| 1. |
- Malm, Magdalena, 1983-, et al.
(author)
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Evolution from adherent to suspension: systems biology of HEK293 cell line development
- 2020
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
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Journal article (peer-reviewed)abstract
- The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.
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| 2. |
- Saghaleyni, Rasool, 1987, et al.
(author)
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Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells
- 2022
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In: Cell Reports. - : Elsevier BV. - 2211-1247. ; 39:11
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Journal article (peer-reviewed)abstract
- Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories.
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| 3. |
- Bastin, G., et al.
(author)
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Metabolic flux analysis of VERO cells under various culture conditions
- 2021
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In: Processes. - : MDPI AG. - 2227-9717. ; 9:12
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Journal article (peer-reviewed)abstract
- Although the culture of VERO cells in bioreactors is an important industrial bioprocess for the production of viruses and vaccines, surprisingly few reports on the analysis of the flux distribution in the cell metabolism have been published. In this study, an attempt is made to fill this gap by providing an analysis of relatively simple metabolic networks, which are constructed to describe the cell behavior in different culture conditions, e.g., the exponential growth phase (availability of glucose and glutamine), cell growth without glutamine, and cell growth without glucose and glutamine. The metabolic networks are kept as simple as possible in order to avoid underdeterminacy linked to the lack of extracellular measurements, and a unique flux distribution is computed in each case based on a mild assumption that the macromolecular composition of the cell is known. The result of this computation provides some insight into the metabolic changes triggered by the culture conditions, which could support the design of feedback control strategies in fed batch or perfusion bioreactors where the lactate concentration is measured online and regulated by controlling the delivery rates of glucose and, possibly, of some essential amino acids.
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| 4. |
- Brechmann, Nils A., et al.
(author)
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Antibody capture process based on magnetic beads from very high cell density suspension
- 2021
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In: Biotechnology and Bioengineering. - : John Wiley and Sons Inc. - 0006-3592 .- 1097-0290. ; 118:9, s. 3499-3510
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Journal article (peer-reviewed)abstract
- Cell clarification represents a major challenge for the intensification through very high cell density in the production of biopharmaceuticals such as monoclonal antibodies (mAbs). The present report proposes a solution to this challenge in a streamlined process where cell clarification and mAb capture are performed in a single step using magnetic beads coupled with protein A. Capture of mAb from non-clarified CHO cell suspension showed promising results; however, it has not been demonstrated that it can handle the challenge of very high cell density as observed in intensified fed-batch cultures. The performances of magnetic bead-based mAb capture on non-clarified cell suspension from intensified fed-batch culture were studied. Capture from a culture at density larger than 100 × 106 cells/ml provided an adsorption efficiency of 99% and an overall yield of 93% with a logarithmic host cell protein (HCP) clearance of ≈2–3 and a resulting HCP concentration ≤≈5 ppm. These results show that direct capture from very high cell density cell suspension is possible without prior processing. This technology, which brings significant benefits in terms of operational cost reduction and performance improvements such as low HCP, can be a powerful tool alleviating the challenge of process intensification.
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| 5. |
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| 6. |
- Brechmann, Nils Arnold, et al.
(author)
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Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture
- 2019
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In: Biotechnology progress (Print). - : AIChE. - 8756-7938 .- 1520-6033.
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Journal article (peer-reviewed)abstract
- High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the developmentof a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarifiedCHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum bindingcapacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for anIgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture stepfrom 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions wereobtained in cell broth containing 40 Å~ 106 cells/mL as in clarified supernatant. Two pilot-scalepurification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L,where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single proteinA capture step, the mAb purity was similar to the one obtained by column chromatography, whilethe host cell protein content was very low, <10 ppm. Our results showed that this magnetic beadmAb purification process, using a dedicated pilot-scale separation device, was a highly efficientsingle step, which directly connected the culture to the downstream process without cell clarification.Purification of mAb directly from non-clarified cell broth without cell separation can providesignificant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step.
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| 7. |
- Brechmann, Nils A., et al.
(author)
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Proof-of-Concept of a Novel Cell Separation Technology Using Magnetic Agarose-Based Beads
- 2022
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In: MAGNETOCHEMISTRY. - : MDPI AG. - 2312-7481. ; 8:3, s. 34-
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Journal article (peer-reviewed)abstract
- The safety of the cells used for Advanced Therapy Medicinal Products is crucial for patients. Reliable methods for the cell purification are very important for the commercialization of those new therapies. With the large production scale envisioned for commercialization, the cell isolation methods need to be efficient, robust, operationally simple and generic while ensuring cell biological functionality and safety. In this study, we used high magnetized magnetic agarose-based beads conjugated with protein A to develop a new method for cell separation. A high separation efficiency of 91% yield and consistent isolation performances were demonstrated using population mixtures of human mesenchymal stem cells and HER2(+) SKBR3 cells (80:20, 70:30 and 30:70). Additionally, high robustness against mechanical stress and minimal unspecific binding obtained with the protein A base conjugated magnetic beads were significant advantages in comparison with the same magnetic microparticles where the antibodies were covalently conjugated. This study provided insights on features of large high magnetized microparticles, which is promising for the large-scale application of cell purification.
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| 8. |
- Colin, Kevin, et al.
(author)
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Gaussian process modeling of macroscopic kinetics : a better-tailored kernel for Monod-type kinetics
- 2022
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In: 10th Vienna International Conference on Mathematical Modelling MATHMOD 2022 Vienna Austria, 27–29 July 2022. - : Elsevier BV. ; , s. 397-402
-
Conference paper (peer-reviewed)abstract
- In bioprocesses, it is important to model the kinetics of the macroscopic rates of reactions since these are required to catch the dynamical aspects of a process. In [Wang et al. 2020], a modeling method involving Gaussian processes has been developed, using a kernel especially designed for the modeling of Monod-type kinetics (activation, inhibition, double component, neutral effect). However, as will be illustrated in this paper, when the number of training data is limited or the metabolite concentration data do not have large variations (which is generally the case for real-life data), this kernel can yield inaccurate models for the kinetics. In this paper, we develop a new kernel better tailored for the modeling of Monod-type kinetics and we show that it has good modeling performances in the case of a limited number of data. The idea is to use the particular structure of Monod-type functions in the design of the kernel, i.e., we incorporate prior knowledge in the modeling.
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| 9. |
- Dewasme, Laurent, et al.
(author)
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Multivariable robust tube-based nonlinear model predictive control of mammalian cell cultures
- 2024
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In: Computers and Chemical Engineering. - : Elsevier BV. - 0098-1354 .- 1873-4375. ; 183
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Journal article (peer-reviewed)abstract
- In this paper, the application of a robust nonlinear model predictive control (NMPC) framework to mammalian cell cultures is proposed, dealing with possible large kinetic parameter uncertainties. Industrial constraints formulated in view of good manufacturing practice and quality-by-design approach are also considered, namely the assurance that all state trajectories are contained within a corridor defined by lower and upper safety bounds. The latter are assimilated to the well-known tube-based paradigm which is used to formulate the corresponding robust NMPC problem. Both classical and tube-based NMPC performances are assessed in numerical simulations where specific key-species are regulated while dealing with an uncertain plant model. The capability of the tube-based method to reduce the impact of the parameter variations on the state trajectories and the violation of the constraints is highlighted, suggesting the transfer of the method on a real pharmaceutical process.
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| 10. |
- Dewasme, L., et al.
(author)
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Practical data-driven modeling and robust predictive control of mammalian cell fed-batch process
- 2023
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In: Computers and Chemical Engineering. - : Elsevier BV. - 0098-1354 .- 1873-4375. ; 171
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Journal article (peer-reviewed)abstract
- Even if the performances of bioprocesses can be significantly improved by model-based control, there often remains a tradeoff between model complexity and control robustness. This paper proposes an original data -driven strategy for fast design of dynamic bioprocess models with minimal complexity (i.e., minimal number of bioreactions). Maximum likelihood principal component analysis (MLPCA) is applied to infer the minimal reaction scheme from a 25-state mammalian cell culture database. Then, a systematic algorithm is used to provide a continuous kinetic model formulation assuming all rates to occur simultaneously, which may be far from true cell metabolic conditions sometimes presenting discontinuous metabolic switches. A robust model predictive formulation is therefore adopted to reduce the impact of model structural uncertainty on the process performances. Additional numerical results show that the proposed strategy presents excellent performances in presence of unexpected metabolic switches.
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| 11. |
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| 12. |
- Gomis-Fons, Joaquín, et al.
(author)
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Model-based design and control of a small-scale integrated continuous end-to-end mAb platform
- 2020
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In: Biotechnology progress (Print). - : John Wiley and Sons Inc.. - 8756-7938 .- 1520-6033.
-
Journal article (peer-reviewed)abstract
- A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small-scale drug candidates. The process presented in this article is a proof-of-concept of a continuous end-to-end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model-based design and control. The downstream process, consisting of a periodic twin-column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real-time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4-log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained.
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| 13. |
- Hagrot, Erika, et al.
(author)
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Identification of experimentally relevant elementary flux mode subsets in a genome-scale metabolic network of CHO cell metabolism using column generation
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Other publication (other academic/artistic)abstract
- An elementary flux mode (EFM) is a stoichiometrically balanced pathway through a metabolic network that links extracellular substrates to products. For large and complex networks, finding all such pathways is a computational challenge due to the combinatorial explosion of possible modes. Herein, we show how a new algorithm based on the column generation optimization technique can be applied to efficiently identify small sets of pathways in a genome-scale metabolic network. We examine the metabolism of Chinese hamster ovary (CHO) cells by identifying pathways that are relevant to data obtained in a pseudo-perfusion cell culture experiment. Based on the identified pathways, we examine the intracellular metabolism behind the uptake and utilization of extracellular medium components for the biomass and mAb synthesis, and the generation of extracellular metabolic by-products.
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| 14. |
- Hagrot, Erika
(author)
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Macroscopic models of Chinese hamster ovary cell cultures
- 2019
-
Doctoral thesis (other academic/artistic)abstract
- Biopharmaceuticals treat a range of diseases, and is a growing sector within the pharmaceutical industry. A majority of these complex molecules are produced by genetically modified mammalian cells in large-scale cell cultures. Biopharmaceutical process development is costly and labor intensive, and has often been based on time-consuming empirical methods and trial-and-error. Mathematical modeling has great potential to speed up this work. A central question however, engaging researchers from various fields, is how to translate these complex biological systems into feasible and useful models.For biopharmaceutical production, macroscopic kinetic flux modeling has been proposed. This model type is derived from typical data obtained in the industry, and has been able to simulate cell growth and the uptake/secretion of important metabolites. Often, however, their scope is limited to specific culture conditions due to e.g. the lack of information on reaction kinetics, limited data sets, and simplifications to achieve calculability.In this thesis, the macroscopic kinetic model type is the starting point, but the goal is to capture a variety of culture conditions, as will be necessary for future applications in process optimization. The effects of varied availability of amino acids in the culture medium on cell growth, uptake/secretion of metabolites, and product secretion were studied in cell cultures.In Paper I, the established methodology of Metatool was tested: (i) a simplified metabolic network of approx. 30 reactions was defined; (ii) all possible so-called elementary flux modes (EFMs) through the network were identified using an established mathematical algorithm; and (iii) the effect on each flux was modelled by a simplified generalized kinetic equation. A limitation was identified; the Metatool algorithm could only handle simple networks, and therefore several reactions had to be discarded. In this paper, a new strategy for the kinetics was developed. A pool of alternative kinetic equations was created, from which a smaller set could be given higher weight as determined via data-fitting. This improved the simulations.The identification of EFMs was further studied in papers II–IV. In Paper II, a new algorithm was developed based on the column generation optimization technique, that in addition to the network also accounts for the data from one of the parallel cultures. The method identifies a subset of the EFMs that can optimally fit the data, even in more complex metabolic networks.In Paper III, a kinetic model based on EFM subsets in a 100 reaction network was generated, which further improved the simulations. Finally, in Paper IV, the algorithm was extended to EFM identification in a genome-scale network. Despite the high complexity, small subsets of EFMs relevant to the experimental data could be e ciently identified.
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| 15. |
- Ihling, Nina, et al.
(author)
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Scale‐down of CHO cell cultivation from shake flasks based on oxygen mass transfer allows application of parallelized, non‐invasive, and time‐resolved monitoring of the oxygen transfer rate in 48‐well microtiter plates
- 2023
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In: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 18:11
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Journal article (peer-reviewed)abstract
- Cultivating Chinese hamster ovary (CHO) cells in microtiter plates (MTPs) with time-resolved monitoring of the oxygen transfer rate (OTR) is highly desirable to provide process insights at increased throughput. However, monitoring of the OTR in MTPs has not been demonstrated for CHO cells, yet. Hence, a CHO cultivation process was transferred from shake flasks to MTPs to enable monitoring of the OTR in each individual well of a 48-well MTP. For this, the cultivation of an industrially relevant, antibody-producing cell line was transferred from shake flask to MTP based on the volumetric oxygen mass transfer coefficient (kLa). Culture behavior was well comparable (deviation of the final IgG titer less than 10%). Monitoring of the OTR in 48-well MTPs was then used to derive the cytotoxicity of dimethyl sulfoxide (DMSO) based on a dose–response curve in a single experiment using a second CHO cell line. Logistic fitting of the dose–response curve determined after 100 h was used to determine the DMSO concentration that resulted in a cytotoxicity of 50% (IC50). A DMSO concentration of 2.70% ± 0.25% was determined, which agrees with the IC50 previously determined in shake flasks (2.39% ± 0.1%). Non-invasive, parallelized, and time-resolved monitoring of the OTR of CHO cells in MTPs was demonstrated and offers excellent potential to speed up process development and assess cytotoxicity.
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| 16. |
- Ladd, Brian, et al.
(author)
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Proof-of-Concept of Continuous Transfection for Adeno-Associated Virus Production in Microcarrier-Based Culture
- 2022
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In: Processes. - : MDPI AG. - 2227-9717. ; 10:3
-
Journal article (peer-reviewed)abstract
- Adeno-associated virus vectors (AAV) are reported to have a great potential for gene therapy, however, a major bottleneck for this kind of therapy is the limitation of production capacity. Higher specific AAV vector yield is often reported for adherent cell systems compared to cells in suspension, and a microcarrier-based culture is well established for the culture of anchored cells on a larger scale. The purpose of the present study was to explore how microcarrier cultures could provide a solution for the production of AAV vectors based on the triple plasmid transfection of HEK293T cells in a stirred tank bioreactor. In the present study, cells were grown and expanded in suspension, offering the ease of this type of operation, and were then anchored on microcarriers in order to proceed with transfection of the plasmids for transient AAV vector production. This process was developed in view of a bioreactor application in a 200 mL stirred-tank vessel where shear stress aspects were studied. Furthermore, amenability to a continuous process was studied. The present investigation provided a proof-of-concept of a continuous process based on microcarriers in a stirred-tank bioreactor.
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| 17. |
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| 18. |
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| 19. |
- Malm, Magdalena, 1983-, et al.
(author)
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Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins
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Other publication (other academic/artistic)abstract
- Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify bottlenecks limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant overexpression. Surprisingly, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, genes with significant expression variation were identified. Among these, ATF4, SRP9, JUN, PDIA3 and HSPA8 were validated as productivity boosters in CHO. Further, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components improving recombinant protein yield in HEK293 and CHO.
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| 20. |
- Malm, Magdalena, 1983-, et al.
(author)
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Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins
- 2022
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In: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 171-187
-
Journal article (peer-reviewed)abstract
- Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.
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| 21. |
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| 22. |
- Mikkonen, Saara, et al.
(author)
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Capillary and microchip electrophoresis method development for amino acid monitoring during biopharmaceutical cultivation
- 2022
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In: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314.
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Journal article (peer-reviewed)abstract
- The increased use of biopharmaceuticals calls for improved means of bioprocess monitoring. In this work, capillary electrophoresis (CE) and microchip electrophoresis (MCE) methods were developed and applied for the analysis of amino acids (AAs) in cell culture supernatant. In samples from different days of a Chinese hamster ovary cell cultivation process, all 19 proteinogenic AAs containing primary amine groups could be detected using CE, and 17 out of 19 AAs using MCE. The relative concentration changes in different samples agreed well with those measured by high-performance liquid chromatography (HPLC). Compared to the more commonly employed HPLC analysis, the CE and MCE methods resulted in faster analysis, while significantly lowering both the sample and reagent consumption, and the cost per analysis.
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| 23. |
- Moradi, Mona, et al.
(author)
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Autophagy and intracellular product degradation genes identified by systems biology analysis reduce aggregation of bispecific antibody in CHO cells
- 2022
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In: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 68, s. 68-76
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Journal article (peer-reviewed)abstract
- Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of these products. Pairs of stable Chinese hamster ovary (CHO) cell lines produced two difficult-to-express bispecific antibodies with different levels of aggregated product (10-75% aggregate) in a miniaturised bioreactor system. Here, transcriptome analysis was used to interpret the biological causes for the aggregation and to identify strategies to improve product yield and quality. Differential expression-and gene set analysis revealed upregulated proteasomal degradation, unfolded protein response and autophagy processes to be correlated with reduced protein aggregation. Fourteen candidate genes with the potential to reduce aggregation were co expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AKT1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased titres of the anti-HER2 monoclonal antibody trastuzumab by 50% during transient expression. It is suggested that this approach could be of general use for defining aggregation bottlenecks in CHO cells.
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| 24. |
- Moradi, Mona, et al.
(author)
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Autophagy and intracellular product degradation genes reduce aggregation of bispecific antibody in CHO cells with a high translational burden
-
Other publication (other academic/artistic)abstract
- Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of the product. Pairs of stable Chinese hamster ovary cell lines produced two difficult- to-express bispecific antibodies with different levels of aggregated product (10-75% aggregate) in a miniaturized bioreactor system. Here, we analyse the cellular response and link to product aggregation by comparative transcriptome analysis of these CHO cells, to define biological causes and infer strategies to improve yield and quality. Differential expression- and gene set analysis revealed upregulated proteosomal degradation, unfolded protein response and autophagy processes to be correlated with reduction of protein aggregation. Fourteen candidate genes with potential to reduce aggregation were co-expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AK1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased trastuzumab titres by 50% each during transient expression. We suggest our approach to be of general use for defining aggregation bottlenecks in CHO.
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| 25. |
- Pappenreiter, Magdalena, et al.
(author)
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Product sieving of mAb and its high molecular weight species in different modes of ATF and TFF perfusion cell cultures
- 2023
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In: Journal of chemical technology and biotechnology (1986). - : Wiley. - 0268-2575 .- 1097-4660. ; 98:7, s. 1658-1672
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Journal article (peer-reviewed)abstract
- BACKGROUND: Tangential flow filtration (TFF) systems are widely used cell retention devices in perfusion cultures, but significant challenges occur with their prolonged operation. A well-known and common issue includes membrane fouling, which leads to reduced permeate flow and increased product retention. The fouling behavior in hollow fibers have been studied widely and the application of alternating flow profiles, cell lysis, shear stress, and membrane pore size have been found to be major contributors to this phenomenon. RESULTS: In this work, different process set ups and conditions were tested using magnetic levitation pumps for low shear TFF systems in small-scale perfusion bioreactors (200 mL). A novel concept based on the application of reverse flow across the hollow fiber using two magnetically levitating pumps was validated with a Chinese Hamster Ovary cells (CHO) cell line producing a recombinant monoclonal antibody. Product sieving could be improved by 30% when a dynamic recirculation flow was applied. Furthermore, minimal product retention was achieved by reversing the flow of two alternating pumps with short cycle times. Besides this, a correlation was found between the passage of high molecular weight species to the harvest stream of the perfusion process and the flow direction, as well as the degree of product sieving. CONCLUSION: TFF with a reverse flow is a valuable alternative to an alternating tangential flow (ATF) system for overcoming antibody retention and it can be used at various scales and at a constant bioreactor volume. The comparison of ATF and TFF showed differences in product yield and purity and is, therefore, an important point for process design.
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| 26. |
- Pasquini, Mirko, 1991-, et al.
(author)
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A Lyapunov based heuristic to speed up convergence of a feedback optimization framework with experiment batches-application to bioprocess manufacturing
- 2022
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In: IFAC PAPERSONLINE. - : Elsevier BV. - 2405-8963. ; , s. 135-140
-
Conference paper (peer-reviewed)abstract
- In this work a heuristic to speed up the convergence of a feedback-based optimization scheme, when experiments can be run in batches, is discussed. The proposed approach allows to select the most promising experiment in the batch, as the one maximising the decrease of an associated Lyapunov function, and to define the inputs for the next batch, based on this. We suggest the application of the scheme to a biological setting, with the goal of maximizing the concentration of a product of interest in a bioreactor under a continuous perfusion framework, while at the same time minimizing the yield of a toxic byproduct. The potential of the approach is exposed by means of a simple synthetic example.
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| 27. |
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| 28. |
- Pinto, Ines Fernandes, et al.
(author)
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Microfluidic Cartridge for Bead-Based Affinity Assays
- 2024
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In: Methods in Molecular Biology. - : Springer Nature. - 1064-3745 .- 1940-6029. ; 2804, s. 127-138
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Journal article (peer-reviewed)abstract
- Within the vast field of medical biotechnology, the biopharmaceutical industry is particularly fast-growing and highly competitive, so reducing time and costs associated to process optimization becomes instrumental to ensure speed to market and, consequently, profitability. The manufacturing of biopharmaceutical products, namely, monoclonal antibodies (mAbs), relies mostly on mammalian cell culture processes, which are highly dynamic and, consequently, difficult to optimize. In this context, there is currently an unmet need of analytical methods that can be integrated at-line in a bioreactor, for systematic monitoring and quantification of key metabolites and proteins. Microfluidic-based assays have been extensively and successfully applied in the field of molecular diagnostics; however, this technology remains largely unexplored for Process Analytical Technology (PAT), despite holding great potential for the at-line measurement of different analytes in bioreactor processes, combining low reagent/molecule consumption with assay sensitivity and rapid turnaround times.Here, the fabrication and handling of a microfluidic cartridge for protein quantification using bead-based affinity assays is described. The device allows geometrical multiplexed immunodetection of specific protein analytes directly from bioreactor samples within 2.5 h and minimal hands-on time. As a proof-of-concept, quantification of Chinese hamster ovary (CHO) host cell proteins (HCP) as key impurities, IgG as product of interest, and lactate dehydrogenase (LDH) as cell viability marker was demonstrated with limits of detection (LoD) in the low ng/mL range. Negligible matrix interference and no cross-reactivity between the different immunoassays on chip were found. The results highlight the potential of the miniaturized analytical method for PAT at reduced cost and complexity in comparison with sophisticated instruments that are currently the state-of-the-art in this context.
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| 29. |
- Rodrigues, Diogo, et al.
(author)
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An Integrated Approach for Modeling and Identification of Perfusion Bioreactors via Basis Flux Modes
- 2021
-
In: Computers and Chemical Engineering. - : Elsevier BV. - 0098-1354 .- 1873-4375. ; 149
-
Journal article (peer-reviewed)abstract
- This paper discusses the challenges associated with the reliable and optimal operation of perfusion bioreactors and presents methods for modeling and identification of perfusion bioreactors as well as the vision for their integration. After presenting ageneric model of perfusion bioreactors, the paper shows how to use the concept of basis flux modes to uniquely compute reaction rates. The advantage of this concept with respect to elementary flux nodes and similar concepts in metabolic flux analysis is the reduced number of flux modes that need to be modeled. In addition, a procedure to identify the model and estimate the parameters for each reaction using Monod-type kinetics is presented. It is shown that the rational structure of these kinetic models results in optimization problems that are amenable to tractable computation of globally optimal parameter estimates. The methods are illustrated via examples with simulated or experimental data.
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| 30. |
- Scheffel, Julia, et al.
(author)
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Design of an integrated continuous downstream process for acid-sensitive monoclonal antibodies based on a calcium-dependent Protein A ligand
- 2022
-
In: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1664, s. 462806-462806
-
Journal article (peer-reviewed)abstract
- Monoclonal antibodies (mAb) are used as therapeutics and for diagnostics of a variety of diseases, and novel antibodies are continuously being developed to find treatments for new diseases. Therefore, the manufacturing process must accommodate a range of mAb characteristics. Acid-sensitive mAbs can severely compromise product purity and yield in the purification process due to the potential formation of aggregates. To address this problem, we have developed an integrated downstream process for the purification of pH-sensitive mAbs at mild conditions. A calcium-dependent Protein A-based ligand, called ZCa, was used in the capture step in a 3-column periodic counter-current chromatography operation. The binding of ZCa to antibodies is regulated by calcium, meaning that acidic conditions are not needed to break the interaction and elute the antibodies. Further, the virus inactivation was achieved by a solvent/detergent method, where the pH could remain unchanged. The polishing steps included a cation and an anion exchange chromatography step, and screening of the capture and polishing steps was performed to allow for a seamless integration of the process steps. The process was implemented at laboratory scale for 9 days obtaining a high yield, and a consistently pure drug substance, including high reduction values of the host cell protein and DNA concentrations, as well as aggregate levels below the detection limit, which is attributed to the mild conditions used in the process.
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| 31. |
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| 32. |
- Schwarz, Hubert, et al.
(author)
-
Monitoring of amino acids and antibody N-glycosylation in high cell density perfusion culture based on Raman spectroscopy
- 2022
-
In: Biochemical engineering journal. - : Elsevier BV. - 1369-703X .- 1873-295X. ; 182, s. 108426-
-
Journal article (peer-reviewed)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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| 33. |
- Schwarz, Hubert, et al.
(author)
-
Optimization of medium with perfusion microbioreactors for high density CHO cell cultures at very low renewal rate aided by design of experiments
- 2023
-
In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 120:9, s. 2523-2541
-
Journal article (peer-reviewed)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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| 34. |
- Schwarz, Hubert, et al.
(author)
-
Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin
-
Other publication (other academic/artistic)abstract
- Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 108 cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be disadvantageous compared with human cell lines.In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). Firstly, a small-scale perfusion system from commercial bench-top screening bioreactors was developed for <250 mL working volume. Then, after the first trial runs with CHO cells, the system was modified for HEK293 cells (more sensitive than CHO cells) to achieve a higher oxygen transfer under mild aeration and agitation conditions. Steady states for medium (20 x 106 cells/mL) and high cell densities (80 x 106 cells/mL), normal process temperature (37 °C) and mild hypothermia (33 °C) as well as different cell specific perfusion rates (CSPR) from 10 to 60 pL/cell/day were applied to study the performance of the culture. The volumetric productivity was maximized for the high cell density steady state but decreased when an extremely low CSPR of 10 pL/cell/day was applied. The shift from high to low CSPR strongly reduced the nutrient uptake rates. The results from our study show that human cell lines, such as HEK293 can be used for intensified perfusion processes.
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| 35. |
- Särnlund, Sigrid, et al.
(author)
-
Process intensification to produce a difficult-to-express therapeutic enzyme by high cell density perfusion or enhanced fed-batch
- 2021
-
In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 118:9, s. 3533-3544
-
Journal article (peer-reviewed)abstract
- Intensified bioprocesses have caught industrial interest in the field of biomanufacturing in recent years. Thanks to new technology, intensified processes can support high cell densities, higher productivities and longer process times, which together can offer lower cost of goods. In this study two different intensified process modes, high cell density perfusion and enhanced fed-batch, were evaluated and compared with a conventional fed-batch process for a difficult-to-express therapeutic enzyme. The intensified process modes were cultivated with a target cell density of 100 × 106 cells/ml and with alternating tangential flow filtration, ATF, as cell retention device. The processes were designed to resemble an established optimized fed-batch process using the knowledge of this process without new dedicated optimization for the intensified modes. The design strategy included decision of the ratio of feed concentrate to base medium and glucose supplementation, which were based on target cell-specific consumption rates of key amino acids and glucose, using a targeted feeding approach (TAFE). A difficult-to-express therapeutic enzyme with multiple glycosylation sites was expressed and analyzed in the different production processes. The two new intensified processes both achieved 10 times higher volumetric productivity (mg/L/day) with retained protein quality and minor changes to the glycan profile compared to the fed-batch process. The study demonstrates the potential of using intensified processes for sensitive complex enzymes. It is shown here that it is possible to transfer a developed fed-batch process into high cell density processes either in intensified fed-batch or steady-state perfusion without new dedicated optimization. The results demonstrated as well that these intensified modes significantly increase the productivity while maintaining the desired product quality, for instance the same amount of product was obtained in 1 day during the perfusion process than in a whole fed-batch run. Without any prior optimization of the perfusion rate, the high cell density perfusion process resulted in only 1.2 times higher medium cost per gram produced protein.
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| 36. |
- van der Burg, D., et al.
(author)
-
Method development for mono- and disaccharides monitoring in cell culture medium by capillary and microchip electrophoresis
- 2022
-
In: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 43:9-10, s. 922-929
-
Journal article (peer-reviewed)abstract
- The rapidly growing, competitive biopharmaceutical market requires tight bioprocess monitoring. An integrated, automated platform for the routine online/at-line monitoring of key factors in the cell culture medium could greatly improve process monitoring. Mono- and disaccharides, as the main energy and carbon source, are one of these key factors. A CE-LIF method was developed for the analysis of several mono- and disaccharides, considering requirements and restrictions for analysis in an integrated, automated monitoring platform, such as the possibility for miniaturization to microchip electrophoresis. Analysis was performed after fluorescent derivatization with 8-aminopyrene-1,3,6-trisulfonic acid. The derivatisation reaction and the separation BGE were optimized using design of experiments. The developed method is applicable to the complex matrix of cell culture medium and proved transferable to microchip electrophoresis.
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| 37. |
- Wang, Mingliang, et al.
(author)
-
A multi-step least-squares method for nonlinear rational models
- 2019
-
In: Proceedings of the American Control Conference. - : Institute of Electrical and Electronics Engineers (IEEE). - 9781538679265 ; , s. 4509-4514
-
Conference paper (peer-reviewed)abstract
- Models rational in the parameters arise frequently in biosystems and other applications. As with all models that are non-linear in the parameters, direct parameter estimation, using e.g. nonlinear least-squares, can become challenging due to the issues of local minima and finding good initial estimates. Here we propose a multi-step least-squares method for a class of nonlinear rational models. The proposed method is applied to an extended Monod-type model. Numerical simulations indicate that the proposed method is consistent.
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| 38. |
- Wang, Mingliang, et al.
(author)
-
Estimation of Heteroscedastic Multilinear Systems
- 2020
-
In: Proceedings of the IEEE Conference on Decision and Control. - : Institute of Electrical and Electronics Engineers Inc.. ; , s. 2875-2880
-
Conference paper (peer-reviewed)abstract
- In this paper, we propose an estimation method for heteroscedastic multilinear systems. The system consists of a multilinear map of latent functions and an input-dependent noise process. We assume Gaussian-process priors on the unknowns to embed non-parametric models. This leads to a hierarchical model called heteroscedastic multilinear Gaussian processes which do not admit closed-form posterior and predictive distributions. The model is treated in an empirical Bayes fashion where the hyperparameters are estimated by maximizing the marginal distribution. To achieve that, we use a Monte Carlo expectation maximization method based on a Gibbs sampling algorithm. The predictive inference is also introduced where the mean is used as an approximation of the unknown functions. The performance of the proposed method is illustrated in a simulation study.
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| 39. |
- Wang, Mingliang, et al.
(author)
-
Unscented Bayes Methods for Hierarchical Gaussian Processes
- 2020
-
In: 2020 Australian and New Zealand control conference (ANZCC 2020). - : IEEE. ; , s. 137-142
-
Conference paper (peer-reviewed)abstract
- In this paper, we propose an unscented Bayes method for hierarchical Gaussian processes. The hierarchical Gaussian process consists of multiple layers of Gaussian process, which leads to intractable marginal likelihood and posterior distributions. Instead of resorting to the traditional sampling approach, we use the unscented transform to compute the intractable quantities in a hierarchical model, which allows us to optimize the hyperparameters using a gradient based approach and to obtain the predictive distributions. We develop the proposed approach to different application scenarios. The performance of the proposed method is validated in two experiments with comparison to the state-of-art methods.
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| 40. |
- Wang, Yu, et al.
(author)
-
Iterative learning robust optimization - with application to medium optimization of CHO cell cultivation in continuous monoclonal antibody production
- 2024
-
In: Journal of Process Control. - : Elsevier BV. - 0959-1524 .- 1873-2771. ; 137
-
Journal article (peer-reviewed)abstract
- In the presence of uncertainty, the optimum obtained based on a nominal identified model can neither provide any performance guarantee nor ensure that critical constraints are satisfied, which is crucial for e.g., bioprocess applications characterized by a high degree of complexity combined with costly experiments. Hence, uncertainty should be considered in the optimization and, furthermore, experiments designed to reduce the uncertainty most important for optimization. Herein, we propose a general framework that combines model-based robust optimization with optimal experiment design. The proposed framework can take advantage of prior knowledge in the form of a mechanistic model structure, and the importance of this is demonstrated by comparing it to more standard black-box models typically employed in learning. Through optimal experiment design, we repeatedly reduce the uncertainty most relevant for optimization so as to maximize the potential for improving the worst-case performance by balancing between exploration and exploitation. This makes the proposed method an efficient model-based robust optimization framework, especially in cases with limited experimental resources. The main part of the paper focuses on the case with modeling uncertainty that can be reduced with the availability of more experimental data. Towards the end of the paper, we consider extending the method to also include inherent uncertainty, such as input uncertainty and unmeasured disturbances. The effectiveness of the method is illustrated through a realistic simulation case study of medium optimization of Chinese hamster ovary cell cultivation in continuous monoclonal antibody production, where the metabolic network consists of 23 extracellular metabolites and 126 reactions.
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| 41. |
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| 42. |
- Wang, Yu, et al.
(author)
-
Robust optimization with optimal experiment design - with application to continuous biopharmaceutical production
- 2022
-
In: IFAC PAPERSONLINE. - : Elsevier BV. - 2405-8963. ; , s. 234-241
-
Conference paper (peer-reviewed)abstract
- Model-based optimization typically obtains the optimum based on a nominal identified model. However, in the presence of uncertainty, the nominal optimum leads to suboptimal operating conditions that furthermore can be highly sensitive to uncertainties. Hence, uncertainty should be considered in the optimization and, furthermore, experiments should be designed to reduce the uncertainty most important for optimization. Herein, we propose a general framework that combines model-based robust optimization with optimal experiment design. The proposed framework can take advantage of prior knowledge in the form of a mechanistic model structure, and the importance of this is demonstrated by comparing to more standard black-box models typically employed in learning. Through optimal experiment design, we repeatedly reduce uncertainties in the region where the likelihood of improvement on the worst-case performance is maximized This makes the proposed method an efficient model-based robust optimization framework, especially with limited experiment resources. The effectiveness of the method is illustrated by a cell culture development example in continuous biopharmaceutical production.
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| 43. |
- Yousefi-Darani, Abdolrahim, et al.
(author)
-
Generic Chemometric Models for Metabolite Concentration Prediction Based on Raman Spectra
- 2022
-
In: Sensors. - : MDPI AG. - 1424-8220. ; 22:15
-
Journal article (peer-reviewed)abstract
- Chemometric models for on-line process monitoring have become well established in pharmaceutical bioprocesses. The main drawback is the required calibration effort and the inflexibility regarding system or process changes. So, a recalibration is necessary whenever the process or the setup changes even slightly. With a large and diverse Raman dataset, however, it was possible to generate generic partial least squares regression models to reliably predict the concentrations of important metabolic compounds, such as glucose-, lactate-, and glutamine-indifferent CHO cell cultivations. The data for calibration were collected from various cell cultures from different sites in different companies using different Raman spectrophotometers. In testing, the developed "generic" models were capable of predicting the concentrations of said compounds from a dilution series in FMX-8 mod medium, as well as from an independent CHO cell culture. These spectra were taken with a completely different setup and with different Raman spectrometers, demonstrating the model flexibility. The prediction errors for the tests were mostly in an acceptable range (<10% relative error). This demonstrates that, under the right circumstances and by choosing the calibration data carefully, it is possible to create generic and reliable chemometric models that are transferrable from one process to another without recalibration.
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| 44. |
- Zamani, Leila, et al.
(author)
-
High Cell Density Perfusion Culture has a Maintained Exoproteome and Metabolome
- 2018
-
In: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 13:10
-
Journal article (peer-reviewed)abstract
- The optimization of bioprocesses for biopharmaceutical manufacturing by Chinese hamster ovary (CHO) cells can be a challenging endeavor and, today, heavily relies on empirical methods treating the bioreactor process and the cells as black boxes. Multi-omics approaches have the potential to reveal otherwise unknown characteristics of these systems and identify culture parameters to more rationally optimize the cultivation process. Here, the authors have applied both metabolomic and proteomic profiling to a perfusion process, using CHO cells for antibody production, to explore how cell biology and reactor environment change as the cell density reaches 200x10(6)cellsmL(-1). The extracellular metabolic composition obtained in perfusion mode shows a markedly more stable profile in comparison to fed-batch, despite a far larger range of viable cell densities in perfusion. This stable profile is confirmed in the extracellular proteosome. Furthermore, the proteomics data shows an increase of structural proteins as cell density increases, which could be due to a higher shear stress and explain the decrease in cell diameter at very high cell densities. Both proteomic and metabolic results shows signs of oxidative stress and changes in glutathione metabolism at very high cell densities. The authors suggest the methodology presented herein to be a powerful tool for optimizing processes of recombinant protein production.
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| 45. |
- Zhan, Caijuan, 1985-, et al.
(author)
-
Hydrodynamic shear stress in hollow filter for perfusion culture of human cells
-
Other publication (other academic/artistic)abstract
- High cell density perfusion process is an economical way to produce biopharmaceuticals at high yield. To achieve high density of healthy cells, the cell culture conditions should be free from mechanically detriment. Human embryonic kidney (HEK) K293 cells, interesting for the production of therapeutic glycoproteins, are known as shear sensitive. In order to obtain the optimal hydrodynamics conditions with reduced mechanical damage, we investigated the fact of the shear stress compatible with HEK293 cells. We reviewed hollow filter based tangential flow filtration strategies, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). We studied shear stress introduced by these two flow filtration methods. By theoretical study, we obtained that lower shear stress introduced by alternating tangential flow filtration result in lower average shear stress comparing to tangential flow filtration with same flow rate. In our experimental runs, we achieved different shear stress levels by applying different flow rates. 5-Days batch cultivations were performed to examine the influence of shear stress on cell growing and metabolic behaviour. We identified that the shear stress potentially reduce the growth rate and productivity of HEK293 cells and found the cell metabolism associated with shear stress levels. By documenting these cell responses to shear stress, we confirmed our theoretical results and could further optimize the hydrodynamic conditions for perfusion process of HEK 293 cells.
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| 46. |
- Zhan, Caijuan, 1985-
(author)
-
Hydrodynamics considerations in cells systems from ocean flow to perfusion cultivation process
- 2019
-
Doctoral thesis (other academic/artistic)abstract
- Microorganisms and animal cells are grown surrounded by fluid, which is providing them with nutrients and removing their waste products. In nature and industry processes, cells/microbes can be subject to aggressive environments, such as turbulent flow or shear flow. Hydrodynamics force generated in these flows can affect the distribution of cells/microbes and even lead to cell damage. Understanding the mechanism and exploring the effect of hydrodynamic force in these environments could make the prediction of cells’ hydrodynamic response more systematic. In pharmaceutical industry, perfusion process is recognized as an attractive option for biologics production due to its high productivity. However, there are still some challenges and limitations for further process improvement due to lack of information of cell response to hydrodynamic force and nutrients. In both cases, hydrodynamics plays an important role and similar tool can be used to achieve a deeper understanding of these processes. This thesis is mainly aiming to elucidate the influence of hydrodynamic forces on microorganisms or cells in nature and during bioprocesses. In particular, shear stress in a natural environment and in a bioreactor operated in perfusion mode is studied.This work mainly investigates hydrodynamics in nature and bioprocess including three flow cases. The first study investigates the effect of turbulence on marine life by performing direct numerical simulations (DNS) of motile micro-organisms in isotropic homogeneous turbulence. The clustering level of micro-organisms with one preferential swimming direction (e.g. gyrotaxis) is examined. The second study uses Computation Fluid Dynamics (CFD) to simulate the fluid flow inside a Wave bioreactor bag. The phenomenon of mixing, oxygen transfer rate and shear stress in nine different operating conditions of rocking speeds and angles are discussed. In the third study, the cellular response to shear force including growth and metabolism in a cell retention device such as hollow fiber filters during a perfusion process is analyzed. Theoretical calculations and experiment validation is performed to compare two filtration modes, tangential flow filtration (TFF) or alternating tangential flow filtration (ATF). Further optimizations regarding mixing and feeding are performed in a screening scale of in a perfusion system of stirred tank bioreactor with cell separation device.The main findings can be summarized as that spherical gyrotaxis swimmers show significant clustering, whereas prolate swimmers remain more uniformly distributed due to their large sensitivity to the local shear. These results could explain how pure hydrodynamic effects can alter the ecology of micro-organisms for instance by varying shape and their preferential orientation (paper I). The simulations of Wave bioreactors show that the mixing and shear stress increase with the rocking angle but that increasing rocking speeds are not systematically associated with increasing mixing and shear stress. A resonance phenomenon is responsible for the fact that the lowest studied rocking speed generates the highest fluid velocity, mixing and shear stress (paper II). Theoretical velocity profile-based calculations suggested a lower shear stress for ATF by a factor 0.637 compared to TFF. This is experimentally validated by cultures of HEK (human embryonic kidney) 293 cells subjected to shear stress by a perfusion system that affects growth and metabolism using these cell separation devices (paper III). Thanks to optimization of mixing and oxygen transfer in a screening system for perfusion process, very high cell densities above 100 x 106 cells/mL of mammalian cells were achieved (paper IV).
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| 47. |
- Zhang, Liang, et al.
(author)
-
Control of IgG glycosylation in CHO cell perfusion cultures by GReBA mathematical model supported by a novel targeted feed, TAFE
- 2021
-
In: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 65, s. 135-145
-
Journal article (peer-reviewed)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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| 48. |
- Zhang, Liang
(author)
-
Development of mathematical modelling for the glycosylation of IgG in CHO cell cultures
- 2020
-
Doctoral thesis (other academic/artistic)abstract
- Chinese hamster ovary (CHO) cells are the most popular expression system for the production of biopharmaceuticals. More than 80% of the approved monoclonal antibodies (mAbs) or immunoglobulin G (IgG) are produced with these cells. Glycosylation is a usual post- translational modification important for therapeutic mAbs. It affects their stability, half-life and immunological activities. Substantial studies have shown that glycosylation can be affected by the culture conditions in manufacturing, e.g. pH, temperature and media components. To achieve a good control of the glycosylation, a number of mathematical models have been developed. However, most of them have been developed for the cell line engineering, while very few can be used to design the media components for matching a given glycoprofile.This thesis presents developments of mathematical modelling for glycosylation prediction and experimental design of feeding different combinations of carbon sources in CHO cell cultures. The first study investigates the impacts of mannose, galactose, fructose and fucose to the IgG glycoprofile. Specifically, we look at intracellular nucleotide sugars in fed-batch cultures, where glucose is absent and lactate is used as complementary carbon source. The second study is based on the concept of elementary flux mode (EFM) and the mass balance of the glycan residues. A mathematical model named Glycan Residue Balance Analysis (GReBA) is developed for the prediction of the glycosylation profiles of IgG in pseudo perfusion cultures by feeding combinations of glucose, mannose, galactose and lactate. The model is further optimized for a feeding strategy design of perfusion cell cultures to obtain a desired glycoprofile. In the last study, a probabilistic graphic model based on Bayesian network (BN) is developed for glycosylation prediction in cultures under different multiple variable factors affecting the glycosylation.The results show that the manipulation of different sugars in the media can be used to control the glycosylation. Both the GReBA and PGM models exhibit abilities for glycosylation prediction and experimental design.
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| 49. |
- Zhang, Liang, et al.
(author)
-
Glycan Residues Balance Analysis : A novel model for the N-linked glycosylation of IgG produced by CHO cells.
- 2020
-
In: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 57, s. 118-128
-
Journal article (peer-reviewed)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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| 50. |
- Zhang, Liang, et al.
(author)
-
Probabilistic model by Bayesian network for the prediction of antibody glycosylation in perfusion and fed-batch cell cultures
- 2021
-
In: Biotechnology and Bioengineering. - : John Wiley and Sons Inc. - 0006-3592 .- 1097-0290. ; 118:9, s. 3447-3459
-
Journal article (peer-reviewed)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 (BN) 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 Chinese hamster ovary 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 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 BN model can be a powerful tool in upstream process and medium development for glycoprotein productions.
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