| 1. |
- Schwarz, Hubert, et al.
(författare)
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Integrated continuous biomanufacturing on pilot scale for acid-sensitive monoclonal antibodies
- 2022
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290.
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we demonstrated the first, to our knowledge, integrated continuous bioprocess (ICB) designed for the production of acid-sensitive monoclonal antibodies, prone to aggregate at low pH, on pilot scale. A high cell density perfusion culture, stably maintained at 100 × 106 cells/ml, was integrated with the downstream process, consisting of a capture step with the recently developed Protein A ligand, ZCa; a solvent/detergent-based virus inactivation; and two ion-exchange chromatography steps. The use of a mild pH in the downstream process makes this ICB suitable for the purification of acid-sensitive monoclonal antibodies. Integration and automation of the downstream process were achieved using the Orbit software, and the same equipment and control system were used in initial small-scale trials and the pilot-scale downstream process. High recovery yields of around 90% and a productivity close to 1 g purified antibody/L/day were achieved, with a stable glycosylation pattern and efficient removal of impurities, such as host cell proteins and DNA. Finally, negligible levels of antibody aggregates were detected owing to the mild conditions used throughout the process. The present work paves the way for future industrial-scale integrated continuous biomanufacturing of all types of antibodies, regardless of acid stability.
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| 6. |
- Schwarz, Hubert, et al.
(författare)
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Monitoring of amino acids and antibody N-glycosylation in high cell density perfusion culture based on Raman spectroscopy
- 2022
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Ingår i: Biochemical engineering journal. - : Elsevier BV. - 1369-703X .- 1873-295X. ; 182, s. 108426-
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Tidskriftsartikel (refereegranskat)abstract
- Raman spectrum based predictive models provide a process analytical technology (PAT) tool for monitoring and control of culture parameters in bioprocesses. Steady-state perfusion cultures generate a relatively stable metabolite profile, which is not conducive to modeling due to the absence of variations of culture parameters. Here we present an approach where different steady-states obtained by variation of the cell specific perfusion rate (CSPR) between 10 and 40 pL/(cell * day) with cell densities up to 100 × 106 cells/mL during the process development provided a dynamic culture environment, favorable for the model calibration. The cell density had no effect on the culture performance at similar CSPR, however a variation in the CSPR had a strong influence on the metabolism, mAb productivity and N-glycosylation. Predictive models were developed for multiple culture parameters, including cell density, lactate, ammonium and amino acids; and then validated with new runs performed at multiple or single steady-states, showing high prediction accuracy. The relationship of amino acids and antibody N-glycosylation was modeled to predict the glycosylation pattern of the product in real time. The present efficient process development approach with integration of Raman spectroscopy provides a valuable PAT tool for later implementation in steady-state perfusion production processes.
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| 7. |
- Schwarz, Hubert, et al.
(författare)
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Optimization of medium with perfusion microbioreactors for high density CHO cell cultures at very low renewal rate aided by design of experiments
- 2023
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 120:9, s. 2523-2541
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Tidskriftsartikel (refereegranskat)abstract
- A novel approach of design of experiment (DoE) is developed for the optimization of key substrates of the culture medium, amino acids, and sugars, by utilizing perfusion microbioreactors with 2 mL working volume, operated in high cell density continuous mode, to explore the design space. A mixture DoE based on a simplex-centroid is proposed to test multiple medium blends in parallel perfusion runs, where the amino acids concentrations are selected based on the culture behavior in presence of different amino acid mixtures, and using targeted specific consumption rates. An optimized medium is identified with models predicting the culture parameters and product quality attributes (G0 and G1 level N-glycans) as a function of the medium composition. It is then validated in runs performed in perfusion microbioreactor in comparison with stirred-tank bioreactors equipped with alternating tangential flow filtration (ATF) or with tangential flow filtration (TFF) for cell separation, showing overall a similar process performance and N-glycosylation profile of the produced antibody. These results demonstrate that the present development strategy generates a perfusion medium with optimized performance for stable Chinese hamster ovary (CHO) cell cultures operated with very high cell densities of 60 × 106 and 120 × 106 cells/mL and a low cell-specific perfusion rate of 17 pL/cell/day, which is among the lowest reported and is in line with the framework recently published by the industry.
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| 8. |
- Velut, Stephane, et al.
(författare)
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Influence of bioreactor scale and complex medium on probing control of glucose feeding in cultivations of recombinant strains of Escherichia coli
- 2007
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 97:4, s. 816-824
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this work was to evaluate the performance of a feedback glucose control strategy (the probing strategy) in production relevant bioreactors with complex and mineral media. Experimental results from fedbatch cultivations with two recombinant Escherichia coli constructs expressing two different human therapeutic proteins were used to assess the performance and limitations of the glucose probing technique. Even though the performance of the probing strategy was affected by scale and complex media, this methodology rapidly identified a glucose feed protocol similar to an experimentally derived feed regime. This methodology may serve as a powerful tool for industrial process development and in optimization of glucose feed regimes when transferring process technology from one bioreactor system to another.
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| 9. |
- Zhang, Liang, et al.
