Publications by authors named "Veronique Chotteau"

30 Publications

  • Page 1 of 1

Design of an integrated continuous downstream process for acid-sensitive monoclonal antibodies based on a calcium-dependent Protein A ligand.

J Chromatogr A 2022 Jan 8;1664:462806. Epub 2022 Jan 8.

Department of Chemical Engineering, Lund University, SE-211 00 Lund, Sweden. Electronic address:

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 Z, was used in the capture step in a 3-column periodic counter-current chromatography operation. The binding of Z 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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http://dx.doi.org/10.1016/j.chroma.2022.462806DOI Listing
January 2022

Method development for mono- and disaccharides monitoring in cell culture medium by capillary and microchip electrophoresis.

Electrophoresis 2021 Sep 12. Epub 2021 Sep 12.

Kantisto BV, Baarn, The Netherlands.

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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http://dx.doi.org/10.1002/elps.202100213DOI Listing
September 2021

Knowing more from less: miniaturization of ligand-binding assays and electrophoresis as new paradigms for at-line monitoring and control of mammalian cell bioprocesses.

Curr Opin Biotechnol 2021 10 7;71:55-64. Epub 2021 Jul 7.

KTH Royal Institute of Technology, Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Stockholm, Sweden; AdBIOPRO, Competence Centre for Advanced BioProduction by Continuous Processing, KTH, Stockholm, Sweden. Electronic address:

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http://dx.doi.org/10.1016/j.copbio.2021.06.018DOI Listing
October 2021

Process intensification to produce a difficult-to-express therapeutic enzyme by high cell density perfusion or enhanced fed-batch.

Biotechnol Bioeng 2021 09 13;118(9):3533-3544. Epub 2021 May 13.

AdBIOPRO, Competence Centre for Advanced Bioproduction by Continuous Processing, Stockholm, Sweden.

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 × 10  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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http://dx.doi.org/10.1002/bit.27806DOI Listing
September 2021

Antibody capture process based on magnetic beads from very high cell density suspension.

Biotechnol Bioeng 2021 09 4;118(9):3499-3510. Epub 2021 May 4.

AdBIOPRO, VINNOVA Competence Centre for Advanced Bioproduction by Continuous Processing, Stockholm, Sweden.

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 × 10 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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http://dx.doi.org/10.1002/bit.27776DOI Listing
September 2021

Probabilistic model by Bayesian network for the prediction of antibody glycosylation in perfusion and fed-batch cell cultures.

Biotechnol Bioeng 2021 09 3;118(9):3447-3459. Epub 2021 May 3.

Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH-Royal Institute of Technology, Stockholm, Sweden.

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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http://dx.doi.org/10.1002/bit.27769DOI Listing
September 2021

Multiplexed Microfluidic Cartridge for At-Line Protein Monitoring in Mammalian Cell Culture Processes for Biopharmaceutical Production.

ACS Sens 2021 03 16;6(3):842-851. Epub 2021 Mar 16.

KTH Royal Institute of Technology, Division of Nanobiotechnology, Department of Protein Science, Science for Life Laboratory, 171 21 Solna, Sweden.

The biopharmaceutical market has been rapidly growing in recent years, creating a highly competitive arena where R&D is critical to strike a balance between clinical safety and profitability. Toward process optimization, the recent development and adoption of new process analytical technologies (PAT) highlight the dynamic complexity of mammalian/human cell culture processes, as well as the importance of fine-tuning and modeling key metabolites and proteins. In this context, simple, rapid, and cost-effective devices allowing routine at-line monitoring of specific proteins during process development and production are currently lacking. Here, we report the development of a versatile microfluidic protein analysis cartridge allowing the multiplexed bead-based immunodetection of specific proteins directly from complex mixtures with minimal hands-on time. Colorimetric quantification of Chinese hamster ovary (CHO) host cell proteins as key impurities, monoclonal antibodies as target biopharmaceuticals, and lactate dehydrogenase as a marker of cell viability was achieved with limits of detection in the 1-10 ng/mL range and analysis times as short as 30 min. The device was further demonstrated for the monitoring of a Rituximab-producing CHO cell bioreactor over the course of 8 days, providing comparable recoveries to standard enzyme-linked immunosorbent assay (ELISA) kits. The high sensitivity combined with robustness to matrix interference highlights the potential of the device to perform at-line measurements spanning from the bioreactor to the downstream processing.
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http://dx.doi.org/10.1021/acssensors.0c01884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034812PMC
March 2021

Author Correction: Evolution from adherent to suspension: systems biology of HEK293 cell line development.

Sci Rep 2021 Mar 2;11(1):5407. Epub 2021 Mar 2.

KTH ‑ School of Engineering Sciences in Chemistry, Biotechnology, and Health, Dept. of Protein Science, Royal Institute of Technology, 106 91, Stockholm, Sweden.

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http://dx.doi.org/10.1038/s41598-021-85105-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925574PMC
March 2021

Control of IgG glycosylation in CHO cell perfusion cultures by GReBA mathematical model supported by a novel targeted feed, TAFE.

Metab Eng 2021 05 5;65:135-145. Epub 2020 Nov 5.

Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH-Royal Institute of Technology, Sweden; AdBIOPRO, VINNOVA Competence Centre for Advanced Bioproduction by Continuous Processing, KTH, Sweden. Electronic address:

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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http://dx.doi.org/10.1016/j.ymben.2020.11.004DOI Listing
May 2021

Evolution from adherent to suspension: systems biology of HEK293 cell line development.

Sci Rep 2020 11 4;10(1):18996. Epub 2020 Nov 4.

KTH - School of Engineering Sciences in Chemistry, Biotechnology, and Health, Dept. of Protein Science, Royal Institute of Technology, 106 91, Stockholm, Sweden.

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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http://dx.doi.org/10.1038/s41598-020-76137-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642379PMC
November 2020

Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells.

iScience 2020 Nov 7;23(11):101653. Epub 2020 Oct 7.

KTH - Cell Technology Group (CETEG), Department of Industrial Biotechnology, 106 91, Stockholm, Sweden.

Human embryonic kidney cells HEK293 can be used for the production of therapeutic glycoproteins requiring human post-translational modifications. High cell density perfusion processes are advantageous for such production but are challenging due to the shear sensitivity of HEK293 cells. To understand the impact of hollow filter cell separation devices, cells were cultured in bioreactors operated with tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) at various flow rates. The average theoretical velocity profile in these devices showed a lower shear stress for ATF by a factor 0.637 compared to TFF. This was experimentally validated and, furthermore, transcriptomic evaluation provided insights into the underlying cellular processes. High shear caused cellular stress leading to apoptosis by three pathways, i.e. endoplasmic reticulum stress, cytoskeleton reorganization, and extrinsic signaling pathways. Positive effects of mild shear stress were observed, with increased recombinant erythropoietin production and increased gene expression associated with transcription and protein phosphorylation.
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http://dx.doi.org/10.1016/j.isci.2020.101653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593556PMC
November 2020

Model-based design and control of a small-scale integrated continuous end-to-end mAb platform.

Biotechnol Prog 2020 07 6;36(4):e2995. Epub 2020 Apr 6.

Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Royal Institute of Technology, Stockholm, Sweden.

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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http://dx.doi.org/10.1002/btpr.2995DOI Listing
July 2020

Assembly of FN-silk with laminin-521 to integrate hPSCs into a three-dimensional culture for neural differentiation.

Biomater Sci 2020 May 26;8(9):2514-2525. Epub 2020 Mar 26.

Dept. of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH-Royal Institute of Technology, SE-10691, Stockholm, Sweden.

Three-dimensional (3D) neural tissue cultures recapitulate the basic concepts during development and disease better than what can be obtained using conventional two-dimensional cultures. Here, we use a recombinant spider silk protein functionalized with a cell binding motif from fibronectin (FN-silk) in combination with a human recombinant laminin 521 (LN-521) to create a fully defined stem cell niche in 3D. A novel method to assemble silk blended with LN-521 together with human pluripotent stem cells (hPSC) is used to create centimeter-sized foams, which upon cultivation develop into 3D cell constructs supported by a microfibrillar network. After initial cell expansion, neural differentiation was induced to form a homogenous layer of continuous neuroectodermal tissue that allows further differentiation into neuronal subtypes. The silk-supported 3D cell constructs could then be detached from the bottom of the well and cultured as floating entities, where cells appeared in distinctive radial organization resembling early neural tube. This shows that the neural progenitors retain their cellular self-organization ability in the FN-silk/LN-521-supported 3D culture. Calcium imaging demonstrated spontaneous activity, which is important for the formation of neuronal networks. Together, the results show that hPSCs integrated into FN-silk/LN-521 foam develop into neural progenitors and that these stay viable during long-term differentiations. FN-silk/LN-521 also supports morphogenesis mimicking the human brain development and can serve as base for engineering of hPSC-derived neural tissue.
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http://dx.doi.org/10.1039/c9bm01624dDOI Listing
May 2020

Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin.

J Biotechnol 2020 Feb 28;309:44-52. Epub 2019 Dec 28.

School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Dept. of Industrial Biotechnology, Cell Technology Group (CETEG), Royal Institute of Technology (KTH), Stockholm, Sweden; Wallenberg Centre for Protein Research (WCPR), Stockholm, Sweden; Centre for Advanced Bioproduction by Continuous Processing (AdBIOPRO), Stockholm, Sweden. Electronic address:

Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 10 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 × 10 cells/mL) and high cell densities (80 × 10 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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http://dx.doi.org/10.1016/j.jbiotec.2019.12.017DOI Listing
February 2020

Glycan Residues Balance Analysis - GReBA: A novel model for the N-linked glycosylation of IgG produced by CHO cells.

Metab Eng 2020 01 17;57:118-128. Epub 2019 Sep 17.

Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH-Royal Institute of Technology, Sweden; AdBIOPRO, VINNOVA Competence Centre for Advanced Bioproduction by Continuous Processing, KTH, Sweden. Electronic address:

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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http://dx.doi.org/10.1016/j.ymben.2019.08.016DOI Listing
January 2020

Novel column generation-based optimization approach for poly-pathway kinetic model applied to CHO cell culture.

Metab Eng Commun 2019 Jun 25;8:e00083. Epub 2018 Dec 25.

Cell Technology Group, Department of Industrial Biotechnology/Bioprocess Design, School of Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden.

Mathematical modelling can provide precious tools for bioprocess simulation, prediction, control and optimization of mammalian cell-based cultures. In this paper we present a novel method to generate kinetic models of such cultures, rendering complex metabolic networks in a poly-pathway kinetic model. The model is based on subsets of elementary flux modes (EFMs) to generate macro-reactions. Thanks to our column generation-based optimization algorithm, the experimental data are used to identify the EFMs, which are relevant to the data. Here the systematic enumeration of all the EFMs is eliminated and a network including a large number of reactions can be considered. In particular, the poly-pathway model can simulate multiple metabolic behaviors in response to changes in the culture conditions. We apply the method to a network of 126 metabolic reactions describing cultures of antibody-producing Chinese hamster ovary cells, and generate a poly-pathway model that simulates multiple experimental conditions obtained in response to variations in amino acid availability. A good fit between simulated and experimental data is obtained, rendering the variations in the growth, product, and metabolite uptake/secretion rates. The intracellular reaction fluxes simulated by the model are explored, linking variations in metabolic behavior to adaptations of the intracellular metabolism.
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http://dx.doi.org/10.1016/j.mec.2018.e00083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6376161PMC
June 2019

Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture.

Biotechnol Prog 2019 05 30;35(3):e2775. Epub 2019 Jan 30.

AdBIOPRO, VINNOVA Competence Centre for Advanced BioProduction by Continuous Processing, Stockholm, Sweden.

High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding capacity of 65 mg/mL and an adsorption capacity of 25-42 mg IgG/mL bead in suspension for an IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were obtained in cell broth containing 40 × 10 cells/mL as in clarified supernatant. Two pilot-scale purification 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 protein A capture step, the mAb purity was similar to the one obtained by column chromatography, while the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient single 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 provide significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2775, 2019.
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http://dx.doi.org/10.1002/btpr.2775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617771PMC
May 2019

Combined effects of glycosylation precursors and lactate on the glycoprofile of IgG produced by CHO cells.

