Biopharmaceuticals, and specifically monoclonal antibodies, are of exceptional importance in modern medicine. As such, the implementation of process analytical technologies to optimize process control can have many benefits, such as a reduction in costs and time needed for their development and during their application in production. This thesis focuses on the evaluation of an online variable length spectrophotometer and inline multi angle light scattering (MALS) and refractive index (RI) detector, with the objective to test their reliability compared to offline measurements and applicability in an ultrafiltration diafiltration and chromatographic processes. The idea was to monitor the concentration in real time for the ultrafiltration diafiltration process and to assess the ability of the MALS and RI detectors to detect the presence of aggregates during a chromatographic step. Indeed, aggregates constitute a major issue in the production of protein-based pharmaceuticals, due to their ability to elicit an immunologic reaction. Additionally, in the chromatographic process, various parameters such as buffer pH, storage temperature of the intermediate for load preparation, bed height and load ratio were analyzed, to find whether they had an influence on the formation of aggregates. For the ultrafiltration diafiltration process step, besides the comparison of the inline and offline concentrations, additional product quality parameters were tested, to analyze different buffer formulations and their effect on the stability of the monoclonal antibody solution in analysis. These were the turbidity, the aggregate content and the viscosity. The experimental results obtained for the UF/DF process showed that the technology is promising in the real-time concentration monitoring, though the influence of the position of the spectrophotometer in the flowpath might result in a delay in the measurements and must be considered. Addressing the different buffer formulations, the diafiltrations buffers used showed a stabilization effect on the monoclonal antibody compared to the matrix buffer. Furthermore, the alteration of the diafiltration buffer with arginine resulted in a lower viscosity of the antibody solution. An increase in turbidity was found at high concentration, which could not be explained by the number of aggregates found. It was thus hypothesized that there might be an increase in the content of transient aggregates. An interesting feature was found in the diafiltrated protein solution. Using the diafiltration buffer not altered with arginine, the solution showed shear thinning behavior, which might be exploitable for the formulation of drugs delivered through subcutaneous injection. From the results of the MALS and RI experiments, the MALS detector was not able to reveal any aggregates. The results from offline measurements were used to find a possible reason for that and the most plausible hypothesis is that the highly concentrated native antibody shielded the signal of the low concentrated aggregates. The storage temperature of the load material was found to influence the aggregates formation, suggesting that it is of outmost importance to keep the final drug at the designated temperature.
I farmaci biologici, nello specifico gli anticorpi monoclonali, sono di estrema importanza nella medicina moderna. L’utilizzo delle cosiddette process analytical technologies nell’ottimizzazione del processo di sviluppo e produzione, e nel suo controllo, può comportare vantaggi, come la riduzione dei costi e dei tempi necessari. Questa tesi verterà sulla valutazione di uno spettrofotometro (variable length spectrophotometer) posizionato online e dei detector che misurano la dispersione della luce a diverse angolazioni (multi angle light scattering, MALS) e l’indice di rifrazione (refractive index, RI) posizionati inline. L’obiettivo è testare la loro affidabilità rispetto alle misure ottenute offline e la loro applicabilità in processi quali la ultrafiltrazione diafiltrazione (UF/DF) e la cromatografia. L’idea di fondo è, nel processo di UF/DF, di monitorare la concentrazione in real-time e, nel caso del processo cromatografico, di valutare la capacità dei detector MALS e RI di rilevare la presenza di aggregati durante il processo cromatografico. Gli aggregati, infatti, costituiscono un problema rilevante nella produzione di farmaci biologici basati su proteine, a causa della loro capacità di scatenare una risposta immunitaria. In aggiunta, durante il processo cromatografico, numerosi parametri quali il pH dei buffer, la temperatura di conservazione dell’intermedio usato per la preparazione del load, l’altezza del letto della resina e la load ratio sono stati analizzati, allo scopo di verificare se essi influiscano sulla formazione di aggregati. Nel caso dell’UF/DF, oltre alla comparazione tra le misure della concentrazione prese inline e offline, sono stati analizzati ulteriori parametri di qualità del prodotto, allo scopo di analizzare diverse formulazioni e il loro effetto sulla stabilità della soluzione contente l’anticorpo monoclonale in analisi. I parametri sono i seguenti: torbidità, viscosità e presenza di aggregati. I risultati ottenuti per il processo di UF/DF hanno dimostrato