In the modern era, biopharmaceuticals are becoming critical treatments for chronic and autoimmune human diseases. The possibility to dive deeply into the root causes and deliver genetic materials able to regulate and modulate the transcription of proteins involved in a specific disease opens up the fascinating opportunity to treat conditions that cannot be addressed through conventional therapies. Currently, state-of-art production for biopharmaceuticals revolves around batch or fed-batch fermentations, due to their simple process control, low capital expenditures, and well-established knowledge. However, the ever-increasing market pressure as well as the need for reducing process development time are making these platforms obsolescent, underlining the urgency for more efficient units. In this context, optimized perfusion bioreactors represent a valid solution to achieve improved process throughput, high-quality biotherapeutics, minimization of footprint and both economic and environmental sustainability. Hence, this study aims at developing, both at small and bench-scale, optimized perfusion systems for the manufacturing of two different classes of therapeutics, namely an oligonucleotide and adeno-associated viruses. At first, a comprehensive overview of the latest applications, of the current industrial scenario and the main economic and environmental implications of perfusion cell cultures is presented. Starting from the operational fundamentals, the discussion highlights the sever-al advantages associated to this setup. The process flexibility, proved by the variety of bioproducts successfully manufactured, the major economic gain, and the environmental benefit represent some of the strengths of perfusion operation. Nonetheless, a final discussion will concern present challenges, which still limit the industrial spread. Then, Chapter 2 reports the first scale-up, from small- to liter-scale, of a perfusion bacterial fermentation for the production of oligonucleotides. Based on previously collected outcomes, the dynamics of main process variables is assessed and finally compared against small-scale results. From here, the medium formulation was further refined in Chapter 3 to optimize cell growth and product titer, with specific reference to the biomanufacturing of the recombinant human pre-micro-RNA-29b-1. Interestingly, a trade-off between bacteria proliferation and extracellular product concentration as a function of sodium chloride concentration in the medium was established in small-scale perfusion devices and then validated at bench-scale. The upgrade to a continuous system allowed to simultaneously increase volumetric productivity while minimizing resource utilization in comparison to unoptimized batch systems. Furthermore, in Chapter 4, the development of an alternative, cutting-edge perfusion system was conducted as proof of concept for the manufacturing of very labile biologics. In particular, first steps were conducted towards a packed-bed bioreactor, whose the stationary phase consisted of bacteria-encapsulated polymer microbeads. The formulation of extracellular polymeric matrix, followed by investigation on microbead synthesis parameters and batch-like fermentation, indicated a potential manufacturing platform able to compete against traditional suspension set-ups. Finally, in Chapter 5, the focus shifted onto triple transient transfection-based manufacturing of a serotype 5 adeno-associated virus (AAV) by means of systematic investigations of the perfusion design space. The influence of transfecting cell density and plasmid concentration was explored, emphasizing the crucial role of these parameters. Moreover, a diverse transfection strategy was adopted and compared to literature results. Eventually, a scale-up to a 2 L perfusion bioreactor enabled a continuous harvest of extracellularly released AAV5 for 10 consecutive days, demonstrating not only a stable cell culture but also the absence of membrane fouling as well as a balanced release expression.
Nei tempi attuali, i biofarmaci stanno diventando trattamenti importanti per la cura di malattie croniche ed autoimmuni. La possibilità di far luce sulle cause fondamentali della malattia e di inserire materiale genetico, capace di regolare e modulare la trascrizione delle proteine coinvolte, apre l’opportunità di trattare condizioni non attualmente risolvibili tramite terapie convenzionali. Attualmente, la produzione allo stato dell’arte per biofarmaci ruota attorno a operazioni batch o fed-batch, dovuto ad un semplice controllo di preciso, minor capitale, e conoscenza di processo ben consolidata. Tuttavia, le crescenti pressioni del mercato, così come il bisogno di ridurre il tempo di sviluppo, rendono obsolete queste piattaforme, sottolineando l’urgenza di unità più efficienti. In questo contesto, bioreattori a perfusione rappresentano una valida soluzione per ottenere rese maggiori, bioterapeutici con alta qualità, impatti minori and sostenibilità economica ed ambientale. Pertanto, questo studio mira allo sviluppo, sia su piccola scala che laboratoriale, sistemi a perfusione ottimizzati per due diverse classi di terapeutici, nello specifico un oligonucleotide e virus adeno-associati. In primis, viene presentata una visione d’insieme delle ultime applicazioni, dell’attuale scenario industriale e implicazioni principali in termini di impatto ambientale ed economico, della tecnologia di perfusione. Flessibilità di processo, provata dalla varietà di bioprodotti sintetizzati con successo, il maggior rientro economico e il beneficio ambientale rappresentano alcuni dei punti di forza dell’operazione in perfusione. Nondimeno, una discussione finale presenterà le sfide presenti, che tuttora limitano la diffusione a livello industriale. Successivamente, il Capitolo 2 riporta il primo scale-up, da piccola scala al litro, di una fermentazione batterica in perfusione per la produzione di oligonucleotidi. Sulla base di risultati precedenti, la dinamica delle principali variabili di processo è stimata and infine confrontata rispetto ai risultati della piccola scala. Da qui, l’analisi della formulazione del mezzo è stata ulteriormente rifinita nel Capitolo 3 per ottimizzare la crescita cellulare e la concentrazione di prodotto, con