Comparative Economic Analysis Between Endogenous and Recombinant Production of Hyaluronic Acid

Hyaluronic acid (HA) is a biopolymer with a wide range of applications, mainly in the cosmetic and pharmaceutical sectors. Typical industrial-scale production utilizes organisms that generate HA during their developmental cycle, such as Streptococcus equi sub. zooepidemicus. However, a significant disadvantage of using Streptococcus equi sub. zooepidemicus is that it is a zoonotic pathogen, which use at industrial scale can create several risks. This creates opportunities for heterologous, or recombinant, production of HA. At an industrial scale, the recovery and purification of HA follow a series of precipitation and filtration steps. Current recombinant approaches are developing promising alternatives, although their industrial implementation has yet to be adequately assessed. The present study aims to create a theoretical framework to forecast the advantages and disadvantages of endogenous and recombinant strains in production with the same downstream strategy. The analyses included a selection of the best cost-related recombinant and endogenous production strategies, followed by a sensitivity analysis of different production variables in order to identify the three most critical parameters. Then, all variables were analyzed by varying them simultaneously and employing multiple linear regression. Results indicate that, regardless of HA source, production titer, recovery yield and bioreactor scale are the parameters that affect production costs the most. Current results indicate that recombinant production needs to improve current titer at least 2-fold in order to compete with costs of endogenous production. This study serves as a platform to inform decision-making for future developments and improvements in the recombinant production of HA

Antibiofilm surfaces based on the immobilization of a novel recombinant antimicrobial multidomain protein using self-assembled monolayers

The constant increase of microorganisms resistant to antibiotics has been classified as a global health emergency, which is especially challenging when biofilms are formed. Herein, novel biofunctionalized gold surfaces with the antimicrobial multidomain recombinant protein JAMF1, both in the soluble form and nanostructured as nanoparticles, were developed. The interaction between His-tag termination of the protein and a nitriloacetic acid-Ni complex formed on mixed self-assembled monolayers (mixed SAMs) was exploited. The obtained antibiofilm surfaces based on the immobilization of the novel JAMF1 protein using self-assembled monolayers were characterized using a multi-technique approach including: cyclic voltammetry, X-ray photoelectron spectroscopy, atomic force microscopy and fluorescence, proving that the modification and immobilization of JAMF1 were successfully done. The antibiofilm activity against Escherichia coli and carbapenem-resistant Klebsiella pneumoniae showed that the immobilized antimicrobial proteins were able to reduce biofilm formation of both microorganisms. This strategy opens up new possibilities for controlled biomolecule immobilization for fundamental biological studies and biotechnological applications, at the interface of materials science and molecular biology.This work has been developed under the Biochemistry and Biomedicine and Materials Science Program of Universitat Autònoma de Barcelona (UAB). The characterization has been performed by the ICTS ‘‘NANBIOSIS’’, more specifically by the Biomaterial Processing and Nanostructuring Unit (U6), Unit of CIBER-BBN located at ICMAB-CSIC. Authors are grateful for the financial support received from MICINN (PID2020-115296RA-I00 and PID2019–105622RB-I00), the Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), GenCat (grant 2017-SGR-918, SGR Cat 2021-00438 and CERCA programme), the European Social Fund, and Fundació La Marató de TV3 (Nr. 201812). This project has received funding from the European Union's Horizon 2020 research and innovation program under the HORIZON-RIA project NABIHEAL (GA number 101092269), the Marie Skłodowska-Curie grant agreement No. 801342 (Tecniospring INDUSTRY) and the Government of Catalonia's Agency for Business Competitiveness (ACCIÓ; TECSPR19-1-0065). J.G. is grateful to MICINN for a ‘‘Ramón y Cajal’’ fellowship (RYC-2017-22614) as well as to the Max Planck Society through the Max Planck Partner Group “Dynamic Biomimetics for Cancer Immunotherapy” in collaboration with the Max Planck Institute for Medical Research (Heidelberg, Germany). R.R-P received a PhD fellowship from Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (AGAUR) and E.G-F a post-doctoral fellowship from INIA (DOC-INIA). ICMAB-CSIC acknowledges the support from the Severo Ochoa Programme for Centres of Excellence in R&D (FUNFUTURE, CEX2019-000917-S). Table of contents entry and Fig. 2 were designed with BioRender.com.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Estrategias para la Purificación de Proteínas Pegiladas Utilizando Cromatografía de Interacción Hidrofóbica: Ribonucleasa A como Modelo de Estudio

La PEGilación es la unión covalente de una proteína a una o varias cadenas de poli(etilen glicol) (PEG). Esta técnica ha sido utilizada para mejorar las propiedades fisicoquímicas de varias proteínas utilizadas como drogas terapéuticas. Durante la reacción de PEGilación se forman diferentes bioconjugados variando en el nÚmero de cadenas de PEG añadidas y el sitio de unión. La purificación de proteínas PEGiladas consiste en remover todas las especies que no formen parte del producto de interés, lo que involucra dos retos principalmente: 1) la separación de las proteínas PEGiladas del resto de los productos de la reacción y 2) el sub-fraccionamiento de las proteínas PEGiladas en base al grado de PEGilación y a los isómeros posicionales. Los métodos cromatográficos han sido frecuentemente utilizados para resolver las mezclas de la reacción de PEGilación, la cromatografía de exclusión molecular (SEC) y de intercambio iónico (IEC) son las más utilizadas. Comparativamente muy poco trabajo ha sido realizado para explorar otras técnicas como la cromatografía de interacción hidrofóbica (HIC). Por otro lado, una revisión detallada de la literatura muestra que las técnicas no-cromatográficas (ultrafiltración, sistemas de dos fases acuosas, electroforesis, etc.) son básicamente utilizadas para la caracterización de los productos de la reacción de PEGilación. Dentro de este contexto, en este trabajo se presenta el estudio de la separación de los productos de la reacción de PEGilación de RNasa A utilizando diferentes condiciones y tipos de resinas en HIC. Se demuestra que el uso de un soporte ligeramente hidrofóbico como CH sefarosa 4B cubierta con Tris, puede ser utilizado como una alternativa para la separación de las proteínas PEGiladas de la proteína nativa. Adicionalmente, los productos de la reacción de PEGilación fueron separados utilizando tres resinas con diferente grado de hidrofobicidad: butil, octil y fenil sefarosa. Se evaluaron los efectos del tipo de resina, el tipo y la concentración de sal (sulfato de amonio y NaCl) y la longitud del gradiente sobre el proceso de separación. Se calcularon la pureza y el rendimiento empleando el modelo de platos. Bajo todas las condiciones analizadas, la proteína nativa es completamente separada de las especies PEGiladas. Las mejores condiciones para la purificación de RNasa A monoPEGilada se dan cuando se utiliza la resina butil sefarosa, sulfato de amonio 1M y un gradiente de elución de 35 CVs. Con esto es posible obtener un rendimiento del 85% y una pureza del 97%. Este proceso representa una alternativa viable para la separación de proteínas PEGiladas

Laccase-luminol chemiluminescence system: an investigation of substrate inhibition

Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase-luminol system, determine its kinetic parameters, and analyze the effects of protein and OH- concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase-luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate-inhibition behaviour. A two-site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 μM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase-luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification.Translational Omics and Bioengineering and Regenerative Medicine strategic focus groups of Tecnologico de Monterrey; CONACyT, Grant/Award Number: 492276With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe