José González-Valdez | Tecnológico de Monterrey (original) (raw)

Papers by José González-Valdez

Research paper thumbnail of State-of-the-Art and Opportunities for Bioactive Pentacyclic Triterpenes from Native Mexican Plants

Plants

Native Mexican plants are a wide source of bioactive compounds such as pentacyclic triterpenes. P... more Native Mexican plants are a wide source of bioactive compounds such as pentacyclic triterpenes. Pentacyclic triterpenes biosynthesized through the mevalonate (MVA) and the 2-C-methyl-D-erythritol-phosphate (MEP) metabolic pathways are highlighted by their diverse biological activity. Compounds belonging to the oleanane, ursane, and lupane groups have been identified in about 33 Mexican plants, located geographically in the southwest of Mexico. The works addressing these findings have reported 45 compounds that mainly show antimicrobial activity, followed by anti-inflammatory, cytotoxic, anxiolytic, hypoglycemic, and growth-stimulating or allelopathic activities. Extraction by maceration and Soxhlet with organic solvents and consecutive chromatography of silica gel have been used for their whole or partial purification. Nanoparticles and nanoemulsions are the vehicles used in Mexican formulations for drug delivery of the pentacyclic triterpenes until now. Sustainable extraction, form...

Research paper thumbnail of Mono-PEGylated lysozyme purification with increased productivity and isomer differentiation through heparin monolith chromatography

Journal of Chromatography B

Research paper thumbnail of Recovery of roseoflavin from a recombinant Streptomyces davaonensis strain by using biphasic aqueous systems

Journal of Chemical Technology & Biotechnology, 2021

This study aims to explain the brand image in mediating the effect of word of mouth on purchase i... more This study aims to explain the brand image in mediating the effect of word of mouth on purchase intention. This research was conducted in Denpasar City using sample size ofi150irespondents, using a non-probability method in the form of purposive sampling. The data analysis techniques used in this study were path analysis and the Sobel test. The results of this study indicate that word of mouth has a positive and significant effect on purchase intention, word of mouth has a positive and significant effect on brand image, brand image has a positive and significant effect on purchase intention, and brand image can mediate the effect of word of mouth on purchase intention.

Research paper thumbnail of Reactive aqueous two-phase systems for the production and purification of PEGylated proteins

Electronic Journal of Biotechnology, 2021

Research paper thumbnail of Cost-effective serological test to determine exposure to SARS-CoV-2: ELISA based on the receptor-binding domain of the spike protein (Spike-RBDN318-V510) expressed in Escherichia coli

Massive worldwide serological testing for SARS-CoV-2 is needed to determine the extent of virus e... more Massive worldwide serological testing for SARS-CoV-2 is needed to determine the extent of virus exposure in a particular region, the ratio of symptomatic to asymptomatic infected persons, and the duration and extent of immunity after infection. To achieve this aim, the development and production of reliable and cost-effective SARS-CoV-2 antigens is critical.Here, we report the bacterial production of the peptide S-RBDN318-V510, which contains the receptor binding domain of the SARS-CoV-2 spike protein. We purified this peptide using a straightforward approach involving bacterial lysis, his-tag mediated affinity chromatography, and imidazole-assisted refolding. The antigen performances of S-RBDN318-V510 and a commercial full-length spike protein were compared in two distinct ELISAs. In direct ELISAs, where the antigen was directly bound to the ELISA surface, both antigens discriminated sera from non-exposed and exposed individuals. However, the discriminating resolution was better in...

Research paper thumbnail of Insights into the application of polyhydroxyalkanoates derivatives from the combination of experimental and simulation approaches

Journal of Molecular Structure, 2019

The combination of spectroscopy studies with simulation models can be a useful tool to predict th... more The combination of spectroscopy studies with simulation models can be a useful tool to predict the applications of macromolecules. However, it is currently unknown if applications of polyhydroxyalkanoate (PHA) derivatives can be determined with this methodology. Here, we used a new nuclear magnetic resonance (NMR) technique to structurally characterize poly (3-hydroxybutyrate) grafted with 2-aminoethyl methacrylate and combined the experimental results with those of molecular dynamic simulations. The NMR results show the possible grafting mechanism and final structure of the grafted polymer. Quantum and molecular dynamic simulations of the proposed structures were carried out to determine the reactive sites, hydrogen bonds, and physical movements of atoms. Using the information gathered, the application of the polymer as a scaffold for cell growth was discussed. It was concluded that this approach would be advantageous to achieve considerable savings in cost and time while developing potential applications of these bio-derived polyesters.

Research paper thumbnail of Perspectives, Tendencies, and Guidelines in Affinity-Based Strategies for the Recovery and Purification of PEGylated Proteins

Advances in Polymer Technology, 2020

In recent years, the effective purification of PEGylated therapeutic proteins from reaction media... more In recent years, the effective purification of PEGylated therapeutic proteins from reaction media has received particular attention. Although several techniques have been used, affinity-based strategies have been scarcely explored despite the fact that, after PEGylation, marked changes in the molecular affinity parameters of the modified molecules are observed. With this in mind, future contributions in the bioseparation of these polymer-protein conjugates are expected to exploit affinity in chromatographic and nonchromatographic techniques which will surely derive in the integration of different operations. However, this will only occur as novel ligands which are simultaneously found. As it will be mentioned, these novel ligands may be screened or designed. In both cases, computer-aided tools will support their identification or development. Additionally, ligand discovery by high-throughput screening (HTS) is believed to become a fast, economic, and informative technology that will...

Research paper thumbnail of Development and Characterization of PEGylated Chromatographic Monoliths as a Novel Platform for the Separation of PEGylated RNase a Isomers

Advances in Polymer Technology, 2019

PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in differ... more PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in different diseases. However, the major drawback in their procurement is the purification process to separate unreacted proteins and the PEGylated species. Several efforts have been done to separate PEGylation reactions by chromatography using different stationary phases and modified supports. In this context, this study presents the use of chromatographic monoliths modified with polyethylene glycol (PEG) to separate PEGylated Ribonuclease A (RNase A). To do this, Convective Interaction Media (CIM) Ethylenediamine (EDA) monolithic disks were PEGylated using three PEG molecular weights (1, 10, and 20 kDa). The PEGylated monoliths were used to separate PEGylated RNase A modified, as well, with three PEG molecular weights (5, 20, and 40 kDa) by hydrophobic interaction chromatography. Performance results showed that Bovine Serum Albumin (BSA) can bind to PEGylated monoliths and the amount of bound ...

Research paper thumbnail of Electrokinetically Driven Exosome Separation and Concentration Using Dielectrophoretic-Enhanced PDMS-Based Microfluidics

Analytical Chemistry, 2019

Research paper thumbnail of Practical experiences from the bench-scale implementation of a bioprocess for fucoxanthin production

Journal of Chemical Technology & Biotechnology, 2017

BACKGROUND: A laboratory scale protocol for the production, primary recovery and partial purifica... more BACKGROUND: A laboratory scale protocol for the production, primary recovery and partial purification of fucoxanthin via biomass production at low light intensities was previously reported. This proposed approach exploited the use of ethanol salt aqueous twophase systems (ATPS) and ultrafiltration (UF) to deliver a 45% total recovery yield if all steps were implemented in a bioprocess. In this study, practical experiences derived from the bench-scale implementation of the previously characterized lab protocol are presented. RESULTS: After the implementation of the process, a 47.5% fucoxanthin recovery was obtained, while 87.2% of the contaminant proteins were removed. CONCLUSION: The

Research paper thumbnail of New Trends in Biopolymer-Based Membranes for Pervaporation

Molecules, 2019

Biopolymers are currently the most convenient alternative for replacing chemically synthetized po... more Biopolymers are currently the most convenient alternative for replacing chemically synthetized polymers in membrane preparation. To date, several biopolymers have been proposed for such purpose, including the ones derived from animal (e.g., polybutylene succinate, polylactic acid, polyhydroxyalcanoates), vegetable sources (e.g., starch, cellulose-based polymers, alginate, polyisoprene), bacterial fermentation products (e.g., collagen, chitin, chitosan) and specific production processes (e.g., sericin). Particularly, these biopolymer-based membranes have been implemented into pervaporation (PV) technology, which assists in the selective separation of azeotropic water-organic, organic-water, organic-organic mixtures, and specific separations of chemical reactions. Thereby, the aim of the present review is to present the current state-of-the-art regarding the different concepts on preparing membranes for PV. Particular attention is paid to the most relevant insights in the field, highl...

Research paper thumbnail of Detailed characterization of the solution kinetics and thermodynamics of biotin, biocytin and HABA binding to avidin and streptavidin

PLOS ONE, 2019

The high affinity (K D~1 0 −15 M) of biotin for avidin and streptavidin is the essential componen... more The high affinity (K D~1 0 −15 M) of biotin for avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (k on) is approximately diffusion limited (10 9 M-1 s-1) but recent single molecule and surface binding studies indicate that they are slower than expected (10 5 to 10 7 M-1 s-1). In this study, we asked whether these reactions in solution are diffusion controlled, which reaction model and thermodynamic cycle describes the complex formation, and if there are any functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4'-hydroxyazobenzene)-benzoic acid. Both native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The k on values of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the k on values revealed strong temperature dependence with large activation energies (6-15 kcal/mol) that do not correspond to a diffusion limited process (3-4 kcal/mol). Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle, in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics, and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications.

Research paper thumbnail of Protein A chromatography: Challenges and progress in the purification of monoclonal antibodies

Journal of Separation Science, 2019

Antibodies for therapeutic use are being continuously approved and their demand has been steadily... more Antibodies for therapeutic use are being continuously approved and their demand has been steadily growing. As known, the golden standard for monoclonal antibody purification is Protein A affinity chromatography a technology that has gained high interest because of its great performance and capabilities. The main concerns are the elevated resins costs and their limited lifetime compared to other resins (e.g. ion exchange chromatography). Great efforts have been carried out to improve purification conditions, such as resin characterization and designing alkali/acid stable resins with a longer lifetime. Modification of Protein A ligands and alternative formats such as monoliths membranes and microshperes have been tested to increase the purification performance. New technology has been proposed to improve the large-scale separation; in addition, alternative ligands have been suggested to capture mAbs instead of Protein A ligand; however, most of the information is locked by pharmaceutical companies. This mini review summarizes and describes the advances, results, and impact on the Protein A chromatography purification processing.

Research paper thumbnail of Aqueous Two-Phase Systems at Large Scale: Challenges and Opportunities

Biotechnology journal, Jan 7, 2018

Aqueous two-phase systems (ATPS) have proved to be an efficient and integrative operation to enha... more Aqueous two-phase systems (ATPS) have proved to be an efficient and integrative operation to enhance recovery of industrially relevant bioproducts. After ATPS discovery, a variety of works have been published regarding their scaling from 10 to 1000 L. Although ATPS have achieved high recovery and purity yields, there is still a gap between their bench-scale use and potential industrial applications. In this context, this review paper critically analyzes ATPS scale-up strategies to enhance the potential industrial adoption. In particular, large-scale operation considerations, different phase separation procedures, the available optimization techniques (univariate, response surface methodology, and genetic algorithms) to maximize recovery and purity and economic modeling to predict large-scale costs, are discussed. ATPS intensification to increase the amount of sample to process at each system, developing recycling strategies and creating highly efficient predictive models, are still ...

Research paper thumbnail of “Smart” Polymers: Physicochemical Characteristics and Applications in Bio-Separation Strategies

Separation & Purification Reviews, 2017

In recent years, smart polymers (SP) which are also referred as bio-responsive polymers have gain... more In recent years, smart polymers (SP) which are also referred as bio-responsive polymers have gained considerable attention as a unique class of polymers and their applications have been increasing significantly. These so-called "smart" polymers, either synthetic or biological, have been defined as "polymers designed to respond or undergo physical and structural conformational changes/rearrangement in response to slight changes in their surrounding environment". They are categorized as thermo-, pH-, electro-, and magneto-responsive polymers. The advances in upstream bio-production stages and the high cost associated with downstream chromatographic techniques have pushed the development of new alternatives. In this context, the use of SPs, in combination with non-chromatographic technologies, represents a useful approach to the development of new downstream operation units. With the key scientific advancements, SPs have become the "next generation" of bio-separation tool for eco-friendlier and cost-effective purification. The review describes the different characteristics and classifications of various "smart" polymers available for their use in bio-separation strategy. The focus is also given to the recent advances in SP inclusion in the improvement of alternative nonchromatographic methods in downstream bioprocessings.

Research paper thumbnail of Refolding of laccase from Trametes versicolor using aqueous two phase systems: Effect of different additives

Journal of Chromatography A, 2017

Highlights.  Refolding of laccase using PEG-phosphate ATPS was studied.  The use of different a... more Highlights.  Refolding of laccase using PEG-phosphate ATPS was studied.  The use of different additives was studied with the aim of increase the refolding.  The cysteamine with Cu +2 have a synergic effect in refolding.  First time that the use of additives to enhance the refolding in ATPS is reported.

Research paper thumbnail of Cover Image, Volume 92, Issue 1

Journal of Chemical Technology & Biotechnology, 2016

Research paper thumbnail of Monolithic chromatography: insights and practical perspectives

Journal of Chemical Technology & Biotechnology, 2016

The use of monoliths, one-piece porous separation materials, as column packaging media is known a... more The use of monoliths, one-piece porous separation materials, as column packaging media is known as monolithic chromatography and this technology has been widely exploited to improve the separation profile of high-valued biomolecules from complex biological matrices. Monoliths possess a wide range of processing advantages over commonly used chromatographical resins, including: fast flow rates, short processing times, sufficient surface area interactions, and enhanced thoroughness. The application of three different types of monolithic supports (hydrophobic, ion exchange and affinity) as an alternative to overcome traditional drawbacks in bioseparations will be presented as a practical perspective for the recovery of PEGylated proteins, stem cells or industrial enzymes. In our experience, the use of monolithic matrixes in the separation of high-valued biological materials has greatly simplified the usual problems observed in traditional chromatographic separations, resulting in a highly efficient downstream separation procedure. © 2016 Society of Chemical Industry

Research paper thumbnail of Modelling of electrokinetic phenomena for capture of PEGylated ribonuclease A in a microdevice with insulating structures

Biomicrofluidics, 2016

Synthesis of PEGylated proteins results in a mixture of protein-polyethylene glycol (PEG) conjuga... more Synthesis of PEGylated proteins results in a mixture of protein-polyethylene glycol (PEG) conjugates and the unreacted native protein. From a ribonuclease A (RNase A) PEGylation reaction, mono-PEGylated RNase A (mono-PEG RNase A) has proven therapeutic effects against cancer, reason for which there is an interest in isolating it from the rest of the reaction products. Experimental trapping of PEGylated RNase A inside an electrokinetically driven microfluidic device has been previously demonstrated. Now, from a theoretical point of view, we have studied the electrokinetic phenomena involved in the dielectrophoretic streaming of the native RNase A protein and the trapping of the mono-PEG RNase A inside a microfluidic channel. To accomplish this, we used two 3D computational models, a sphere and an ellipse, adapted to each protein. The effect of temperature on parameters related to trapping was also studied. A temperature increase showed to rise the electric and thermal conductivities of the suspending solution, hindering dielectrophoretic trapping. In contrast, the dynamic viscosity of the suspending solution decreased as the temperature rose, favoring the dielectrophoretic manipulation of the proteins. Also, our models were able to predict the magnitude and direction of the velocity of both proteins indicating trapping for the PEGylated conjugate or no trapping for the native protein. In addition, a parametric sweep study revealed the effect of the protein zeta potential on the electrokinetic response of the protein. We believe this work will serve as a tool to improve the design of electrokinetically driven microfluidic channels for the separation and recovery of PEGylated proteins in one single step.

Research paper thumbnail of Synthesis of Adsorbents with Dendronic Structures for Protein Hydrophobic Interaction Chromatography

Journal of Chromatography A, 2016

Here, we introduced a new technology based on the incorporation of dendrons-branched chemical str... more Here, we introduced a new technology based on the incorporation of dendrons-branched chemical structures-onto supports for synthesis of HIC adsorbents. In doing so we studied the synthesis and performance of these novel HIC dendron-based adsorbents. The adsorbents were synthesized in a facile two-step reaction. First, Sepharose 4FF (R) was chemically modified with polyester dendrons of different branching degrees i.e. third (G3) or fifth (G5) generations. Then, butyl-end valeric acid ligands were coupled to dendrons via ester bond formation. UV-vis spectrophotometry and FTIR analyses of the modified resins confirmed the presence of the dendrons and their ligands on them. Inclusion of dendrons allowed the increment of ligand density, 82.5 ± 11 and 175.6 ± 5.7 mol ligand/mL resin for RG3 and RG5, respectively. Static adsorption capacity of modified resins was found to be ∼60 mg BSA/mL resin. Interestingly, dynamic binding capacity was higher at high flow rates, 62.5 ± 0.8 and 58.0 ± 0.5 mg/mL for RG3 and RG5, respectively. RG3 was able to separate lipase, ␤-lactoglobulin and ␣-chymotrypsin selectively as well as fractionating of a whole proteome from yeast. This innovative technology will improve the existing HIC resin synthesis methods. It will also allow the reduction of the amount of adsorbent used in a chromatographic procedure and thus permit the use of smaller columns resulting in faster processes. Furthermore, this method could potentially be considered as a green technology since both, dendrons and ligands, are formed by ester bonds that are more biodegradable allowing the disposal of used resin waste in a more ecofriendly manner when compared to other exiting resins.

Research paper thumbnail of State-of-the-Art and Opportunities for Bioactive Pentacyclic Triterpenes from Native Mexican Plants

Plants

Native Mexican plants are a wide source of bioactive compounds such as pentacyclic triterpenes. P... more Native Mexican plants are a wide source of bioactive compounds such as pentacyclic triterpenes. Pentacyclic triterpenes biosynthesized through the mevalonate (MVA) and the 2-C-methyl-D-erythritol-phosphate (MEP) metabolic pathways are highlighted by their diverse biological activity. Compounds belonging to the oleanane, ursane, and lupane groups have been identified in about 33 Mexican plants, located geographically in the southwest of Mexico. The works addressing these findings have reported 45 compounds that mainly show antimicrobial activity, followed by anti-inflammatory, cytotoxic, anxiolytic, hypoglycemic, and growth-stimulating or allelopathic activities. Extraction by maceration and Soxhlet with organic solvents and consecutive chromatography of silica gel have been used for their whole or partial purification. Nanoparticles and nanoemulsions are the vehicles used in Mexican formulations for drug delivery of the pentacyclic triterpenes until now. Sustainable extraction, form...

Research paper thumbnail of Mono-PEGylated lysozyme purification with increased productivity and isomer differentiation through heparin monolith chromatography

Journal of Chromatography B

Research paper thumbnail of Recovery of roseoflavin from a recombinant Streptomyces davaonensis strain by using biphasic aqueous systems

Journal of Chemical Technology & Biotechnology, 2021

This study aims to explain the brand image in mediating the effect of word of mouth on purchase i... more This study aims to explain the brand image in mediating the effect of word of mouth on purchase intention. This research was conducted in Denpasar City using sample size ofi150irespondents, using a non-probability method in the form of purposive sampling. The data analysis techniques used in this study were path analysis and the Sobel test. The results of this study indicate that word of mouth has a positive and significant effect on purchase intention, word of mouth has a positive and significant effect on brand image, brand image has a positive and significant effect on purchase intention, and brand image can mediate the effect of word of mouth on purchase intention.

Research paper thumbnail of Reactive aqueous two-phase systems for the production and purification of PEGylated proteins

Electronic Journal of Biotechnology, 2021

Research paper thumbnail of Cost-effective serological test to determine exposure to SARS-CoV-2: ELISA based on the receptor-binding domain of the spike protein (Spike-RBDN318-V510) expressed in Escherichia coli

Massive worldwide serological testing for SARS-CoV-2 is needed to determine the extent of virus e... more Massive worldwide serological testing for SARS-CoV-2 is needed to determine the extent of virus exposure in a particular region, the ratio of symptomatic to asymptomatic infected persons, and the duration and extent of immunity after infection. To achieve this aim, the development and production of reliable and cost-effective SARS-CoV-2 antigens is critical.Here, we report the bacterial production of the peptide S-RBDN318-V510, which contains the receptor binding domain of the SARS-CoV-2 spike protein. We purified this peptide using a straightforward approach involving bacterial lysis, his-tag mediated affinity chromatography, and imidazole-assisted refolding. The antigen performances of S-RBDN318-V510 and a commercial full-length spike protein were compared in two distinct ELISAs. In direct ELISAs, where the antigen was directly bound to the ELISA surface, both antigens discriminated sera from non-exposed and exposed individuals. However, the discriminating resolution was better in...

Research paper thumbnail of Insights into the application of polyhydroxyalkanoates derivatives from the combination of experimental and simulation approaches

Journal of Molecular Structure, 2019

The combination of spectroscopy studies with simulation models can be a useful tool to predict th... more The combination of spectroscopy studies with simulation models can be a useful tool to predict the applications of macromolecules. However, it is currently unknown if applications of polyhydroxyalkanoate (PHA) derivatives can be determined with this methodology. Here, we used a new nuclear magnetic resonance (NMR) technique to structurally characterize poly (3-hydroxybutyrate) grafted with 2-aminoethyl methacrylate and combined the experimental results with those of molecular dynamic simulations. The NMR results show the possible grafting mechanism and final structure of the grafted polymer. Quantum and molecular dynamic simulations of the proposed structures were carried out to determine the reactive sites, hydrogen bonds, and physical movements of atoms. Using the information gathered, the application of the polymer as a scaffold for cell growth was discussed. It was concluded that this approach would be advantageous to achieve considerable savings in cost and time while developing potential applications of these bio-derived polyesters.

Research paper thumbnail of Perspectives, Tendencies, and Guidelines in Affinity-Based Strategies for the Recovery and Purification of PEGylated Proteins

Advances in Polymer Technology, 2020

In recent years, the effective purification of PEGylated therapeutic proteins from reaction media... more In recent years, the effective purification of PEGylated therapeutic proteins from reaction media has received particular attention. Although several techniques have been used, affinity-based strategies have been scarcely explored despite the fact that, after PEGylation, marked changes in the molecular affinity parameters of the modified molecules are observed. With this in mind, future contributions in the bioseparation of these polymer-protein conjugates are expected to exploit affinity in chromatographic and nonchromatographic techniques which will surely derive in the integration of different operations. However, this will only occur as novel ligands which are simultaneously found. As it will be mentioned, these novel ligands may be screened or designed. In both cases, computer-aided tools will support their identification or development. Additionally, ligand discovery by high-throughput screening (HTS) is believed to become a fast, economic, and informative technology that will...

Research paper thumbnail of Development and Characterization of PEGylated Chromatographic Monoliths as a Novel Platform for the Separation of PEGylated RNase a Isomers

Advances in Polymer Technology, 2019

PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in differ... more PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in different diseases. However, the major drawback in their procurement is the purification process to separate unreacted proteins and the PEGylated species. Several efforts have been done to separate PEGylation reactions by chromatography using different stationary phases and modified supports. In this context, this study presents the use of chromatographic monoliths modified with polyethylene glycol (PEG) to separate PEGylated Ribonuclease A (RNase A). To do this, Convective Interaction Media (CIM) Ethylenediamine (EDA) monolithic disks were PEGylated using three PEG molecular weights (1, 10, and 20 kDa). The PEGylated monoliths were used to separate PEGylated RNase A modified, as well, with three PEG molecular weights (5, 20, and 40 kDa) by hydrophobic interaction chromatography. Performance results showed that Bovine Serum Albumin (BSA) can bind to PEGylated monoliths and the amount of bound ...

Research paper thumbnail of Electrokinetically Driven Exosome Separation and Concentration Using Dielectrophoretic-Enhanced PDMS-Based Microfluidics

Analytical Chemistry, 2019

Research paper thumbnail of Practical experiences from the bench-scale implementation of a bioprocess for fucoxanthin production

Journal of Chemical Technology & Biotechnology, 2017

BACKGROUND: A laboratory scale protocol for the production, primary recovery and partial purifica... more BACKGROUND: A laboratory scale protocol for the production, primary recovery and partial purification of fucoxanthin via biomass production at low light intensities was previously reported. This proposed approach exploited the use of ethanol salt aqueous twophase systems (ATPS) and ultrafiltration (UF) to deliver a 45% total recovery yield if all steps were implemented in a bioprocess. In this study, practical experiences derived from the bench-scale implementation of the previously characterized lab protocol are presented. RESULTS: After the implementation of the process, a 47.5% fucoxanthin recovery was obtained, while 87.2% of the contaminant proteins were removed. CONCLUSION: The

Research paper thumbnail of New Trends in Biopolymer-Based Membranes for Pervaporation

Molecules, 2019

Biopolymers are currently the most convenient alternative for replacing chemically synthetized po... more Biopolymers are currently the most convenient alternative for replacing chemically synthetized polymers in membrane preparation. To date, several biopolymers have been proposed for such purpose, including the ones derived from animal (e.g., polybutylene succinate, polylactic acid, polyhydroxyalcanoates), vegetable sources (e.g., starch, cellulose-based polymers, alginate, polyisoprene), bacterial fermentation products (e.g., collagen, chitin, chitosan) and specific production processes (e.g., sericin). Particularly, these biopolymer-based membranes have been implemented into pervaporation (PV) technology, which assists in the selective separation of azeotropic water-organic, organic-water, organic-organic mixtures, and specific separations of chemical reactions. Thereby, the aim of the present review is to present the current state-of-the-art regarding the different concepts on preparing membranes for PV. Particular attention is paid to the most relevant insights in the field, highl...

Research paper thumbnail of Detailed characterization of the solution kinetics and thermodynamics of biotin, biocytin and HABA binding to avidin and streptavidin

PLOS ONE, 2019

The high affinity (K D~1 0 −15 M) of biotin for avidin and streptavidin is the essential componen... more The high affinity (K D~1 0 −15 M) of biotin for avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (k on) is approximately diffusion limited (10 9 M-1 s-1) but recent single molecule and surface binding studies indicate that they are slower than expected (10 5 to 10 7 M-1 s-1). In this study, we asked whether these reactions in solution are diffusion controlled, which reaction model and thermodynamic cycle describes the complex formation, and if there are any functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4'-hydroxyazobenzene)-benzoic acid. Both native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The k on values of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the k on values revealed strong temperature dependence with large activation energies (6-15 kcal/mol) that do not correspond to a diffusion limited process (3-4 kcal/mol). Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle, in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics, and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications.

Research paper thumbnail of Protein A chromatography: Challenges and progress in the purification of monoclonal antibodies

Journal of Separation Science, 2019

Antibodies for therapeutic use are being continuously approved and their demand has been steadily... more Antibodies for therapeutic use are being continuously approved and their demand has been steadily growing. As known, the golden standard for monoclonal antibody purification is Protein A affinity chromatography a technology that has gained high interest because of its great performance and capabilities. The main concerns are the elevated resins costs and their limited lifetime compared to other resins (e.g. ion exchange chromatography). Great efforts have been carried out to improve purification conditions, such as resin characterization and designing alkali/acid stable resins with a longer lifetime. Modification of Protein A ligands and alternative formats such as monoliths membranes and microshperes have been tested to increase the purification performance. New technology has been proposed to improve the large-scale separation; in addition, alternative ligands have been suggested to capture mAbs instead of Protein A ligand; however, most of the information is locked by pharmaceutical companies. This mini review summarizes and describes the advances, results, and impact on the Protein A chromatography purification processing.

Research paper thumbnail of Aqueous Two-Phase Systems at Large Scale: Challenges and Opportunities

Biotechnology journal, Jan 7, 2018

Aqueous two-phase systems (ATPS) have proved to be an efficient and integrative operation to enha... more Aqueous two-phase systems (ATPS) have proved to be an efficient and integrative operation to enhance recovery of industrially relevant bioproducts. After ATPS discovery, a variety of works have been published regarding their scaling from 10 to 1000 L. Although ATPS have achieved high recovery and purity yields, there is still a gap between their bench-scale use and potential industrial applications. In this context, this review paper critically analyzes ATPS scale-up strategies to enhance the potential industrial adoption. In particular, large-scale operation considerations, different phase separation procedures, the available optimization techniques (univariate, response surface methodology, and genetic algorithms) to maximize recovery and purity and economic modeling to predict large-scale costs, are discussed. ATPS intensification to increase the amount of sample to process at each system, developing recycling strategies and creating highly efficient predictive models, are still ...

Research paper thumbnail of “Smart” Polymers: Physicochemical Characteristics and Applications in Bio-Separation Strategies

Separation & Purification Reviews, 2017

In recent years, smart polymers (SP) which are also referred as bio-responsive polymers have gain... more In recent years, smart polymers (SP) which are also referred as bio-responsive polymers have gained considerable attention as a unique class of polymers and their applications have been increasing significantly. These so-called "smart" polymers, either synthetic or biological, have been defined as "polymers designed to respond or undergo physical and structural conformational changes/rearrangement in response to slight changes in their surrounding environment". They are categorized as thermo-, pH-, electro-, and magneto-responsive polymers. The advances in upstream bio-production stages and the high cost associated with downstream chromatographic techniques have pushed the development of new alternatives. In this context, the use of SPs, in combination with non-chromatographic technologies, represents a useful approach to the development of new downstream operation units. With the key scientific advancements, SPs have become the "next generation" of bio-separation tool for eco-friendlier and cost-effective purification. The review describes the different characteristics and classifications of various "smart" polymers available for their use in bio-separation strategy. The focus is also given to the recent advances in SP inclusion in the improvement of alternative nonchromatographic methods in downstream bioprocessings.

Research paper thumbnail of Refolding of laccase from Trametes versicolor using aqueous two phase systems: Effect of different additives

Journal of Chromatography A, 2017

Highlights.  Refolding of laccase using PEG-phosphate ATPS was studied.  The use of different a... more Highlights.  Refolding of laccase using PEG-phosphate ATPS was studied.  The use of different additives was studied with the aim of increase the refolding.  The cysteamine with Cu +2 have a synergic effect in refolding.  First time that the use of additives to enhance the refolding in ATPS is reported.

Research paper thumbnail of Cover Image, Volume 92, Issue 1

Journal of Chemical Technology & Biotechnology, 2016

Research paper thumbnail of Monolithic chromatography: insights and practical perspectives

Journal of Chemical Technology & Biotechnology, 2016

The use of monoliths, one-piece porous separation materials, as column packaging media is known a... more The use of monoliths, one-piece porous separation materials, as column packaging media is known as monolithic chromatography and this technology has been widely exploited to improve the separation profile of high-valued biomolecules from complex biological matrices. Monoliths possess a wide range of processing advantages over commonly used chromatographical resins, including: fast flow rates, short processing times, sufficient surface area interactions, and enhanced thoroughness. The application of three different types of monolithic supports (hydrophobic, ion exchange and affinity) as an alternative to overcome traditional drawbacks in bioseparations will be presented as a practical perspective for the recovery of PEGylated proteins, stem cells or industrial enzymes. In our experience, the use of monolithic matrixes in the separation of high-valued biological materials has greatly simplified the usual problems observed in traditional chromatographic separations, resulting in a highly efficient downstream separation procedure. © 2016 Society of Chemical Industry

Research paper thumbnail of Modelling of electrokinetic phenomena for capture of PEGylated ribonuclease A in a microdevice with insulating structures

Biomicrofluidics, 2016

Synthesis of PEGylated proteins results in a mixture of protein-polyethylene glycol (PEG) conjuga... more Synthesis of PEGylated proteins results in a mixture of protein-polyethylene glycol (PEG) conjugates and the unreacted native protein. From a ribonuclease A (RNase A) PEGylation reaction, mono-PEGylated RNase A (mono-PEG RNase A) has proven therapeutic effects against cancer, reason for which there is an interest in isolating it from the rest of the reaction products. Experimental trapping of PEGylated RNase A inside an electrokinetically driven microfluidic device has been previously demonstrated. Now, from a theoretical point of view, we have studied the electrokinetic phenomena involved in the dielectrophoretic streaming of the native RNase A protein and the trapping of the mono-PEG RNase A inside a microfluidic channel. To accomplish this, we used two 3D computational models, a sphere and an ellipse, adapted to each protein. The effect of temperature on parameters related to trapping was also studied. A temperature increase showed to rise the electric and thermal conductivities of the suspending solution, hindering dielectrophoretic trapping. In contrast, the dynamic viscosity of the suspending solution decreased as the temperature rose, favoring the dielectrophoretic manipulation of the proteins. Also, our models were able to predict the magnitude and direction of the velocity of both proteins indicating trapping for the PEGylated conjugate or no trapping for the native protein. In addition, a parametric sweep study revealed the effect of the protein zeta potential on the electrokinetic response of the protein. We believe this work will serve as a tool to improve the design of electrokinetically driven microfluidic channels for the separation and recovery of PEGylated proteins in one single step.

Research paper thumbnail of Synthesis of Adsorbents with Dendronic Structures for Protein Hydrophobic Interaction Chromatography

Journal of Chromatography A, 2016

Here, we introduced a new technology based on the incorporation of dendrons-branched chemical str... more Here, we introduced a new technology based on the incorporation of dendrons-branched chemical structures-onto supports for synthesis of HIC adsorbents. In doing so we studied the synthesis and performance of these novel HIC dendron-based adsorbents. The adsorbents were synthesized in a facile two-step reaction. First, Sepharose 4FF (R) was chemically modified with polyester dendrons of different branching degrees i.e. third (G3) or fifth (G5) generations. Then, butyl-end valeric acid ligands were coupled to dendrons via ester bond formation. UV-vis spectrophotometry and FTIR analyses of the modified resins confirmed the presence of the dendrons and their ligands on them. Inclusion of dendrons allowed the increment of ligand density, 82.5 ± 11 and 175.6 ± 5.7 mol ligand/mL resin for RG3 and RG5, respectively. Static adsorption capacity of modified resins was found to be ∼60 mg BSA/mL resin. Interestingly, dynamic binding capacity was higher at high flow rates, 62.5 ± 0.8 and 58.0 ± 0.5 mg/mL for RG3 and RG5, respectively. RG3 was able to separate lipase, ␤-lactoglobulin and ␣-chymotrypsin selectively as well as fractionating of a whole proteome from yeast. This innovative technology will improve the existing HIC resin synthesis methods. It will also allow the reduction of the amount of adsorbent used in a chromatographic procedure and thus permit the use of smaller columns resulting in faster processes. Furthermore, this method could potentially be considered as a green technology since both, dendrons and ligands, are formed by ester bonds that are more biodegradable allowing the disposal of used resin waste in a more ecofriendly manner when compared to other exiting resins.

Research paper thumbnail of Practical aspects for the development of ATPS-Based processes for protein recovery

Aqueous Two-Phase Systems for Bioprocess Development for the Recovery of Biological Products, 2017

Successful implementation of aqueous two-phase system (ATPS) strategies requires a complete under... more Successful implementation of aqueous two-phase system (ATPS) strategies requires a complete understanding of the different physical and chemical phenomena hap- pening within a particular system. This said, the design of ATPS involves the correct selection of parameters such as phase-forming chemicals (i.e., polymers, salts, ionic liquids, alcohols, etc.), pH, tie-line length (TLL), and phase volume ratio (VR) for the particular product or products being recovered (Rito-Palomares 2004). The mixed interactions of these parameters upon the physicochemical characteristics of the products being fractionated will cause the molecules of interest to partition to either of the phases either by concentration or by separation from the rest of the contaminants in the mixtures (González-Valdez et al. 2013).
In most cases, the appropriate selection of system design parameters requires a thorough experimentation of all these variables since, to date, models fail to predict the behavior of solutes (especially in mixtures) in ATPS (Mistry et al. 1996). This is particularly important because, being a primary recovery operation, ATPS are usu- ally involved in the separation of a particular set of products from streams carrying mixtures of contaminants that must be eliminated (Rito-Palomares 2004). More importantly, because of its characteristics, a well-designed ATPS operation is usually regarded as a viable option for substituting other more complicated or time- consuming procedures including chromatography (Mayolo-Deloisa et al. 2011). With this in mind, the engineering design of a biphasic separation should be opti- mal, and in doing so, ATPS should be completely characterized and understood from an intrinsic (i.e., the physicochemical interactions affecting it) and operational point of view. This chapter aims to present in a logical manner the different proce- dures to characterize the physicochemical properties of an ATPS and the solutes that are partitioned within them and to present novel and potential new uses where ATPS strategies could be successfully implemented.

Research paper thumbnail of Characterization of Aqueous Two-Phase Systems and their potential new applications

Aqueous Two-Phase Systems for Bioprocess Development for the Recovery of Biological Products , 2017

Successful implementation of aqueous two-phase system (ATPS) strategies requires a complete under... more Successful implementation of aqueous two-phase system (ATPS) strategies requires a complete understanding of the different physical and chemical phenomena hap- pening within a particular system. This said, the design of ATPS involves the correct selection of parameters such as phase-forming chemicals (i.e., polymers, salts, ionic liquids, alcohols, etc.), pH, tie-line length (TLL), and phase volume ratio (VR) for the particular product or products being recovered (Rito-Palomares 2004). The mixed interactions of these parameters upon the physicochemical characteristics of the products being fractionated will cause the molecules of interest to partition to either of the phases either by concentration or by separation from the rest of the contaminants in the mixtures (González-Valdez et al. 2013).
In most cases, the appropriate selection of system design parameters requires a thorough experimentation of all these variables since, to date, models fail to predict the behavior of solutes (especially in mixtures) in ATPS (Mistry et al. 1996). This is particularly important because, being a primary recovery operation, ATPS are usu- ally involved in the separation of a particular set of products from streams carrying mixtures of contaminants that must be eliminated (Rito-Palomares 2004). More importantly, because of its characteristics, a well-designed ATPS operation is usually regarded as a viable option for substituting other more complicated or time- consuming procedures including chromatography (Mayolo-Deloisa et al. 2011). With this in mind, the engineering design of a biphasic separation should be opti- mal, and in doing so, ATPS should be completely characterized and understood from an intrinsic (i.e., the physicochemical interactions affecting it) and operational point of view. This chapter aims to present in a logical manner the different proce- dures to characterize the physicochemical properties of an ATPS and the solutes that are partitioned within them and to present novel and potential new uses where ATPS strategies could be successfully implemented.

Research paper thumbnail of Case studies in the application of aqueous two-phase processes for the recovery of high value biological products