Maria Elisa Rodrigues

Technological progresses in monoclonal antibody production systems

Monoclonal antibodies (mAbs) have become vitally important to modern medicine and are currently one of the major biopharmaceutical products in development. However, the high clinical dose requirements of mAbs demand a greater biomanufacturing capacity, leading to the development of new technologies for their large‐scale production, with mammalian cell culture dominating the scenario. Although some companies have tried to meet these demands by creating bioreactors of increased capacity, the optimization of cell culture productivity in normal bioreactors appears as a better strategy. This review describes the main technological progresses made with this intent, presenting the advantages and limitations of each production system, as well as suggestions for improvements. New and upgraded bioreactors have emerged both for adherent and suspension cell culture, with disposable reactors attracting increased interest in the last years. Furthermore, the strategies and technologies used to control culture parameters are in constant evolution, aiming at the on‐line multiparameter monitoring and considering now parameters not seen as relevant for process optimization in the past. All progresses being made have as primary goal the development of highly productive and economic mAb manufacturing processes that will allow the rapid introduction of the product in the biopharmaceutical market at more accessible prices.

Glycosylation: impact, control and improvement during therapeutic protein production

Abstract The emergence of the biopharmaceutical industry represented a major revolution for modern medicine, through the development of recombinant therapeutic proteins that brought new hope for many patients with previously untreatable diseases. There is a ever-growing demand for these therapeutics that forces a constant technological evolution to increase product yields while simultaneously reducing costs. However, the process changes made for this purpose may also affect the quality of the product, a factor that was initially overlooked but which is now a major focus of concern. Of the many properties determining product quality, glycosylation is regarded as one of the most important, influencing, for example, the biological activity, serum half-life and immunogenicity of the protein. Consequently, monitoring and control of glycosylation is now critical in biopharmaceutical manufacturing and a requirement of regulatory agencies. A rapid evolution is being observed in this context, concerning the influence of glycosylation in the efficacy of different therapeutic proteins, the impact on glycosylation of a diversity of parameters/processes involved in therapeutic protein production, the analytical methodologies employed for glycosylation monitoring and control, as well as strategies that are being explored to use this property to improve therapeutic protein efficacy (glycoengineering). This work reviews the main findings on these subjects, providing an up-to-date source of information to support further studies.

Novel strategies to fight Candida species infection

Abstract In recent years, there has been a significant increase in the incidence of human fungal infections. The increase in cases of infection caused by Candida species, and the consequent excessive use of antimicrobials, has favored the emergence of resistance to conventional antifungal agents over the past decades. Consequently, Candida infections morbidity and mortality are also increasing. Therefore, new approaches are needed to improve the outcome of patients suffering from Candida infections, because it seems unlikely that the established standard treatments will drastically lower the morbidity of mucocutaneous Candida infections and the high mortality associated with invasive candidiasis. This review aims to present the last advances in the traditional antifungal therapy, and present an overview of novel strategies that are being explored for the treatment of Candida infections, with a special focus on combined antifungal agents, antifungal therapies with alternative compounds (plant extracts and essential oils), adjuvant immunotherapy, photodynamic therapy and laser therapy.

The impact of cell adaptation to serum-free conditions on the glycosylation profile of a monoclonal antibody produced by Chinese hamster ovary cells

N-glycosylation is one of the most crucial parameters affecting the biological activity of therapeutic monoclonal antibodies (mAbs), and should therefore be closely monitored and controlled to guarantee a consistent and high-quality product in biopharmaceutical processes. In the present work, the effect of the time-consuming step of gradual cell adaptation to serum-free conditions on the glycosylation profile of a mAb produced by CHO-K1 cells was evaluated. High-performance liquid chromatography analysis revealed important changes in mAb glycosylation patterns in all steps of serum reduction. These changes could be grouped in two distinct phases of the process of adaptation: middle (2.5 to 0.15% serum) and final (0.075 and 0% serum). For intermediate levels of serum, a desirable increase of galactosylation and decrease of fucosylation, but an undesirable increase in sialylation were observed; while the inverse was obtained at the final stages of adaptation. These divergences may be related to the reduction of serum supplementation, to variations in the levels of cell density and viability achieved at these stages, and to the natural shift of the cell growth mode during adaptation from adherent to suspended. The divergent glycan profiles obtained in this study demonstrate a strong influence of the adaptation process on mAb glycosylation, suggesting that control and monitoring of product quality should be implemented at the early stages of process development.

Comparison of commercial serum-free media for CHO-K1 cell growth and monoclonal antibody production.

The selection of a serum-free medium for a particular process of production using mammalian cells is a critical step for its success. In this study, seven commercially available serum-free media (EX-CELL, ISF-I, CD CHO, CDM4CHO, CHO-III-A, Octomed and HybridoMed) were evaluated and compared for cell growth and monoclonal antibody (mAb) production of a transfected CHO-K1 cell line. In the conditions assayed, EX-CELL and particularly CDM4CHO are the most recommended media for extended biopharmaceutical processes, on account of inducing superior levels of cell proliferation and mAb production, accentuated by a tendency to improve over time. Furthermore, the less positive results obtained with some media emphasize the importance and the impact of the correct medium selection.

