The establishment of efficient and reliable downstream operations in biotechnology processes is crucial to deliver biopharmaceuticals with the required high purity and affordable costs. Chromatography is often the technique of choice for the purification of these biomolecules, mainly due to the high selectivity that this operation offers. Although capable of meeting stringent purity requirements, the single-column chromatographic operations currently in use often result in poor product recovery, which has a tremendous impact on the process efficiency and, in turn, its economics. To cover this gap, this thesis encompasses the development, optimization, and economic evaluation of continuous countercurrent chromatographic operations, specifically focused on the purification of polyethylene glycol (PEG)−protein conjugates. In particular, multicolumn countercurrent solvent gradient purification (MCSGP) is demonstrated as a viable alternative to batch chromatography in enhancing yield and productivity while maintaining the purity specification. Its notable feature of an automated internal recycling of product-containing side fractions, typically discarded in batch processes, was investigated. An ad hoc design strategy was established for the optimization of the batch purification of PEGylated lysozyme and an industrially relevant PEGylated protein, used as case studies. The optimal set of process parameters derived from this strategy were then transferred to MCSGP for the improvement of yield and productivity. Next, the role of gradient slope within the recycling stage of MCSGP was elucidated. Various gradient configurations were investigated to understand their impact on the yield and productivity of the process, introducing, for the first time, the concept of dual gradient elution as a solution for maximizing product recovery. Despite the promise of these dual slope MCSGP operations, challenges related to higher buffer consumption and lower productivity emerged. These were primarily associated with the need for extensive inline dilution of the side fractions before their recycling to the downstream column and operational constraints of the column flowrate. To address these challenges, the integration of an ultrafiltration/diafiltration (UF/DF) unit into the inline dilution process was proposed. The implementation of UF/DF was evaluated through a feasibility study, testing its application on the recycling fractions. The resulting process offered promising advancements in terms of productivity and reduced solvent consumption. Collectively, this body of work advances the field of biopharmaceutical purification, delivering optimized MCSGP processes and proposing an expansion of its versatility by investigating modifications to the elution strategy through a second gradient slope and a compression in the inline dilution through UF/DF. It presents not only a deep understanding of the MCSGP process but also its economic viability, process flexibility, and environmental implications.
La creazione di operazioni a valle efficienti e affidabili nei processi biotecnologici è fondamentale per fornire biofarmaci con l'elevata purezza richiesta e costi accessibili. La cromatografia è spesso la tecnica di scelta per la purificazione di queste biomolecole, principalmente a causa dell'elevata selettività che questa operazione offre. Sebbene siano in grado di soddisfare severi requisiti di purezza, le operazioni cromatografiche a colonna singola attualmente in uso spesso si traducono in uno scarso recupero del prodotto, che ha un enorme impatto sull'efficienza del processo e, a sua volta, sulla sua economia. Per colmare questa lacuna, questa tesi comprende lo sviluppo, l'ottimizzazione e la valutazione economica di operazioni cromatografiche continue in controcorrente, specificamente focalizzate sulla purificazione di coniugati polietilenglicole (PEG)-proteine. In particolare, la purificazione in gradiente di solvente in controcorrente multicolonna (MCSGP) è dimostrata come una valida alternativa alla cromatografia batch per migliorare la resa e la produttività mantenendo le specifiche di purezza. È stata studiata la sua notevole caratteristica di un riciclaggio interno automatizzato delle frazioni laterali contenenti il prodotto, tipicamente scartate nei processi batch. È stata stabilita una strategia di progettazione ad hoc per l'ottimizzazione della purificazione batch del lisozima PEGilato e di una proteina PEGilata rilevante a livello industriale, utilizzata come caso di studio. L'insieme ottimale di parametri di processo derivati da questa strategia è stato poi trasferito a MCSGP per il miglioramento della resa e della produttività. Successivamente, è stato chiarito il ruolo della pendenza del gradiente all'interno della