Amélioration de la production et de la qualité des protéines thérapeutiques recombinantes produites en culture en mode cuvée-alimentée de cellules de mammifère

La production de protéines thérapeutiques recombinantes en cellule de mammifère représente aujourd’hui une part importante des revenus des industries biopharmaceutiques. Des avancées considérables ont été réalisées ces 30 dernières années dans l’amélioration des rendements des cultures, notamment grâce au développement des procédés en mode cuvée-alimentée (fed-batch). Cependant, malgré des rendements de plus en plus élevés, la qualité des protéines produites est souvent faible, inconsistante et sujette à une grande variabilité d’une production à l’autre. La glycosylation est l’attribut de qualité des glycoprotéines considérée comme la plus critique car celle-ci influence de manière significative l’efficacité et l’immunogénicité du biothérapeutique. Plus particulièrement, la sialylation a un impact majeur sur le temps de demi-vie du biothérapeutique dans le sérum sanguin. Pour ces raisons, les agences de régulation tels que la Food and Drug Administration (FDA) et l’agence européenne des médicaments (EMA) imposent une caractérisation rigoureuse de la qualité des glycoprotéines, rendant ainsi le contrôle et l’optimisation de la glycosylation un enjeu primordial pour les industries pharmaceutiques. Cela représente un challenge de taille car la glycosylation est influencée par une multitude de facteurs allant du choix de la plateforme cellulaire de production, au type de protéines recombinantes produites ainsi qu’aux conditions de culture. Parmi les facteurs affectant la production et la qualité des protéines recombinantes se trouve l’accumulation des déchets métaboliques parmi lesquels le lactate et l’ammonium sont considérés les plus nocifs. Notamment, l’accumulation de l’ammonium contribue à la dégradation de la glycosylation des protéines recombinantes. Dans cette étude, nous avons visé la réduction de l’accumulation de ces métabolites toxiques dans le but d’améliorer la production et la qualité d’une glycoprotéine thérapeutique modèle, l’interféron α2b (IFNα2b), qui est utilisée dans le traitement contre les maladies virales et les cancers. Dans un premier temps, nous avons démontré l’effet sur la qualité de l’IFNα2b d’une stratégie de génie génétique améliorant le métabolisme central du carbone et réduisant l’accumulation de lactate. En combinant plusieurs techniques complémentaires d’analyse de la glycosylation, nous avons montré que la surexpression du gène de la pyruvate carboxylase cytosolique de levure (PYC2) dans la lignée de cellulaire humaine HEK293 a un effet bénéfique sur la viabilité cellulaire, menant ainsi à un prolongement des conditions favorables pour la glycosylation et l’intégrité de l’IFNα2b. Dans un second temps, nous avons combiné la modification PYC2 à des stratégies de substitution de la glutamine dans le but de réduire l’accumulation d’ammonium. Ces expériences nous ont permis de constater l’effet bénéfique du remplacement de la glutamine par le pyruvate sur la croissance et la viabilité cellulaire ainsi que sur la production protéique. L’alimentation des cellules PYC2 en glucose et en pyruvate, combinée à l’utilisation de précurseurs de la glycosylation a permis de maximiser la production et la qualité de l’IFNα2b. En outre, les cellules surexprimant la PYC2 ont démontré une croissance soutenue, une diminution significative de la production d’ammonium ainsi qu’une meilleure production d’IFNα2b en condition sans glutamine. Cette dernière observation a été exploitée pour développer une stratégie de culture en mode fed-batch) à l’aide d’une solution nutritive concentrée ne contenant pas de glutamine. Cela a permis une réduction drastique de l’accumulation de lactate et d’ammonium, ce qui a eu pour conséquence de doubler la production en interféron α2b en culture en flacons. Des performances améliorées ont également été obtenue lors des cultures en bioréacteur conduisant ainsi, malgré l’absence de glutamine, à la production d’IFNα2b hautement glycosylé. Enfin, nous avons investigué sur le développement d’une méthode permettant de suivre la glycosylation en cours de culture. La technique de résonance plasmonique de surface associée aux lectines a été utilisée pour capturer l’IFNα2b à partir du surnageant de culture afin d’analyser sa qualité. Ces travaux ont permis de mettre en évidence les défis liés à cette approche et des pistes d’amélioration ont été proposées. Ce travail apporte une contribution notable dans l’amélioration des procédés de production de protéines thérapeutiques recombinantes. En plus de représenter une première étude sur l’influence de l’altération du métabolisme central du carbone sur la qualité des glycoprotéines recombinantes, cette étude propose des stratégies efficaces et originales pour réduire simultanément le lactate et l’ammonium, les principaux inhibiteurs des procédés de culture de cellules de mammifère. En effet, grâce à leur phénotype avantageux, l’exploitation des cellules PYC2 a permis de concilier une haute production en glycoprotéines avec une haute qualité, offrant ainsi un outil intéressant pour la production de produits médicamenteux plus efficaces et plus sûrs pour le patient. ---------- The production of recombinant therapeutic proteins in mammalian cell lines represents today an important part in the revenue of biopharmaceutical companies. The last 30 years have shown tremendous increase in culture yields, notably through the development of fed-batch cultures. Despite these improvements, the quality of the proteins produced tends to be low, inconsistent and prone to high variability from batch to batch. Glycosylation is considered the most critical quality attribute because of its significant influence on the efficacy and the immunogenicity of the biotherapeutics. Especially, sialylation has a big impact on the protein half-life in the bloodstream. For these reasons, regulatory agencies like the FDA and the EMEA demand a rigorous characterization of glycoproteins quality, thereby making the control and the optimization of glycosylation a major concern for the pharmaceutical industries. This represent a serious challenge because glycosylation is influenced by several factors such as the cell line, the recombinant protein produced and the culture conditions. Among the main elements impacting protein production and quality, lactate and ammonia accumulation are considered the most detrimental. Ammonia accumulation, especially, is a well-known cause of the decrease in recombinant protein glycosylation. In this study, we focused on concomitantly reducing the accumulation of these two toxic metabolic wastes with the goal to improve the production and the quality of a model of therapeutic recombinant protein, the interferon α2b (IFNα2b). This glycosylated protein is used for the treatment of viral diseases and several cancers. Firstly, we studied the influence of a genetic engineering modification aimed to reduce lactate accumulation and to improve the central carbon metabolism on IFNα2b quality. By combining several orthogonal techniques for the analysis of glycosylation, we showed that the overexpression of the yeast pyruvate carboxylase gene (PYC2) in HEK293 cell lines improves cell culture viability which lead to sustained favorable conditions for glycosylation and IFNα2b integrity. Secondly, in order to reduce ammonia accumulation and investigate on potential synergetic effect on protein production and quality, we combined the PYC2 strategy with glutamine substitutions approaches. Theses experiments showed that replacing glutamine by pyruvate is beneficial for cell growth, culture viability and protein production. The supplementation of glucose and pyruvate in combination with glycosylation precursors allowed improving IFNα2b production while sustaining high IFNα2b sialylation. This part of the study also highlighted the singular capability of PYC2 cells to grow in conditions without glutamine while demonstrating reduced ammonia accumulation and enhanced protein production. This characteristic, was then exploited for the development of a glutamine-free fed-batch culture of the PYC2 cells using a concentrated nutrient feed. In addition to reducing lactate and ammonia accumulation, this allowed 2-fold increase in IFNα2b titer in shake flasks. In bioreactor, the glutamine-free fed-batch culture of PYC2 cells also led to drastic reduction in lactate and ammonia accumulation which translated into enhanced IFNα2b production. These improved performances were obtained without impacting IFNα2b quality as evidenced by the similar level of sialylation and glycosylation observed in conditions with and without glutamine. Finally, we investigated on the development of a technique to monitor recombinant protein glycosylation during the culture. Surface plasmon resonance exploiting the affinity of lectins for specific glycan was used to capture the IFNα2b from culture supernatant and then probe the glycans using a specific lectin. This work brought to light some of the challenges posed by this approach and several led to the proposition of several directions of improvement. This work represents an important contribution for the development of quality-focused protein production processes. In addition to present valuable data on the influence of an altered central carbon metabolism on protein quality, this study proposes strategies to concomitantly reduce the accumulation of the two most detrimental inhibitors of mammalian cell culture performances, lactate and ammonia. Indeed, the exploitation of the advantageous phenotype of PYC2 cells allowed to reconcile protein production and quality, thereby offering an attractive and simple approach for the development of efficient cell culture processes for the mass production of high quality therapeutic glycoproteins

Improving the metabolic efficiency of mammalian cells and its impact on glycoproteins quality

Glycosylation is a critical quality attribute for recombinant therapeutic proteins, which can be impacted by a number of process conditions, including waste metabolite accumulation. While fed-batch strategies that consist in substituting or controlling the main substrates at low concentrations have proven generally effective at improving protein titer, they can also adversely affect product glycosylation. Metabolic engineering strategies aiming at reducing by-product formation may thus be beneficial for ensuring product quality consistency. In this work, we have specifically investigated the impact of PYC2 overexpression on the quality of a recombinant glycoprotein of therapeutic interest, the interferon α2b (IFNα2b) that has one O-glycosylation site. To this end, batch and fed-batch cultures were performed and product characteristics were measured for both the PYC expressing HEK293 clone and the parental cells. SDS-PAGE and Western Blot analysis of batch culture harvests revealed two distinct bands corresponding to glycosylated and non-glycosylated fractions of IFNα2b, as subsequently confirmed via SDS-PAGE analysis of purified samples loaded along with a non-glycosylated commercial standard produced in E.coli. As inferred from densitometry analysis of the gels, the cultures with PYC-expressing cells were shown to sustain a significantly higher percentage of glycosylated IFNα2b at the late stage of the culture, which was correlated with the prolonged viability and reduced accumulation of waste metabolites. Differences between the two cell lines in terms of cell viability and protein quality were even more pronounced when performing fed-batch cultures during which glucose was maintained at high levels. To investigate the potential impact of ammonia, batch cultures with various glutamine substitutes were also performed. Among the different substitutes tested, pyruvate led to the lowest ammonia production with no significant impact on protein titer. Of salient interest, the results suggest that substituting glutamine by α-ketoglutarate, glutamate or pyruvate may allow to maintain a higher fraction of glycosylated proteins during late-stage batch cultures

Combining metabolic and process engineering strategies to improve recombinant glycoprotein production and quality

Increasing recombinant protein production while ensuring a high and consistent protein quality remains a challenge in mammalian cell culture process development. In this work, we combined a nutrient substitution approach with a metabolic engineering strategy that improves glucose utilization efficiency. This combination allowed us to tackle both lactate and ammonia accumulation and investigate on potential synergistic effects on protein production and quality. To this end, HEK293 cells overexpressing the pyruvate yeast carboxylase (PYC2) and their parental cells, both stably producing the therapeutic glycoprotein interferon \u3b12b (IFN\u3b12b), were cultured in media deprived of glutamine but containing chosen substitutes. Among the tested substitutes, pyruvate led to the best improvement in growth (integral of viable cell density) for both cell lines in batch cultures, whereas the culture of PYC2 cells without neither glutamine nor any substitute displayed surprisingly enhanced IFN\u3b12b production. The drastic reduction in both lactate and ammonia in the cultures translated into extended high viability conditions and an increase in recombinant protein titer by up to 47% for the parental cells and the PYC2 cells. Product characterization performed by surface plasmon resonance biosensing using Sambucus nigra (SNA) lectin revealed that the increase in yield was however accompanied by a reduction in the degree of sialylation of the product. Supplementing cultures with glycosylation precursors and a cofactor were effective at counterbalancing the lack of glutamine and allowed improvement in IFN\u3b12b quality as evaluated by lectin affinity. Our study provides a strategy to reconcile protein productivity and quality and highlights the advantages of PYC2-overexpressing cells in glutamine-free conditions.Peer reviewed: YesNRC publication: Ye

Altering the central carbon metabolism of HEK293 cells: Impact on recombinant glycoprotein quality

The accumulation of metabolic by-products remains a critical challenge in the development of mammalian cells culture processes as it impacts cellular growth, productivity and product quality. Although the overexpression of the PYC2 gene was shown to significantly improve the nutrient metabolism efficiency of mammalian cells, its impact on recombinant protein quality has not been investigated yet. In this study, we assess the effect of this metabolic engineering strategy on the quality of a recombinant therapeutic glycoprotein, the human interferon \u3b12b (IFN\u3b12b). As inferred from densitometry analysis of SDS-PAGE gels, PYC2-overexpressing cells sustained a higher percentage of intact glycosylated IFN\u3b12b at the late stage of batch cultures, which was correlated with prolonged viability and reduced accumulation of waste metabolites. Contrarily to the IFN\u3b12b produced by the PYC2 cells, LC\u2013MS analysis confirmed the presence of less glycosylated IFN\u3b12b as well as the occurrence of proteolytic cleavage in the IFN\u3b12b produced in the parental cells. Taken together, these results indicate that PYC2-overexpression in mammalian cells leads to extended favorable conditions for glycosylation and offer an attractive approach to mass-produce high-quality recombinant proteins.Peer reviewed: YesNRC publication: Ye

Combining metabolic and process engineering strategies to improve recombinant glycoprotein production and quality

Increasing recombinant protein production while ensuring a high and consistent protein quality remains a challenge in mammalian cell culture process development. In this work, we combined a nutrient substitution approach with a metabolic engineering strategy that improves glucose utilization efficiency. This combination allowed us to tackle both lactate and ammonia accumulation and investigate on potential synergistic effects on protein production and quality. To this end, HEK293 cells overexpressing the pyruvate yeast carboxylase (PYC2) and their parental cells, both stably producing the therapeutic glycoprotein interferon \u3b12b (IFN\u3b12b), were cultured in media deprived of glutamine but containing chosen substitutes. Among the tested substitutes, pyruvate led to the best improvement in growth (integral of viable cell density) for both cell lines in batch cultures, whereas the culture of PYC2 cells without neither glutamine nor any substitute displayed surprisingly enhanced IFN\u3b12b production. The drastic reduction in both lactate and ammonia in the cultures translated into extended high viability conditions and an increase in recombinant protein titer by up to 47% for the parental cells and the PYC2 cells. Product characterization performed by surface plasmon resonance biosensing using Sambucus nigra (SNA) lectin revealed that the increase in yield was however accompanied by a reduction in the degree of sialylation of the product. Supplementing cultures with glycosylation precursors and a cofactor were effective at counterbalancing the lack of glutamine and allowed improvement in IFN\u3b12b quality as evaluated by lectin affinity. Our study provides a strategy to reconcile protein productivity and quality and highlights the advantages of PYC2-overexpressing cells in glutamine-free conditions.Peer reviewed: YesNRC publication: Ye