Advances and Drawbacks of the Adaptation to Serum-Free Culture of CHO-K1 Cells for Monoclonal Antibody Production

Currently, mammalian cell technology has become the focus of biopharmaceutical production, with strict regulatory scrutiny of the techniques employed. Major concerns about the presence of animal-derived components in the culture media led to the development of serum-free (SF) culture processes. However, cell adaptation to SF conditions is still a major challenge and limiting step of process development. Thus, this study aims to assess the impact of SF adaptation on monoclonal antibody (mAb) production, identify the most critical steps of cell adaptation to the SF EX-CELL medium, and create basic process guidelines. The success of SF adaptation was dependent on critical steps that included accentuated cell sensitivity to common culture procedures (centrifugation, trypsinization), initial cell concentration, time given at each step of serum reduction, and, most importantly, medium supplements used to support adaptation. Indeed, only one of the five supplement combinations assessed (rhinsulin, ammonium metavanadate, nickel chloride, and stannous chloride) succeeded for the Chinese hamster ovary-K1 cell line used. This work also revealed that the chemically defined EX-CELL medium benefits mAb production in comparison with the general purpose Dulbecco’s Modified Eagle’s Medium, but the complete removal of serum attenuates these positive effects.

The impact of microcarrier culture optimization on the glycosylation profile of a monoclonal antibody

Microcarriers are widely used for the large-scale culture of attachment-dependent cells with increased cell densities and, ultimately, higher product yield. In these processes, the specific culture conditions can affect the quality of the product, which is closely related to its glycosylation pattern. Furthermore, the lack of studies in the area reinforces the need to better understand the effects of microcarrier culture in product glycosylation. Consequently, in this work, the glycosylation profile of a monoclonal antibody (mAb) produced by adherent CHO-K1 cells grown in Cytodex 3 was evaluated under different conditions, and compared to that obtained of typical adherent cultures. It was found that microcarrier cultures result in a glycosylation profile with different characteristics from T-flask cultures, with a general increase in galactosylation and decrease in fucosylation levels, both with a potentially positive impact on mAb activity. Sialylation also varied but without a general tendency. This study then showed that the specific culture conditions used in microcarrier culture influence the mAb glycan profile, and each functional element (galactose, core fucose, sialic acid) is independently affected by these conditions. In particular, great reductions of fucosylation (from 79 to 55%) were obtained when using half volume at inoculation, and notable decreases in sialylation (from 23 to 2%) and glycoform heterogeneity (from 20 to 11 glycoforms) were observed for shake flask culture, potentially associated with the improved cell densities achieved in these culture vessels.

Candida Species Biofilms’ Antifungal Resistance

Candida infections (candidiasis) are the most prevalent opportunistic fungal infection on humans and, as such, a major public health problem. In recent decades, candidiasis has been associated to Candida species other than Candida albicans. Moreover, biofilms have been considered the most prevalent growth form of Candida cells and a strong causative agent of the intensification of antifungal resistance. As yet, no specific resistance factor has been identified as the sole responsible for the increased recalcitrance to antifungal agents exhibited by biofilms. Instead, biofilm antifungal resistance is a complex multifactorial phenomenon, which still remains to be fully elucidated and understood. The different mechanisms, which may be responsible for the intrinsic resistance of Candida species biofilms, include the high density of cells within the biofilm, the growth and nutrient limitation, the effects of the biofilm matrix, the presence of persister cells, the antifungal resistance gene expression and the increase of sterols on the membrane of biofilm cells. Thus, this review intends to provide information on the recent advances about Candida species biofilm antifungal resistance and its implication on intensification of the candidiasis.

Research paperThe impact of cell adaptation to serum-free conditions on the glycosylation profile of a monoclonal antibody produced by Chinese hamster ovary cells

N-glycosylation is one of the most crucial parameters affecting the biological activity of therapeutic monoclonal antibodies (mAbs), and should therefore be closely monitored and controlled to guarantee a consistent and high-quality product in biopharmaceutical processes. In the present work, the effect of the time-consuming step of gradual cell adaptation to serum-free conditions on the glycosylation profile of a mAb produced by CHO-K1 cells was evaluated. High-performance liquid chromatography analysis revealed important changes in mAb glycosylation patterns in all steps of serum reduction. These changes could be grouped in two distinct phases of the process of adaptation: middle (2.5 to 0.15% serum) and final (0.075 and 0% serum). For intermediate levels of serum, a desirable increase of galactosylation and decrease of fucosylation, but an undesirable increase in sialylation were observed; while the inverse was obtained at the final stages of adaptation. These divergences may be related to the reduction of serum supplementation, to variations in the levels of cell density and viability achieved at these stages, and to the natural shift of the cell growth mode during adaptation from adherent to suspended. The divergent glycan profiles obtained in this study demonstrate a strong influence of the adaptation process on mAb glycosylation, suggesting that control and monitoring of product quality should be implemented at the early stages of process development.

Evaluation of the OSCAR system for the production of monoclonal antibodies by CHO-K1 cells.

Biopharmaceutical production of complex recombinant protein therapeutics currently relies on mammalian cells. The development of high-yielding stable cell lines requires processes of transfection, selection and adaptation. With several technologies available, selection has been most frequently based on dihydrofolate reductase or glutamine synthetase systems, which can be very time-consuming. Due to the pressure to reduce development costs and speed up time to market, new technologies are emerging, as the promising OSCAR expression system that could provide more rapid development of high-yielding stable cell lines than the traditional systems. However, further evaluation of its application in a wider range of cell types and media is still necessary. In this study, application of OSCAR for the transfection of a CHO-K1 cell line with a monoclonal antibody was evaluated. OSCAR was reasonably fast and simple, without negative impact on cell growth characteristics. However, minigene selection was critical, with only pDWM128 working for the cell line assessed. Initial relatively high levels of production decreased significantly in the first few weeks of passing, remaining relatively stable although with low yield thereafter. The results suggest that more work is required to develop methodologies and prove that OSCAR has significant value to the bioproduction industry.