fase di riciclaggio di MCSGP. Sono state studiate varie configurazioni di gradiente per comprenderne l'impatto sulla resa e sulla produttività del processo, introducendo, per la prima volta, il concetto di eluizione a doppio gradiente come soluzione per massimizzare il recupero del prodotto.Nonostante la promessa di queste operazioni MCSGP a doppia pendenza, sono emerse sfide legate al maggiore consumo di buffer e alla minore produttività. Questi erano principalmente associati alla necessità di un'ampia diluizione in linea delle frazioni laterali prima del loro riciclaggio nella colonna a valle e ai vincoli operativi della portata della colonna. Per affrontare queste sfide, è stata proposta l'integrazione di un'unità di ultrafiltrazione/diafiltrazione (UF/DF) nel processo di diluizione in linea. L'implementazione di UF/DF è stata valutata attraverso uno studio di fattibilità, testandone l'applicazione sulle frazioni di riciclo. Il processo risultante ha offerto progressi promettenti in termini di produttività e riduzione del consumo di solventi. Collettivamente, questo corpo di lavoro fa avanzare il campo della purificazione biofarmaceutica, offrendo processi MCSGP ottimizzati e proponendo un'espansione della sua versatilità studiando le modifiche alla strategia di eluizione attraverso una seconda pendenza del gradiente e una compressione nella diluizione in linea attraverso UF/DF. Presenta non solo una profonda comprensione del processo MCSGP, ma anche la sua fattibilità economica, la flessibilità del processo e le implicazioni ambientali.
Expansion of versatility of continuous countercurrent chromatography for the separation of PEGylated proteins
KIM, TAE KEUN
2022/2023
Abstract
The establishment of efficient and reliable downstream operations in biotechnology processes is crucial to deliver biopharmaceuticals with the required high purity and affordable costs. Chromatography is often the technique of choice for the purification of these biomolecules, mainly due to the high selectivity that this operation offers. Although capable of meeting stringent purity requirements, the single-column chromatographic operations currently in use often result in poor product recovery, which has a tremendous impact on the process efficiency and, in turn, its economics. To cover this gap, this thesis encompasses the development, optimization, and economic evaluation of continuous countercurrent chromatographic operations, specifically focused on the purification of polyethylene glycol (PEG)−protein conjugates. In particular, multicolumn countercurrent solvent gradient purification (MCSGP) is demonstrated as a viable alternative to batch chromatography in enhancing yield and productivity while maintaining the purity specification. Its notable feature of an automated internal recycling of product-containing side fractions, typically discarded in batch processes, was investigated. An ad hoc design strategy was established for the optimization of the batch purification of PEGylated lysozyme and an industrially relevant PEGylated protein, used as case studies. The optimal set of process parameters derived from this strategy were then transferred to MCSGP for the improvement of yield and productivity. Next, the role of gradient slope within the recycling stage of MCSGP was elucidated. Various gradient configurations were investigated to understand their impact on the yield and productivity of the process, introducing, for the first time, the concept of dual gradient elution as a solution for maximizing product recovery. Despite the promise of these dual slope MCSGP operations, challenges related to higher buffer consumption and lower productivity emerged. These were primarily associated with the need for extensive inline dilution of the side fractions before their recycling to the downstream column and operational constraints of the column flowrate. To address these challenges, the integration of an ultrafiltration/diafiltration (UF/DF) unit into the inline dilution process was proposed. The implementation of UF/DF was evaluated through a feasibility study, testing its application on the recycling fractions. The resulting process offered promising advancements in terms of productivity and reduced solvent consumption. Collectively, this body of work advances the field of biopharmaceutical purification, delivering optimized MCSGP processes and proposing an expansion of its versatility by investigating modifications to the elution strategy through a second gradient slope and a compression in the inline dilution through UF/DF. It presents not only a deep understanding of the MCSGP process but also its economic viability, process flexibility, and environmental implications.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/207609