Micro simulated moving bed chromatography-mass spectrometry as a continuous on-line process analytical tool

Continuous manufacturing is becoming increasingly important in the (bio-)pharmaceutical industry, as more product can be produced in less time and at lower costs. In this context, there is a need for powerful continuous analytical tools. Many established off-line analytical methods, such as mass spectrometry (MS), are hardly considered for process analytical technology (PAT) applications in biopharmaceutical processes, as they are limited to at-line analysis due to the required sample preparation and the associated complexity, although they would provide a suitable technique for the assessment of a wide range of quality attributes. In this study, we investigated the applicability of a recently developed micro simulated moving bed chromatography system (µSMB) for continuous on-line sample preparation for MS. As a test case, we demonstrate the continuous on-line MS measurement of a protein solution (myoglobin) containing Tris buffer, which interferes with ESI-MS measurements, by continuously exchanging this buffer with a volatile ammonium acetate buffer suitable for MS measurements. The integration of the µSMB significantly increases MS sensitivity by removing over 98% of the buffer substances. Thus, this study demonstrates the feasibility of on-line µSMB-MS, providing a versatile PAT tool by combining the detection power of MS for various product attributes with all the advantages of continuous on-line analytics

Photoremovable protecting groups for carbonyl compounds of biological interest

[ES] El espectro de la luz solar está compuesto por una amplia gama de radiaciones electromagnéticas las cuales tienen diferentes impactos sobre la vida en la tierra. Entre ellas, las pertenecientes a la región ultravioleta toman un papel principal cuando nos referimos a la fotobiología, ya que pueden interactuar con las biomoléculas por medio de procesos tanto directos como fotosensibilizados. Como resultado, estas biomoléculas pueden sufrir modificaciones que no siempre tienen efectos beneficiosos. En este contexto, los daños fotoinducidos al ADN son de gran relevancia ya que están estrechamente relacionados con la creciente incidencia de cáncer de piel. Por ello, es necesario investigar tanto los mecanismos involucrados en dichos procesos como el desarrollo de nuevas estrategias para combatirlos. En la presente tesis se da respuesta a estas necesidades mediante el desarrollo y empleo de grupos protectores fotolábiles (PPG). En una primera parte se avanza en el desarrollo de nuevos PPG basados en filtros solares. Estos ofrecen la ventaja de actuar, una vez liberados, como un escudo protector frente a la radiación ultravioleta. En este contexto, en el Capítulo 3 se profundiza en las propiedades fotofísicas y fotoquímicas de los sistemas formados por la avobenzona como PPG de ácidos carboxílicos, más concretamente del ketoprofeno (KP) y del naproxeno (NPX). En este estudio se analiza por medio de modelado molecular y técnicas espectroscópicas la influencia que tiene la energía relativa del triplete de la avobenzona en su forma dicetónica, 3AB(K)*, respecto a la de los compuestos protegidos en el proceso de liberación. Siguiendo en esta misma línea de trabajo, en el Capítulo 4 se ha desarrollado un nuevo PPG capaz de liberar el filtro solar oxibenzona (OB) junto con compuestos carbonílicos. En una segunda parte, el foco de atención se ha puesto en el concepto de "Caballo de Troya", el cual establece que ciertas lesiones del ADN pueden actuar a su vez como fotosensibilizadores endógenos generando así nuevas lesiones en su entorno. En este contexto, en el Capítulo 5 se han estudiado, mediante métodos tanto experimentales como teóricos, las propiedades fotosensibilizantes de dos de los daños oxidativos del ADN, el 5-formiluracilo (ForU) y la 5-formilcitosina (ForC), poniendo especial énfasis en la capacidad de estos para poblar sus estados tripletes, así como de inducir la formación fotosensibilizada de dímeros ciclobutánicos de pirimidina (CPD). Por último, en el Capítulo 6 se ha desarrollado una nueva alternativa sintética para la incorporación del ForU en oligonucleótidos. Debido a la inestabilidad del grupo aldehído, esta síntesis se lleva a cabo generalmente mediante la incorporación de un precursor el cual es posteriormente convertido en el ForU mediante la acción de un agente oxidante. Por el contrario, en la nueva alternativa planteada el aldehído es protegido con un PPG, de manera que una vez insertado en el ODN, el aldehído es liberado de forma selectiva mediante el empleo de luz. Este trabajo supone un avance en el estudio de las propiedades fotosensibilizantes del ForU ofreciendo una nueva herramienta para la evaluación de las mismas en un entorno más cercano al del ADN.[CA] L'espectre de la llum solar està compost per una àmplia gamma de radiacions electromagnètiques les quals tenen diferents impactes sobre la vida en la terra. Entre elles, les pertanyents a la regió ultraviolada prenen un paper principal quan ens referim a la fotobiologia, ja que poden interactuar amb les biomolècules per mitjà de processos tant directes com fotosensibilitzats. Com a resultat, aquestes biomolècules poden patir modificacions que no sempre tenen efectes beneficiosos. En este context, els danys fotoinduits a l'ADN són de gran rellevància ja que estan estretament relacionats amb la creixent incidència de càncer de pell. Per això, és necessari tant d'investigar els mecanismes involucrats en els processos com el desenvolupament de noves estratègies per a combatre'ls. En la present tesi es dóna resposta a aquestes necessitats per mitjà del desenvolupament i ús de grups protectors fotolàbils (PPG). En una primera part s'avança en el desenvolupament de nous PPG basats en filtres solars. Estos ofereixen l'avantatge d'actuar, una vegada alliberats, com un escut protector enfront de la radiació ultraviolada. En este context, en el capítol 3 s'aprofundeix en les propietats fotofísiques i fotoquímiques dels sistemes formats per l'avobenzona com PPG d'àcids carboxílics, més concretament del ketoprofé (KP) i del naproxé (NPX). En este estudi s'analitza per mitjà de modelatge molecular i tècniques espectroscòpiques la influència que té en el procés d'alliberament l'energia relativa del triplet de l'avobenzona en la seua forma dicetònica, 3AB(K)*, respecte a la dels compostos protegits. En esta mateixa línia de treball, en el capítol 4 s'ha desenvolupat un nou PPG capaç d'alliberar el filtre solar oxibenzona (OB) junt amb compostos carbonílics. En una segona part, el focus d'atenció s'ha posat en el concepte de "Cavall de Troia", el qual estableix que certes lesions de l'ADN poden actuar al seu torn com fotosensibilitzadors endògens generant així noves lesions en el seu entorn. En este context, en el capítol 5 s'han estudiat, per mitjà de mètodes tant experimentals com teòrics, les propietats fotosensibilitzants de dos dels danys oxidatius de l'ADN, el 5-formiluracil (ForU) i la 5-formilcitosina (ForC), posant especial èmfasi tant en la capacitat d'estos per a poblar els seus estats triplet, com d'induir la formació fotosensibilitzada de dímers ciclobutànics de pirimidina (CPD). Finalment, en el capítol 6 s'ha desenvolupat una nova alternativa sintètica per a la incorporació del ForU en oligonucleòtids. A causa de la inestabilitat del grup aldehid, esta síntesi es duu a terme generalment per mitjà de la incorporació d'un precursor el qual és posteriorment convertit en el ForU per mitjà de l'acció d'un agent oxidant. Al contrari, en la nova alternativa plantejada l'aldehid és protegit amb un PPG, de manera que una vegada inserit en l'oligonucleòtid, l'aldehid és alliberat de forma selectiva per mitjà de l'ús de llum. Este treball suposa un avanç en l'estudi de les propietats fotosensibilitzants del ForU i ofereix una nova ferramenta per a l'avaluació de les mateixes en un entorn més pròxim al de l'ADN.[EN] The solar spectrum is composed of a wide range of electromagnetic radiations which have different impacts on life on earth. Among them, those belonging to the ultraviolet region are of utmost importance when we refer to photobiology, since they can interact with biomolecules through both direct and photosensitized processes. As a result, these biomolecules can undergo modifications that do not always have beneficial effects. In this context, photoinduced DNA damage is of great relevance as it is closely related to the increasing incidence of skin cancer. Therefore, it is necessary both to investigate the mechanisms involved in these processes and to develop new strategies to avoid them. In this Thesis these issues have been addressed through the development and use of photolabile protecting groups (PPG). The first part of this Thesis involves the development of new PPG based on solar filters. Once released, these PPG offer the advantage of acting as ultraviolet shields. In this context, Chapter 3 looks into the photophysical and photochemical properties of those systems formed by avobenzone as PPG of carboxylic acids, more specifically ketoprofen (KP) and naproxen (NPX). In this study, the influence on the photorelease process of the relative energetic location of the avobenzone triplet manifold in its diketo form, 3AB(K)*, with respect to that of its caged compound, is duly analyzed by means of molecular modeling and spectroscopic techniques. Following this same line of work, a new PPG capable of releasing oxybenzone (OB) solar filter along with carbonyl compounds has been developed in Chapter 4. The second part of this Thesis focuses on the "Trojan Horse" concept, which establishes that certain DNA lesions can act as endogenous photosensitizers, thus generating new lesions in their neighborhood. In this context, in Chapter 5 the photosensitizing properties of two oxidatively generated DNA damages, namely 5-formyluracil (ForU) and 5-formylcytosine (ForC), have been studied by means of experimental and theoretical approaches. Here, special emphasis has been placed on unraveling their capacity to photoinduce the formation of cyclobutane pyrimidine dimers (CPD). Finally, in Chapter 6 a new synthetic alternative for the incorporation of ForU into oligodeoxynucleotides (ODN) has been developed. Due to the instability of the aldehyde group, this synthesis is generally carried out by incorporating a precursor which is subsequently converted into ForU by the action of an oxidative agent. On the contrary, in the new approach, the aldehyde is protected with a PPG, so that once inserted into the ODN, the aldehyde is selectively released through the use of light. This work entails a step forward in the study of the photosensitizing properties of ForU, offering a new tool for their evaluation within the DNA environment.Lineros Rosa, M. (2021). Photoremovable protecting groups for carbonyl compounds of biological interest [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/167764TESI

Fast and versatile chromatography process design and operation optimization with the aid of artificial intelligence

Preparative and process chromatography is a versatile unit operation for the capture, purification, and polishing of a broad variety of molecules, especially very similar and complex compounds such as sugars, isomers, enantiomers, diastereomers, plant extracts, and metal ions such as rare earth elements. Another steadily growing field of application is biochromatography, with a diversity of complex compounds such as peptides, proteins, mAbs, fragments, VLPs, and even mRNA vaccines. Aside from molecular diversity, separation mechanisms range from selective affinity ligands, hydrophobic interaction, ion exchange, and mixed modes. Biochromatography is utilized on a scale of a few kilograms to 100,000 tons annually at about 20 to 250 cm in column diameter. Hence, a versatile and fast tool is needed for process design as well as operation optimization and process control. Existing process modeling approaches have the obstacle of sophisticated laboratory scale experimental setups for model parameter determination and model validation. For a broader application in daily project work, the approach has to be faster and require less effort for non-chromatography experts. Through the extensive advances in the field of artificial intelligence, new methods have emerged to address this need. This paper proposes an artificial neural network-based approach which enables the identification of competitive Langmuir-isotherm parameters of arbitrary three-component mixtures on a previously specified column. This is realized by training an ANN with simulated chromatograms varying in isotherm parameters. In contrast to traditional parameter estimation techniques, the estimation time is reduced to milliseconds, and the need for expert or prior knowledge to obtain feasible estimates is reduced

Generalized calibration across liquid chromatography setups for generic prediction of small-molecule retention times

Accurate prediction of liquid chromatographic retention times from small-molecule structures is useful for reducing experimental measurements and for improved identification in targeted and untargeted MS. However, different experimental setups (e.g., differences in columns, gradients, solvents, or stationary phase) have given rise to a multitude of prediction models that only predict accurate retention times for a specific experimental setup. In practice this typically results in the fitting of a new predictive model for each specific type of setup, which is not only inefficient but also requires substantial prior data to be accumulated on each such setup. Here we introduce the concept of generalized calibration, which is capable of the straightforward mapping of retention time models between different experimental setups. This concept builds on the database-controlled calibration approach implemented in PredRet and fits calibration curves on predicted retention times instead of only on observed retention times. We show that this approach results in substantially higher accuracy of elution-peak prediction than is achieved by setup-specific models

Hybrid Models for the simulation and prediction of chromatographic processes for protein capture

The biopharmaceutical industries are continuously faced with the pressure to reduce the development costs and accelerate development time scales. The traditional approach of heuristic-based or platform process-based optimization is soon getting obsolete, and more generalized tools for process development and optimization are required to keep pace with the emerging trends. Thus, advanced model-based methods that can reduce the can ensure accelerated development of robust processes with minimal experiments are necessary. Though mechanistic models for chromatography are quite popular, their success is limited by the need to have accurate knowledge of adsorption isotherms and mass transfer kinetics. As an alternative, in this work, a hybrid modeling approach is proposed. Thereby, the chromatographic unit behavior is learned by a combination of neural network and mechanistic model while fitting suitable experimental breakthrough curves. Since this approach does not require identifying suitable mechanistic assumptions for all the phenomena, it can be developed with lower effort. Thus, allowing the scientists to concentrate their focus on process development. The performance of the hybrid model is compared with the mechanistic Lumped kinetic Model for in-silico data and experiments conducted on a system of industrial relevance. The flexibility of the hybrid modeling approach results in about three times higher accuracies compared to Lumped Kinetic Model. This is validated for five different isotherm models used to simulate data, with the hybrid model showing about two to three times lower prediction errors in all the cases. Not only in prediction, but we could also show that the hybrid model is more robust in extrapolating across process conditions with about three times lower error than the LKM. Additionally, it could be demonstrated that an appropriately tailored formulation of the hybrid model can be used to generate representations for the underlying principles such as adsorption equilibria and mass transfer kinetics.Fil: Narayanan, Harini. Institute of Chemical and Bioengineering; SuizaFil: Seidler, Tobias. Institute of Chemical and Bioengineering; SuizaFil: Luna, Martín Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo y Diseño. Universidad Tecnológica Nacional. Facultad Regional Santa Fe. Instituto de Desarrollo y Diseño; Argentina. Institute of Chemical and Bioengineering; SuizaFil: Sokolov, Michael. No especifíca;Fil: Morbidelli, Massimo. Politecnico di Milano; ItaliaFil: Butté, Alessandro. No especifíca

Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation

As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data. Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism. We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process. We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants. Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets. Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.National Human Genome Research Institute (U.S.) (HG03067-05

Synthetic Investigation of Natural Products Causing Dopaminergic Neurodegeneration

The terrestrial organisms Streptomyces venezuelae and Rhaponticum repens produce toxic secondary metabolites that likely function as chemical deterrents. The polyketide SV-6 from S. venezuelae and the sesquiterpene lactone repin from R. repens both produce dose-dependent and selective degeneration of dopaminergic neurons. These molecules represent two possible tools that can be used to explore chemotoxic induction of Parkinson’s disease. In the case of SV-6, a newly isolated metabolite, total synthesis was undertaken to confirm its structure and biological activity. The natural product was produced from methacrolein and Roche ester starting materials that were elaborated to their respective fragments, a vinyl iodide and a Weinreb amide. These fragments were coupled to form SV-6 and the synthesis allows for many additional opportunities to easily generate stereoisomers and analogs for further study. In the case of repin, previously isolated material was used as a starting material to create a biotin-labeled probe. There were considerable challenges due to the electrophilic nature of the compound, but a Nicolas reaction folloby copper catalyzed cycloaddition produced an impure biotin-repin probe. Repin was also subjected to screening in multiple biological assays which identified proteins potentially responsible for its neurodegenerative activity

Model based process optimisation of an industrial chromatographic process for separation of lactoferrin from bovine milk

Journal articleModel based process development using predictive mechanistic models is a powerful tool for in-silico downstream process development. It allows to obtain a thorough understanding of the process reducing experimental effort. While in pharma industry, mechanistic modeling becomes more common in the last years, it is rarely applied in food industry. This case study investigates risk ranking and possible optimization of the industrial process of purifying lactoferrin from bovine milk using SP Sepharose Big Beads with a resin particle diameter of 200 µm, based on a minimal number of lab-scale experiments combining traditional scale-down experiments with mechanistic modeling. Depending on the location and season, process water pH and the composition of raw milk can vary, posing a challenge for highly efficient process development. A predictive model based on the general rate model with steric mass action binding, extended for pH dependence, was calibrated to describe the elution behavior of lactoferrin and main impurities. The gained model was evaluated against changes in flow rate, step elution conditions, and higher loading and showed excellent agreement with the observed experimental data. The model was then used to investigate the critical process parameters, such as water pH, conductivity of elution steps, and flow rate, on process performance and purity. It was found that the elution behavior of lactoferrin is relatively consistent over the pH range of 5.5 to 7.6, while the elution behavior of the main impurities varies greatly with elution pH. As a result, a significant loss in lactoferrin is unavoidable to achieve desired purities at pH levels below pH 6.0. Optimal process parameters were identified to reduce water and salt consumption and increase purity, depending on water pH and raw milk composition. The optimal conductivity for impurity removal in a low conductivity elution step was found to be 43 mS/cm, while a conductivity of 95 mS/cm leads to the lowest overall salt usage during lactoferrin elution. Further increasing the conductivity during lactoferrin elution can only slightly lower the elution volume thus can also lead to higher total salt usage. Low flow rates during elution of 0.2 column volume per minute are beneficial compared to higher flow rates of 1 column volume per minute. The, on lab-scale, calibrated model allows predicting elution volume and impurity removal for large-scale experiments in a commercial plant processing over 106 liters of milk per day. The successful model extrapolation was possible without recalibration or detailed knowledge of the manufacturing plant. This study therefore provides a possible pathway for rapid process development of chromatographic purification in the food industries combining traditional scale-down experiments with mechanistic modeling.Lukas Gerstweiler, Paulina Schad, Tatjana Trunzer, Lena Enghauser, Max Mayr, Jagan Billakant