a velocity gradient of RHDV VLP vaccine samples treated at 4°C, 37°C, and 48°C for 7 days.

<p>An emulsion sample taken immediately after formulation was used as control. The average and standard deviation bars for each experimental point are indicated. T0 =  time 0.</p