che questo tipo di spettrofotometro montato inline sia promettente per monitorare la concentrazione in real-time, tuttavia la posizione di esso all’interno del percorso può risultare in un ritardo nelle misurazioni e deve pertanto essere valutata attentamente. Riguardo le differenti formulazioni utilizzate, invece, i buffer di diafiltrazione utilizzati hanno dimostrato di avere un effetto stabilizzante sull’anticorpo rispetto al buffer originario. Inoltre, l’alterazione del buffer di diafiltrazione con arginina ha comportato una diminuzione della viscosità della soluzione contenente l’anticorpo. Alte concentrazioni hanno mostrato un aumento della torbidità, che non è tuttavia spiegabile dal quantitativo di aggregati misurati. È stato quindi ipotizzato che l’aumento possa essere dovuto ad un aumento degli aggregati transienti. D’interesse sono i risultati ottenuti dalla soluzione diafiltrata. Nel caso della soluzione prodotta tramite diafiltrazione utilizzando il buffer non alterato con arginina, la soluzione mostra un comportamento non Newtoniano, di assottigliamento al taglio. Questa caratteristica potrebbe dimostrarsi utile nella formulazione di farmaci amministrabili per via sottocutanea. Dai risultati degli esperimenti, il detector MALS non è stato in grado di rilevare con certezza la presenza di aggregati. I risultati ottenuti offline sono stati analizzati per trovare la possibile causa e l’ipotesi più plausibile è che l’alta concentrazione dell’anticorpo nella sua conformazione nativa abbia coperto il segnale proveniente dalla bassa concentrazione di aggregati. Dato che la temperatura di conservazione del load mostra un’influenza sulla formazione degli aggregati, il mantenimento del farmaco alla corretta temperatura è della massima importanza.
Evaluation of process analytical technologies for inline and online determination of content and quality in downstream processing of biopharmaceuticals
NICOTRA, ASIA
2022/2023
Abstract
Biopharmaceuticals, and specifically monoclonal antibodies, are of exceptional importance in modern medicine. As such, the implementation of process analytical technologies to optimize process control can have many benefits, such as a reduction in costs and time needed for their development and during their application in production. This thesis focuses on the evaluation of an online variable length spectrophotometer and inline multi angle light scattering (MALS) and refractive index (RI) detector, with the objective to test their reliability compared to offline measurements and applicability in an ultrafiltration diafiltration and chromatographic processes. The idea was to monitor the concentration in real time for the ultrafiltration diafiltration process and to assess the ability of the MALS and RI detectors to detect the presence of aggregates during a chromatographic step. Indeed, aggregates constitute a major issue in the production of protein-based pharmaceuticals, due to their ability to elicit an immunologic reaction. Additionally, in the chromatographic process, various parameters such as buffer pH, storage temperature of the intermediate for load preparation, bed height and load ratio were analyzed, to find whether they had an influence on the formation of aggregates. For the ultrafiltration diafiltration process step, besides the comparison of the inline and offline concentrations, additional product quality parameters were tested, to analyze different buffer formulations and their effect on the stability of the monoclonal antibody solution in analysis. These were the turbidity, the aggregate content and the viscosity. The experimental results obtained for the UF/DF process showed that the technology is promising in the real-time concentration monitoring, though the influence of the position of the spectrophotometer in the flowpath might result in a delay in the measurements and must be considered. Addressing the different buffer formulations, the diafiltrations buffers used showed a stabilization effect on the monoclonal antibody compared to the matrix buffer. Furthermore, the alteration of the diafiltration buffer with arginine resulted in a lower viscosity of the antibody solution. An increase in turbidity was found at high concentration, which could not be explained by the number of aggregates found. It was thus hypothesized that there might be an increase in the content of transient aggregates. An interesting feature was found in the diafiltrated protein solution. Using the diafiltration buffer not altered with arginine, the solution showed shear thinning behavior, which might be exploitable for the formulation of drugs delivered through subcutaneous injection. From the results of the MALS and RI experiments, the MALS detector was not able to reveal any aggregates. The results from offline measurements were used to find a possible reason for that and the most plausible hypothesis is that the highly concentrated native antibody shielded the signal of the low concentrated aggregates. The storage temperature of the load material was found to influence the aggregates formation, suggesting that it is of outmost importance to keep the final drug at the designated temperature.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/61188