(författare)
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Combined effects of glycosylation precursors and lactate on the glycoprofile of IgG produced by CHO cells
- 2019
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Ingår i: Journal of Biotechnology. - : Elsevier. - 0168-1656 .- 1873-4863. ; 289, s. 71-79
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Tidskriftsartikel (refereegranskat)abstract
- The glycosylation profile of therapeutic monoclonal antibodies (mAbs) is a crucial quality parameter for industrial Immunoglobulin G (IgG) production. Several alternative carbon sources, which function as glycosylation precursors, have been reported to impact the glycosylation pattern. Since the cells give priority to glucose uptake, the presence of this substrate can lower the effects of alternative sugars on the glycosylation. In order to get a better understanding of the influence of alternative sugars on the glycosylation and to investigate how they impact each other, combinations of mannose, fructose, galactose and fucose were fed to Chinese hamster ovary (CHO) cells in batch culture when the glucose became depleted and the lactate, accumulated in the culture, was used as carbon source. Feeding with a feed containing mannose or glucose decreased by 3-7% the percentage of high mannose glycans compared to a feed without mannose or glucose. Feeding with a feed containing galactose led to 8-20% increase of monogalactoglycans (G1) glycans and 2-6% rise of digalactoglycans (G2) glycans compared to feeding without galactose or glucose. The cells fed with fucose exhibited a significantly higher concentration of intracellular GDP-Fucose. This work indicates that a feeding strategy based on non-glucose sugars and potentially lactate, could be adopted to obtain a targeted glycosylation profile.
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| 10. |
- Zhang, Liang, et al.
(författare)
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Glycan Residues Balance Analysis : A novel model for the N-linked glycosylation of IgG produced by CHO cells.
- 2020
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 57, s. 118-128
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Tidskriftsartikel (refereegranskat)abstract
- The structure of N-linked glycosylation is a very important quality attribute for therapeutic monoclonal antibodies. Different carbon sources in cell culture media, such as mannose and galactose, have been reported to have different influences on the glycosylation patterns. Accurate prediction and control of the glycosylation profile are important for the process development of mammalian cell cultures. In this study, a mathematical model, that we named Glycan Residues Balance Analysis (GReBA), was developed based on the concept of Elementary Flux Mode (EFM), and used to predict the glycosylation profile for steady state cell cultures. Experiments were carried out in pseudo-perfusion cultivation of antibody producing Chinese Hamster Ovary (CHO) cells with various concentrations and combinations of glucose, mannose and galactose. Cultivation of CHO cells with mannose or the combinations of mannose and galactose resulted in decreased lactate and ammonium production, and more matured glycosylation patterns compared to the cultures with glucose. Furthermore, the growth rate and IgG productivity were similar in all the conditions. When the cells were cultured with galactose alone, lactate was fed as well to be used as complementary carbon source, leading to cell growth rate and IgG productivity comparable to feeding the other sugars. The data of the glycoprofiles were used for training the model, and then to simulate the glycosylation changes with varying the concentrations of mannose and galactose. In this study we showed that the GReBA model had a good predictive capacity of the N-linked glycosylation. The GReBA can be used as a guidance for development of glycoprotein cultivation processes.
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| 11. |
- Zhang, Liang, 1990-, et al.
(författare)
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Prediction of IgG glycosylation in CHO cell perfusion cultures by GReBA mathematical model supported by a novel targeted feed, TAFE
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The N-linked glycosylation pattern is an important quality attribute of therapeutic glycoproteins. It has been reported by our group and by others that different carbon sources, such as glucose, mannose and galactose, can differently impact the glycosylation profile of glycoproteins in mammalian cell culture. Acting on the sugar feeding is thus an attractive strategy to tune the glycan pattern. However, in case of feeding of more than one carbon source simultaneously, the cells give priority to the one with the highest uptake rate, which limits the usage of this tuning, e.g. the cells favor consuming glucose in comparison to galactose.We present here a new feeding strategy (named 'TAFE' for targeted feeding) for perfusion culture to adjust the concentrations of fed sugars influencing the glycosylation. The strategy consists in setting the sugar feeding such that the cells are forced to consume these substrates at a target cell specific consumption rate decided by the operator and taking into account the cell specific perfusion rate (CSPR). This strategy is applied in perfusion cultures of Chinese hamster ovary (CHO) cells, illustrated by ten different regimes of sugar feeding, including glucose, galactose and mannose. Applying the TAFE strategy, different glycan profiles were obtained using the different feeding regimes. Furthermore, we successfully forced the cells to consume higher proportions of non-glucose sugars, which have lower transport rates than glucose in presence of this latter, in a controlled way.In previous work, a mathematical model named Glycan Residues Balance Analysis (GReBA) was developed to model the glycosylation profile based on the fed carbon sources. The present data were applied to the GReBA to design a feeding regime targeting a given glycosylation profile. The ability of the model to achieve this objective was confirmed by a multi-round of leave-one- out cross-validation (LOOCV), leading to the conclusion, that the GReBA model can be used to design the feeding regime of a perfusion cell culture to obtain a desired glycosylation profile.
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| 12. |
- Zhang, Liang, 1990-, et al.
(författare)
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Probabilistic Graphic Model by Bayesian Network for the Prediction of Antibody Glycosylation in CHO Cell Cultures
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Glycosylation is a critical quality attribute of therapeutic monoclonal antibodies (mAbs). The glycan pattern can have a large impact on the immunological functions, serum half-life and stability. The medium components and cultivation parameters are known to potentially influence the glycosylation profile. Mathematical modelling provides a strategy for rational design and control of the upstream bioprocess. However, the kinetic models usually contain a very large number of unknown parameters, which limit their practical applications. In this article, we consider the metabolic network of N-linked glycosylation as a Bayesian network and calculate the fluxes of the glycosylation process as joint probability using the culture parameters as inputs. The modelling approach is validated with data of different CHO cell cultures in pseudo perfusion, perfusion and fed batch cultures, all showing very good predictive capacities. In cases where a large number of cultivation parameters is available, it is shown here that principal components analysis (PCA) can efficiently be employed for a dimension reduction of the inputs compared to Pearson correlation analysis and feature importance by decision tree. The present study demonstrates that Bayesian network model can be a powerful tool in upstream process and medium development for glycoprotein productions.
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