Economic evaluation of the primary recovery of tetracycline with traditional and novel aqueous two-phase systems.

Antibiotics are a key pharmaceutical to inhibit growth or kill microorganisms. They represent a profitable market and, in particular, tetracycline has been listed as an essential medicine by the WHO. Therefore it is important to improve their production processes. Recently novel and traditional aqueous two-phase systems for the extraction have been developed with positive results. The present work performs an economic analysis of the production and recovery of tetracycline through the use of several ATPS through bioprocess modeling using specialized software (BioSolve, Biopharm Services Ltd, UK) to determine production costs per gram (CoG/g). First, a virtual model was constructed using published data on the recovery of tetracycline and extended to incorporate uncertainties. To determine how the model behaved, a sensitivity analysis and Monte Carlo simulations were performed. Results showed that ATPS formed by cholinium chloride/K3PO4 was the best option to recover tetracycline, as it had the lowest CoG/g (US$ 672.83/g), offered the highest recovery yield (92.42%), second best sample input capacity (45% of the ATPS composition) and one of the lowest materials contribution to cost. The ionic liquid-based method of ATPS is a promising alternative for recovering tetracycline from fermentation broth.

Genetic manipulation of microalgae for the production of bioproducts.

Microalgae have been used during the past four decades in the Bio-industries for the production of high added value products and development of useful approaches with environmental applications. The fast growing rate, simple growth requirements and using sunlight as the major source of energy are the key factors for usage of algae. In the past 15 years, a considerable progress has been made regarding the use of microalgae for production of proteins, nutraceuticals, food supplements, molecular tags for diagnostics and fixation of greenhouse gases. Nevertheless, genetic manipulation of microalgae still remains a fairly un-explored area which could boost the production of bioproducts. It is anticipated that in the near future use of microalgae will revolutionize its applications in diverse industries. The aim of this work is to present a critical review on potential of microalgae for the production of high-added value molecules, their practical applications, and the role of genetic engineering in its utilization as a unique niche in industry. In addition, current challenges within synthetic biology approaches are discussed.

Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two-phase extraction (with and without PEG recycle).

Uricase is the enzyme responsible for the breakdown of uric acid, the key molecule leading to gout in humans, into allantoin, but it is absent in humans. It has been produced as a PEGylated pharmaceutical where the purification is performed through three sequential chromatographic columns. More recently an aqueous two-phase system (ATPS) was reported that could recover Uricase with high yield and purity. Although the use of ATPS can decrease cost and time, it also generates a large amount of waste. The ability, therefore, to recycle key components of ATPS is of interest. Economic modelling is a powerful tool that allows the bioprocess engineer to compare possible outcomes and find areas where further research or optimization might be required without recourse to extensive experiments and time. This research provides an economic analysis using the commercial software BioSolve of the strategies for Uricase production: chromatographic and ATPS, and includes a third bioprocess that uses material recycling. The key parameters that affect the process the most were located via a sensitivity analysis and evaluated with a Monte Carlo analysis. Results show that ATPS is far less expensive than chromatography, but that there is an area where the cost of production of both bioprocesses overlap. Furthermore, recycling does not impact the cost of production. This study serves to provide a framework for the economic analysis of Uricase production using alternative techniques.

Economic analysis of pilot-scale production of B-phycoerythrin.

ß-Phycoerythrin is a color protein with several applications, from food coloring to molecular labeling. Depending on the application, different purity is required, affecting production cost and price. Different production and purification strategies for B-phycoerythrin have been developed, the most studied are based on the production using Porphyridium cruentum and purified using chromatographic techniques or aqueous two-phase systems. The use of the latter can result in a less expensive and intensive recovery of the protein, but there is lack of a proper economic analysis to study the effect of using aqueous two-phase systems in a scaled-up process. This study analyzed the production of B-Phycoerythrin using real data obtained during the scale-up of a bioprocess using specialized software (BioSolve, Biopharm Services, UK). First, a sensitivity analysis was performed to identify critical parameters for the production cost, then a Monte Carlo analysis to emulate real processes by adding uncertainty to the identified parameters. Next, the bioprocess was analyzed to determine its financial attractiveness and possible optimization strategies were tested and discussed. Results show that aqueous two-phase systems retain their advantages of low cost and intensive recovery (54.56%); the costs of production per gram calculated (before titer optimization: US$15,709 and after optimization: US$2,374) allowed to obtain profit (in the range of US$millions in a 10-year period) for a potential company taking this production method by comparing the production cost against commercial prices. The bioprocess analyzed is a promising and profitable method for the generation of a highly purified B-phycoerythrin. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1472-1479, 2016.

Economic analysis of Royalactin production under uncertainty: Evaluating the effect of parameter optimization.

Royalactin is a protein with several different potential uses in humans. Research, in insects and in mammalian cells, has shown that it can accelerate cell division and prevent apoptosis. The method of action is through the use of the epidermal growth factor receptor, which is present in humans. Potential use in humans could be to lower cholesterolemic levels in blood, and to elicit similar effects to those seen in bees, e.g., increased lifespan. Mass production of Royalactin has not been accomplished, though a recent article presented a Pichia pastoris fermentation and recovery by aqueous two-phase systems at laboratory scale as a possible basis for production. Economic modelling is a useful tool with which compare possible outcomes for the production of such a molecule and in particular, to locate areas where additional research is needed and optimization may be required. This study uses the BioSolve software to perform an economic analysis on the scale-up of the putative process for Royalactin. The key parameters affecting the cost of production were located via a sensitivity analysis and then evaluated by Monte Carlo analysis. Results show that if titer is not optimized the strategy to maintain a low cost of goods is process oriented. After optimization of this parameter the strategy changes to a product-oriented and the target output becomes the critical parameter determining the cost of goods. This study serves to provide a framework for the evaluation of strategies for future production of Royalactin, by analyzing the factors that influence its cost of manufacture.

Specific recognition of influenza A/H1N1/2009 antibodies in human serum: a simple virus-free ELISA method.

BACKGROUND: Although it has been estimated that pandemic Influenza A H1N1/2009 has infected millions of people from April to October 2009, a more precise figure requires a worldwide large-scale diagnosis of the presence of Influenza A/H1N1/2009 antibodies within the population. Assays typically used to estimate antibody titers (hemagglutination inhibition and microneutralization) would require the use of the virus, which would seriously limit broad implementation. METHODOLOGY/PRINCIPAL FINDINGS: An ELISA method to evaluate the presence and relative concentration of specific Influenza A/H1N1/2009 antibodies in human serum samples is presented. The method is based on the use of a histidine-tagged recombinant fragment of the globular region of the hemagglutinin (HA) of the Influenza A H1N1/2009 virus expressed in E. coli. CONCLUSIONS/SIGNIFICANCE: The ELISA method consistently discerns between Inf A H1N1 infected and non-infected subjects, particularly after the third week of infection/exposure. Since it does not require the use of viral particles, it can be easily and quickly implemented in any basic laboratory. In addition, in a scenario of insufficient vaccine availability, the use of this ELISA could be useful to determine if a person has some level of specific antibodies against the virus and presumably at least partial protection.

An influenza A/H1N1/2009 hemagglutinin vaccine produced in Escherichia coli.

BACKGROUND: The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy. METHODOLOGY/PRINCIPAL FINDINGS: We expressed the globular HA receptor binding domain, referred to here as HA(63-286)-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA(63-286)-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model. CONCLUSIONS/SIGNIFICANCE: Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy.