1,720,963 research outputs found
Model-based optimization strategy for intensification in the chromatographic purification of oligonucleotides
Full text linkOligonucleotides (ONs) are acquiring clinical relevance and their demand is expected to grow. However, the ON production capacity is currently limited by high manufacturing costs. Since the purification of the target ON sequence from molecularly similar variants represents a major bottleneck, this work presents a resource-effective strategy for the optimization of their preparative reversed-phase chromatographic purification. First, a model based on the equilibrium-dispersive theory was introduced to describe the chromatographic operation. Considering a deoxyribose nucleic acid with 20 nucleobases as case study, a genetic algorithm was developed to efficiently determine the adsorption isotherm and mass transfer parameters for the target ON and impurities. After the estimation of these parameters, a strategy for the in-silico optimization of the operation was established. The product collection window, gradient duration, and resin loading were considered as process variables and their influence on yield and productivity was investigated after setting a purity specification of 99.0%. The optimal process parameters identified through this analysis were experimentally verified, confirming the reliability of the model, calibrated with only 5 experimental runs. In addition, this optimal setpoint was exploited to design the multicolumn countercurrent solvent gradient purification (MCSGP) of this ON mixture, which allowed to boost the yield of the process and to work at cyclic steady state, while respecting the purity constraint. This study confirmed the potential of this in-silico optimization strategy in both improving the performance of the traditional single-column operations and in the rapid development of multicolumn processes
Hybrid Modeling of the Reversed‐Phase Chromatographic Purification of an Oligonucleotide: Few‐Shot Learning From Differentiable Physics Solver‐in‐the‐Loop
Full text linkHybrid models integrate mechanistic and data-driven components, effectively addressing the challenges of limited process understanding and data availability typical of biopharmaceutical processes. In this study, we applied a hybrid modeling framework named differentiable physics solver-in-the-loop (DP-SOL) to describe the reversed-phase chromatographic purification of an oligonucleotide, overcoming the mentioned limitations of purely mechanistic and data-driven models. The framework establishes a connection between neural networks (NNs) and mechanistic models through differentiable physical operators and their gradients. We first collected a data set comprising six linear gradient elution experiments at different resin loadings and gradient slopes, split in three experiments each for training and testing, for few-shot learning. The hyperparameters were determined through a grid search, resulting in a NN with two hidden layers and 14 nodes. Compared to a calibrated mechanistic model used for initialization of NN, the DP-SOL hybrid model showed significant performance improvement on both training and testing sets, with (Formula presented.) 0.97 for the former. The good predictivity of DP-SOL is attributed to the combination of mechanistic models and NNs at the solver level. As a novel and versatile hybrid modeling paradigm, DP-SOL has the potential to significantly impact modeling approaches in the downstream processing field and the broader biopharmaceutical sector
UV‐based dynamic control improves the robustness of multicolumn countercurrent solvent gradient purification of oligonucleotides
Full text linkTherapeutic oligonucleotides (ONs) have great potential to treat many diseases due to their ability to regulate gene expression. However, the inefficiency of standard purification techniques to separate the target sequence from molecularly similar variants is hindering development of large scale ON manufacturing at a reasonable cost. Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) is a valuable process able to bypass the purity-yield tradeoff typical of single-column operations, and hence to make the ON production more sustainable from both an economic and environmental point of view. However, operating close to the optimum of MCSGP can be challenging, resulting in unstable process performance and in a drift in product quality, especially when running a continuous process for extended periods where process parameters such as temperature are prone to variation. In this work, we demonstrate how greater process robustness is introduced in the design and execution of MCSGP for the purification of a 20mer single-stranded DNA sequence through the implementation of UV-based dynamic control. With this novel approach, the cyclic steady state was reached already in the third cycle and disturbances coming from fluctuations in the feed quality, loading amount and temperature were effectively compensated allowing a stable operation close to the optimum. In response to the perturbations, the controlled process kept the standard deviation on product recovery below 3.4%, while for the non-controlled process it increased up to 27.5%.MCSGP is a twin-column chromatographic process able to by-pass the yield/purity tradeoff typical of single-column operations. In this work, the authors demonstrate the possibility of increasing the robustness of MCSGP through the design and implementation of UV-based dynamic process control, which automatically adjusts the duration of recycling and collection windows based on the UV chromatogram recorded online and user-defined UV thresholds. With this control, MCSGP is 8 times less sensitive to imposed fluctuations in input process parameters compared to the non-controlled operation. imag
Continuous countercurrent chromatographic twin‐column purification of oligonucleotides: The role of the displacement effect
Full text linkOligonucleotides (ONs) are breaking through in the biopharmaceutical industry as a promising class of biotherapeutics. The main success of these molecules is due to their peculiar way of acting in the cellular process, regulating the gene expression and hence influencing the protein synthesis at a pretranslational level. Although the Food and Drug Administration (FDA) already approved a few ON-based therapeutics, their production cost strongly limits large-scale manufacturing: a situation that can be alleviated through process intensification. In this study, we address this problem by developing an efficient and continuous chromatographic purification process for ONs. In particular, we considered the chromatographic purification of an ON crude prepared by chemical synthesis using anion exchange resins. We demonstrate that in this system the competitive adsorption of the various species on the same sites of the resin leads to the displacement of the more weakly adsorbing species by the more strongly adsorbing ones. This phenomenon affects the behavior of the chromatographic units and it has been investigated in detail. Then, we developed a continuous countercurrent solvent gradient purification (MCSGP) process, which can significantly improve the productivity and buffer consumption compared to a classical single-column, batch chromatographic process.Oligonucleotides (ONs) are breaking through in the biopharmaceutical industry as a promising class of biotherapeutics. The main success of these molecules is due to their peculiar way of acting in the cellular process, regulating the gene expression and hence influencing the protein synthesis at a pretranslational level. Although the Food and Drug Administration (FDA) already approved a few ON-based therapeutics, their production cost strongly limits large-scale manufacturing: a situation that can be alleviated through process intensification. In this study, we address this problem by developing an efficient and continuous chromatographic purification process for ONs. In particular, we considered the chromatographic purification of an ON crude prepared by chemical synthesis using anion exchange resins. We demonstrate that in this system the competitive adsorption of the various species on the same sites of the resin leads to the displacement of the more weakly adsorbing species by the more strongly adsorbing ones. This phenomenon affects the behavior of the chromatographic units and it has been investigated in detail. Then, we developed a continuous countercurrent solvent gradient purification (MCSGP) process, which can significantly improve the productivity and buffer consumption compared to a classical single-column, batch chromatographic process
Design and optimization of chromatographic processes based on multicolumn countercurrent solvent gradient purification (MCSGP) for the polishing of an oligonucleotide sequence
No full textDOTTORATOGli oligonucleotidi terapeutici (ON) rappresentano una promettente soluzione per il trattamento di malattie gravi grazie alla loro capacità di modulare l'espressione genica. Tuttavia, ci sono ancora lacune relativamente all’efficacia del processo per purificare la sequenza target dalle impurezze durante la produzione su larga scala, e ciò risulta un aspetto cruciale per garantire una produzione economica e sostenibile. La purificazione tramite MCSGP si propone come una valida alternativa, capace di superare il trade-off tra purezza e resa dei metodi cromatografici a colonna singola. L’obiettivo di questa tesi di dottorato è sviluppare, per la prima volta, un processo MCSGP robusto ed efficiente per la purificazione di un DNA a catena singola composto da 20 nucleotidi.
In una prima parte del lavoro, sono state utilizzate fasi stazionarie a scambio anionico (AIEX) e il processo MCSGP è stato progettato partendo da esperimenti batch per poi valutare i miglioramenti delle performance derivanti dal passaggio all’operazione in continuo. Successivamente, è stato implementato il processo MCSGP con fasi stazionarie a fase inversa (RP) e ne sono state confrontate le performance con quelle ottenute utilizzando AIEX. Considerando la maggiore purezza raggiunta con MCSGP RP, questo processo è stato scelto per lo sviluppo di un controllore dinamico innovativo, finalizzato a migliorarne la robustezza. Infatti, l'ottimizzazione di MCSGP può risultare complessa e portare a instabilità, specialmente durante le operazioni prolungate. Lo studio illustra come l'applicazione di un controllo dinamico basato su UV consenta di regolare i tempi caratteristici per la raccolta del prodotto e per il riciclo delle frazioni impure ciclo dopo ciclo, aumentando la stabilità del processo. Grazie a questa metodologia, si raggiunge uno stato stazionario già al terzo ciclo, e le perturbazioni derivanti da variazioni nella qualità del feed, nella quantità di carico del crudo e nella temperatura vengono efficacemente ridotte, consentendo un'operazione stabile vicino all'ottimo.
Infine, sono state confrontate le performance che si possono ottenere con le fasi stazionarie utilizzate per implementare MCSGP, sia in modalità AIEX che RP, con resine concorrenti. È stato effettuato un confronto approfondito tra la resina Biopro IEX Smartsep (YMC) e due resine concorrenti: la TSK Gel e la SOURCE Q. In particolare, l'analisi si è concentrata sulle condizioni di sovraccarico, nelle quali è stato osservato un fenomeno di adsorbimento irreversibile dell'ON sulle particelle delle resine concorrenti.
In un ulteriore confronto, le performance della resina Triart C18-S (YMC) sono state messe a confronto con quelle della DAISOGEL C18. In questo caso, si è prima analizzato il comportamento di adsorbimento dell'oligonucleotide per entrambe le resine in condizioni diluite, per escludere effetti di competizione per l'adsorbimento nei pori della matrice. Successivamente, è stata effettuata una caratterizzazione in condizioni di sovraccarico per confrontare le prestazioni delle due fasi stazionarie, quando l'adsorbimento è governato da cinetiche non lineari.Therapeutic oligonucleotides (ONs) hold significant promise for treating severe
diseases by regulating gene expression. However, an efficient process for the purification of
the target sequence from molecularly similar variants during large-scale manufacturing is still
missing, which is crucial for cost-effective production. Multicolumn Countercurrent Solvent
Gradient Purification (MCSGP) offers a valuable solution allowing to overcome the purity-
yield trade-off of single-column chromatographic methods, thereby enhancing both economic
and environmental sustainability. The aim of this PhD thesis is to develop, for the first time, an
efficient and robust MCSGP process for the purification of a 20mer single-stranded DNA.
At first, anion-exchange (AIEX) stationary phases were adopted and MCSGP was
designed starting from a batch experiment to comparatively evaluate the gain achieved when
moving to continuous countercurrent operations. The subsequent step involves the
implementation of MCSGP with reversed-phase (RP) stationary phases and the comparison of
its performance with AIEX. Given the enhanced purity achieved with RP MCSGP, this process
was selected for the development of a novel dynamic controller allowing to boost its
robustness. Indeed, optimizing MCSGP can be complex, leading to process instability,
particularly during prolonged runs. This study showcases the enhancement of process
robustness in MCSGP by application of a UV-based dynamic control, which regulates the
characteristic times for product collection and for recycling of the impure side fractions cycle-
by-cycle. By employing this innovative approach, cyclic steady state is achieved by the third
cycle, and disturbances from variations in feed quality, loading amount, and temperature are
effectively mitigated, enabling stable operation near the optimum.
Finally, the performance of the resins exploited for the implementation of MCSGP, both
in AIEX and RP mode, is compared with those of competitor materials. A full side-by-side comparison is made for the Biopro IEX Smartsep (YMC) and two competitor resins: the TSK
Gel and the SOURCE Q. In this case, the focus is more towards the investigation in overloading
conditions, in which a peculiar phenomenon identified as ON irreversible adsorption to the
resin particles has been detected for the two latter stationary phases.
With similar criteria, the Triart C18-S (YMC) performance is compared to the one
obtained with the DAISOGEL C18. Hereby, at first the adsorption behavior of the
oligonucleotide is deepened for both resins. This is done in dilute condition because the affinity
of the molecule of interest towards the stationary phase can be assessed with no competitive
behavior for the adsorption on the pores of the matrix. Afterwards, a characterization in
overloaded conditions is made to compare the performances on both stationary phases once the
adsorption behavior is governed by non-linear kinetics.DIPARTIMENTO DI CHIMICA, MATERIALI E INGEGNERIA CHIMICA "GIULIO NATTA"37METRANGOLO, PIERANGELOCAVALLOTTI, CARLO ALESSANDR
Piattaforme microfluidiche per lo studio della funzionalità piastrinica nel diabete di tipo 2
No full textLAUREA MAGISTRALEIn questo lavoro, sono stati ottimizzati due protocolli sperimentali per studiare la funzionalità piastrinica in pazienti affetti da diabete di tipo 2 utilizzando un dispositivo microfluidico (il single-flow chip) ed il citometro a flusso. Parallelamente è stato sviluppato un nuovo dispositivo microfluidico (il multiplex chip) che permette di studiare la formazione degli aggregati piastrinici in flusso utilizzando simultaneamente sei differenti shear rate.In this work, two experimental protocols were optimized to study the platelet function in patients with type 2 diabetes mellitus using a microfluidic device (the single-flow chip) and the flow cytometer. Moreover, a new microfluidic device (the multiplex chip) was developed to study the thrombus formation under flow using simultaneously six different shear rates
Bi-Langmuir isotherm parameter fitting for the chromatographic purification of an oligonucleotide sequence
No full textLAUREA MAGISTRALEGli oligonucleotidi stanno emergendo nell’industria biofarmaceutica come una classe decisamente promettente di bioterapeutici. Il successo principale di queste molecole è dovuto al loro peculiare modo di agire nel processo cellulare. Gli oligonucleotidi, infatti, sono in grado di influenzare la sintesi proteica a livello pre-traslazionale regolando l’espressione genica.
Esistono, in commercio, delle terapie basate sugli oligonucleotidi, tuttavia il loro costo di produzione ne limita fortemente la produzione su larga scala. Infatti, ai giorni d’oggi, la produzione di oligocnucleotidi è prevalentemente basata sulla sintesi in fase solida, ovvero un processo che genera una miscela contenente la componente target ma anche una quantità non irrilevante di impurezze strutturalmente simili al prodotto target. A causa di questa somiglianza strutturale e molecolare delle impurezze con il prodotto d’interesse, il processo di separazione risulta molto complicato e incide fortemente nel costo di produzione degli oligonucleotidi.
La cromatografia è la tecnica più popolare utilizzata per la purificazione di oligonucleotidi, ma è spesso limitata in termini di trade-off resa-purezza, in particolar modo per la separazione di campioni contenenti un gran numero di impurità legate al prodotto.
In linea con l’intenzione di trovare i parametri di processo che consentono di ottimizzare la performance del modelling approach, è stato utilizzato un modello meccanistico per simulare il processo cromatografico, nel caso specifico di oligonucleotidi. L’obiettivo dell’implementazione del modello è quello di ridurre il tempo, i costi e gli sforzi sperimentali nella fase di ottimizzazione dei parametri di processo, nel caso specifico, i parametri dell’isoterma, che descrivono l’equilibrio di adsorbimento delle specie tra fase mobile e fase stazionaria durante il processo cromatografico di purificazione degli oligonucleotidi. A tal fine, è fondamentale individuare dei buoni parametri utili al modello predittivo.
Durante questo lavoro di tesi, è stato utilizzato l’algoritmo genetico GDE4 che ha permesso, presi in considerazioni i singoli dati ineriti la componente del prodotto di interesse in diverse condizioni sperimentali testate, di trovare i parametri dell’isoterma Bi-Langmuir. Il set individuato è stato successivamente utilizzato per identificare un set di parametri dell’isoterma per tutte le specie coinvolte nella separazione. Sono stati dunque individuati due set di parametri dell’isoterma, ciascuno trovato con una differente strategia e diversi approcci al fitting dei dati sperimentali, in grado di predire il comportamento delle molecole d’interesse.
Al fine di ridurre le operazioni manuali previste per il fitting-multicomponente, la seconda parte del progetto di tesi ha riguardato lo sviluppo teorico e pratico di un algoritmo che sia in grado di simulare un fitting anche per il sistema multi-componente, tale da prendere in considerazione le influenze di tutte le specie sul comportamento di adsorbimento del prodotto e delle impurezze.Oligonucleotides are arising in the biopharmaceutical industry as a highly promising class of biotherapeutics. The primary appeal of these molecules is due to their unique ability to affect cellular processes. Oligonucleotides, in fact, are able to influence protein synthesis at the pre-translational level by regulating gene expression.
There are commercialized therapies based on oligonucleotides but their production cost severely limits their large-scale production. Nowadays, the production of oligonucleotides is mainly based on solid phase synthesis, a process that generates a mixture containing the target component but also a considerable amount of impurities structurally similar to the target product. Due to this structural and molecular similarity of the impurities with the product of interest, the separation process is very challenging and heavily affects the cost of production of oligonucleotides.
Chromatography is the most popular technique used for the purification of oligonucleotides, but it is often limited in terms of yield-purity tradeoff, especially for the separation of samples containing a large number of product-related impurities.
In line with the intention to find the process parameters that optimize the performance of the modelling approach, a mechanistic model was used to simulate the chromatographic process. The goal of the model implementation is to reduce the time, the cost, and the experimental effort in the optimization phase of the separation process parameters, in the specific case, the isotherm parameters that describe, which describe the species adsorption equilibrium between the mobile and stationary phase during the chromatographic separation of oligonucleotides. It is critical, but not trivial, to identify good parameters useful for the predictive model.
During this thesis work, the GDE4 genetic algorithm was used that allowed, considering the individual data inherent in the component of the product of interest in different experimental conditions tested, to find the parameters of the Bi-Langmuir isotherm. The identified set was subsequently used to further identify, a set of parameters for all species involved in the separation. As a result of the experiments performed, two parameter sets were identified that were able to predict the behavior of the molecules of interest.
In order to reduce the manual operations required for the multicomponent fitting, the second part of this thesis project involved the theoretical and practical development of an algorithm that is able to simulate a fitting also for the multi-component system, such as to take into account the influences of all species on the adsorption behavior of the product and impuritie
Role of the gradient slope during the product internal recycling for the multicolumn countercurrent solvent gradient purification of PEGylated proteins
No full textProtein PEGylation, i.e. functionalization with poly(ethylene glycol) chains, has been demonstrated an ef-ficient way to improve the therapeutic index of these biopharmaceuticals. We demonstrated that Multi -column Countercurrent Solvent Gradient Purification (MCSGP) is an efficient process for the separation of PEGylated proteins (Kim et al., Ind. and Eng. Chem. Res. 2021, 60, 29, 10764-10776), thanks to the internal recycling of product-containing side fractions. This recycling phase plays a critical role in the economy of MCSGP as it avoids wasting valuable product, but at the same time impacts its productivity extending the overall process duration. In this study, our aim is to elucidate the role of the gradient slope within this recycling stage on the yield and productivity of MCSGP for two case-studies: PEGylated lysozyme and an industrially relevant PEGylated protein. While all the examples of MCSGP in the literature refer to a single gradient slope in the elution phase, for the first time we systematically investigate three differ-ent gradient configurations: i) a single gradient slope throughout the entire elution, ii) recycling with an increased gradient slope, to shed light on the competition between volume of the recycled fraction and required inline dilution and iii) an isocratic elution during the recycling phase. The dual gradient elution proved to be a valuable solution for boosting the recovery of high-value products, with the potential for alleviating the pressure on the upstream processing.(c) 2023 Elsevier B.V. All rights reserved
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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