The chemical and physical stability of monoclonal antibodies (mAbs) represents a major challenge for their successful development. Aggregation is one of the most problematic possible degradation routes during processing and shelf-life. Liquid formulations at high protein concentration as required for subcutaneous or intramuscular injections are particularly at risk of forming such aggregates. Aggregation must be strictly avoided since aggregates are potentially non-active and immunogenic. Therefore, development of mAb products requires methods that can reliably predict aggregation rates under conditions relevant for drug product storage in order to minimize the risk of aggregate formation. It would be ideal to identify protein parameters that are reporter of aggregation propensity and could be measured in short time and consuming limited amount of sample. Unfortunately, to this day, no single biophysical parameter has been found yet to correlate robustly with aggregation propensity. This project focuses on the correlation between phase separation (PS) associated with native protein-protein interactions and non-native aggregation at high protein concentrations. To this aim, we induce PS of three mAbs in different buffer solutions by adding Polyethylene glycol (PEG) molecules, and we quantify phase equilibrium by measuring mAb concentration in the homogeneous phase. We rationalize the LLPS experiments in the frame of a thermodynamic model to extract quantitative information on the protein-protein interactions in the condensed phase. The condensed phase is further characterized by optical microscopy and fluorescence recovery after photo-bleaching (FRAP). We compare the LLSP results with aggregation rates measured at high mAb concentration and different temperatures (5, 30 and 40 °C). Our comparison shows that, in the absence of salt, there is a partial correlation between native-native interactions measured by LLPS and aggregation rates. However, this correlation does not apply when salt is introduced in the buffer. Moreover, addition of PEG to solutions of mAbs can induce formation of a variety of protein-rich condensed phases, some exhibiting more fluid-like others more gel-like characteristics. Overall, our results indicate that LLPS represents an attractive tool to complement the current set of screening techniques used to assess the developability of monoclonal antibodies during the early stages of the process development. However, further investigations are required to establish which type of information can be extracted from this assay, and how robustly correlates it with the aggregation propensity. This topic has important parallelisms with phase separation and aggregation processes of proteins in living organisms, which underlie both functional and dysfunctional biology, including onset and development of a series of neurodegenerative diseases.
La stabilità chimica e fisica degli anticorpi monoclonali (mAbs) rappresenta una sfida importante per il loro sviluppo. L'aggregazione è una delle degradazioni fisiche più problematiche sia durante la lavorazione che nella fase di conservazione del prodotto stesso. Maggiormente esposte al rischio di formazione di aggregati sono le formulazioni liquide ad alta concentrazione proteica utilizzate per iniezioni sottocutanee o intramuscolari. L'aggregazione deve essere rigorosamente evitata, poiché gli aggregati sono potenzialmente non attivi e immunogenici. Pertanto, lo sviluppo di anticorpi terapeutici richiede metodi per prevedere in modo affidabile la velocità di aggregazione dei farmaci in condizioni rilevanti per la conservazione, al fine di ridurre al minimo il rischio di formazione di aggregati. Sarebbe importante riuscire ad identificare i parametri indicanti la propensione all'aggregazione proteica, che potrebbero essere misurati in breve tempo con un consumo limitato della quantità di campione. Eppure, ad oggi, non è ancora stato trovato nessun singolo parametro biofisico solidamente correlato alla tendenza ad aggregare. Questo progetto si focalizza sulla possibile correlazione tra separazione di fase (PS), associata alle interazioni native proteina-proteina, e l'aggregazione non nativa ad alte concentrazioni proteiche. A questo scopo, viene effettuata la PS di tre mAbs in diverse soluzioni tampone aggiungendo molecole di glicole polietilenico (PEG), e viene quantificata la separazione di fase misurando la concentrazione di proteina nella fase omogenea. Attraverso un modello termodinamico sono stati spiegati gli esperimenti ottenuti con la precedente analisi, estraendo informazioni quantitative sulle interazioni proteina-proteina nella fase condensa. Questa è stata ulteriormente caratterizzata dalla microscopia ottica e dal recupero della fluorescenza dopo il foto-sbiancamento (FRAP). Successivamente, i risultati dall’analisi di LLPS vengono confrontati con le velocità di aggregazione, misurate ad alta concentrazione di mAb e a diverse temperature (5, 30 e 40 °C). Il confronto mostra che in assenza di sale vi è una correlazione parziale tra le interazioni native, misurate da LLPS, e velocità di aggregazione. Tuttavia, questa correlazione non è presente quando il sale viene introdotto nella soluzione iniziale. Inoltre, l'aggiunta di PEG nelle soluzioni di mAbs può indurre la formazione di varie fasi dense di proteine, da quella più liquida a quella più gelatinosa. Nel complesso, i risultati ottenuti indicano che LLPS rappresenta un’interessante strumento per completare l'attuale insieme di tecniche di screening utilizzate per valutare lo sviluppo di anticorpi monoclonali durante le prime fasi del processo. Tuttavia, sono necessarie ulteriori indagini per stabilire quale tipo di informazioni possano essere estratte da questa analisi e quanto sia robusta la correlazione con la propensione all'aggregazione. Questo argomento ha importanti parallelismi con i processi di separazione di fase e di aggregazione delle proteine negli organismi viventi, che sono alla base della biologia funzionale e disfunzionale, tra cui l'insorgenza e lo sviluppo di una serie di malattie neurodegenerative.
Native-state protein-protein interactions evaluated through PEG-precipitation as predictor for long-term stability of high-concentration therapeutic mAbs formulations
FANIZZI, FRANCESCA
2017/2018
Abstract
The chemical and physical stability of monoclonal antibodies (mAbs) represents a major challenge for their successful development. Aggregation is one of the most problematic possible degradation routes during processing and shelf-life. Liquid formulations at high protein concentration as required for subcutaneous or intramuscular injections are particularly at risk of forming such aggregates. Aggregation must be strictly avoided since aggregates are potentially non-active and immunogenic. Therefore, development of mAb products requires methods that can reliably predict aggregation rates under conditions relevant for drug product storage in order to minimize the risk of aggregate formation. It would be ideal to identify protein parameters that are reporter of aggregation propensity and could be measured in short time and consuming limited amount of sample. Unfortunately, to this day, no single biophysical parameter has been found yet to correlate robustly with aggregation propensity. This project focuses on the correlation between phase separation (PS) associated with native protein-protein interactions and non-native aggregation at high protein concentrations. To this aim, we induce PS of three mAbs in different buffer solutions by adding Polyethylene glycol (PEG) molecules, and we quantify phase equilibrium by measuring mAb concentration in the homogeneous phase. We rationalize the LLPS experiments in the frame of a thermodynamic model to extract quantitative information on the protein-protein interactions in the condensed phase. The condensed phase is further characterized by optical microscopy and fluorescence recovery after photo-bleaching (FRAP). We compare the LLSP results with aggregation rates measured at high mAb concentration and different temperatures (5, 30 and 40 °C). Our comparison shows that, in the absence of salt, there is a partial correlation between native-native interactions measured by LLPS and aggregation rates. However, this correlation does not apply when salt is introduced in the buffer. Moreover, addition of PEG to solutions of mAbs can induce formation of a variety of protein-rich condensed phases, some exhibiting more fluid-like others more gel-like characteristics. Overall, our results indicate that LLPS represents an attractive tool to complement the current set of screening techniques used to assess the developability of monoclonal antibodies during the early stages of the process development. However, further investigations are required to establish which type of information can be extracted from this assay, and how robustly correlates it with the aggregation propensity. This topic has important parallelisms with phase separation and aggregation processes of proteins in living organisms, which underlie both functional and dysfunctional biology, including onset and development of a series of neurodegenerative diseases.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/145171