riferimento specifico alla sintesi ricombinante del pre-microRNA-29b-1 umano. Interessatamente, è stato valutato un compromesso tra proliferazione batterica e concentrazione di prodotto extracellulare, funzione della concentrazione salina nel mezzo, nei dispositivi in piccola scala, successivamente validato alla scala del litro. L’upgrade ad un sistema in continuo ha simultaneamente permesso di aumentare la produttività volumetrica e minimizzare l’utilizzo di risorse rispetto a sistemi batch obsoleti. Inoltre, nel Capitolo 4, lo sviluppo di un sistema a perfusione alternativo e all’avanguardia è stato effettuato come prototipo per la manifattura di biologici molto labili. In particolare, sono stati fatti i primi step verso un bioreattore a letto impaccato, la cui fase stazionaria consiste di microsfere con batteri incapsulati. La formulazione della matrice polimerica extracellulare, seguita da un’analisi dei parametri di sintesi delle microsfere e sistemi in batch, ha indicato una potenziale piattaforma di manifattura, capace di compere contro i tradizionali sistemi in sospensione. Infine, nel Capitolo 5, il focus si è spostato sulla produzione, tramite tripla trasfezione, di virus adeno-associati ricombinanti di sierotipo 5 per mezzo di analisi sistematiche dello spazio di design di processi in perfusione. L’influenza della densità cellulare durante la trasfezione e la concentrazione di plasmidi utilizzata è stata esplorata, enfatizzando il ruolo cruciale di queste variabili. Inoltre, una strategia di trasfezione diversa è stata adottata e confrontata con risultati presenti in letteratura. Infine, uno scale-up ad un bioreattore in perfusion di 2 L ha permesso una rimozione continua di virus adeno-associati extracellulari per 10 giorni consecutivi, dimostrando non solo una coltura cellulare stabile ma anche l’assenza di occlusione nella membrana ed un’espressione di rilascio bilanciata.
Development of continuous perfusion cell cultures for the production of biotherapeutics
Iannacci, Francesco
2025/2026
Abstract
In the modern era, biopharmaceuticals are becoming critical treatments for chronic and autoimmune human diseases. The possibility to dive deeply into the root causes and deliver genetic materials able to regulate and modulate the transcription of proteins involved in a specific disease opens up the fascinating opportunity to treat conditions that cannot be addressed through conventional therapies. Currently, state-of-art production for biopharmaceuticals revolves around batch or fed-batch fermentations, due to their simple process control, low capital expenditures, and well-established knowledge. However, the ever-increasing market pressure as well as the need for reducing process development time are making these platforms obsolescent, underlining the urgency for more efficient units. In this context, optimized perfusion bioreactors represent a valid solution to achieve improved process throughput, high-quality biotherapeutics, minimization of footprint and both economic and environmental sustainability. Hence, this study aims at developing, both at small and bench-scale, optimized perfusion systems for the manufacturing of two different classes of therapeutics, namely an oligonucleotide and adeno-associated viruses. At first, a comprehensive overview of the latest applications, of the current industrial scenario and the main economic and environmental implications of perfusion cell cultures is presented. Starting from the operational fundamentals, the discussion highlights the sever-al advantages associated to this setup. The process flexibility, proved by the variety of bioproducts successfully manufactured, the major economic gain, and the environmental benefit represent some of the strengths of perfusion operation. Nonetheless, a final discussion will concern present challenges, which still limit the industrial spread. Then, Chapter 2 reports the first scale-up, from small- to liter-scale, of a perfusion bacterial fermentation for the production of oligonucleotides. Based on previously collected outcomes, the dynamics of main process variables is assessed and finally compared against small-scale results. From here, the medium formulation was further refined in Chapter 3 to optimize cell growth and product titer, with specific reference to the biomanufacturing of the recombinant human pre-micro-RNA-29b-1. Interestingly, a trade-off between bacteria proliferation and extracellular product concentration as a function of sodium chloride concentration in the medium was established in small-scale perfusion devices and then validated at bench-scale. The upgrade to a continuous system allowed to simultaneously increase volumetric productivity while minimizing resource utilization in comparison to unoptimized batch systems. Furthermore, in Chapter 4, the development of an alternative, cutting-edge perfusion system was conducted as proof of concept for the manufacturing of very labile biologics. In particular, first steps were conducted towards a packed-bed bioreactor, whose the stationary phase consisted of bacteria-encapsulated polymer microbeads. The formulation of extracellular polymeric matrix, followed by investigation on microbead synthesis parameters and batch-like fermentation, indicated a potential manufacturing platform able to compete against traditional suspension set-ups. Finally, in Chapter 5, the focus shifted onto triple transient transfection-based manufacturing of a serotype 5 adeno-associated virus (AAV) by means of systematic investigations of the perfusion design space. The influence of transfecting cell density and plasmid concentration was explored, emphasizing the crucial role of these parameters. Moreover, a diverse transfection strategy was adopted and compared to literature results. Eventually, a scale-up to a 2 L perfusion bioreactor enabled a continuous harvest of extracellularly released AAV5 for 10 consecutive days, demonstrating not only a stable cell culture but also the absence of membrane fouling as well as a balanced release expression.| File | Dimensione | Formato | |
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Descrizione: PhD Thesis of Francesco Iannacci
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https://hdl.handle.net/10589/256497