J Biotechnol 2019 Jan 10;289:71-79. Epub 2018 Nov 10.

Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH-Royal Institute of Technology, Sweden; AdBIOPRO, VINNOVA Competence Centre for Advanced Bioproduction by Continuous Processing, KTH, Sweden. Electronic address:

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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http://dx.doi.org/10.1016/j.jbiotec.2018.11.004DOI Listing
January 2019

High Cell Density Perfusion Culture has a Maintained Exoproteome and Metabolome.

Biotechnol J 2018 Oct 13;13(10):e1800036. Epub 2018 Jul 13.

Department Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology, and Health, KTH-Royal Institute of Technology, 106 91 Stockholm, Sweden.

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 ≥200 × 10  cells mL . 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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http://dx.doi.org/10.1002/biot.201800036DOI Listing
October 2018

Retraction notice to "Poly-pathway model, a novel approach to simulate multiple metabolic states by reaction network-based model - Application to amino acid depletion in CHO cell culture".

J Biotechnol 2018 Jan;265:127

Cell Technology Group, Department of Industrial Biotechnology/Bioprocess Design, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.

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http://dx.doi.org/10.1016/j.jbiotec.2017.11.013DOI Listing
January 2018

Poly-pathway model, a novel approach to simulate multiple metabolic states by reaction network-based model - Application to amino acid depletion in CHO cell culture.

J Biotechnol 2017 Oct 5;259:235-247. Epub 2017 Jul 5.

Cell Technology Group, Department of Industrial Biotechnology/Bioprocess Design, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden; AdBIOPRO, Competence Centre for Advanced BioProduction by Continuous Processing, Sweden. Electronic address:

Mammalian cell lines are characterized by a complex and flexible metabolism. A single model that could describe the variations in metabolic behavior triggered by variations in the culture conditions would be a precious tool in bioprocess development. In this paper, we introduce an approach to generate a poly-pathway model and use it to simulate diverse metabolic states triggered in response to removal, reduction or doubling of amino acids in the culture medium of an antibody-producing CHO cell line. Macro-reactions were obtained from a metabolic network via elementary flux mode enumeration and the fluxes were modeled by kinetic equations with saturation and inhibition effects from external medium components. Importantly, one set of kinetic parameters was estimated using experimental data of the multiple metabolic states. A good fit between the model and the data was obtained for the majority of the metabolites and the experimentally observed flux variations. We find that the poly-pathway modeling approach is promising for the simulation of multiple metabolic states.
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http://dx.doi.org/10.1016/j.jbiotec.2017.05.026DOI Listing
October 2017

Human embryonic stem cell dispersion in electrospun PCL fiber scaffolds by coating with laminin-521 and E-cadherin-Fc.

J Biomed Mater Res B Appl Biomater 2018 04 3;106(3):1226-1236. Epub 2017 Jun 3.

School of Biotechnology, Cell Technology Group (CETEG), KTH - Royal Institute of Technology, Stockholm, Sweden.

Advances in human pluripotent cell cultivation and differentiation protocols have led to production of stem cell-derived progenitors as a promising cell source for replacement therapy. Three-dimensional (3-D) culture is a better mimic of the natural niche for stem cells and is widely used for disease modeling. Here, we describe a nonaggregate culture system of human embryonic stem cells inside electrospun polycaprolactone (PCL) fiber scaffolds combined with defined extracellular proteins naturally occurring in the stem cell niche. PCL fiber scaffolds coated with recombinant human laminin-521 readily supported initial stem cell attachment and growth from a single-cell suspension. The combination of recombinant E-cadherin-Fc and laminin-521 further improved cell dispersion rendering a uniform cell population. Finally, we showed that the cells cultured in E-cadherin-Fc- and laminin-521-coated PCL scaffolds could differentiate into all three germ layers. Importantly, we provided a chemically defined 3-D system in which pluripotent stem cells grown and differentiated avoiding the formation of cell aggregates. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1226-1236, 2018.
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http://dx.doi.org/10.1002/jbm.b.33928DOI Listing
April 2018

RETRACTED: Poly-pathway model, a novel approach to simulate multiple metabolic states by reaction network-based model - Application to amino acid depletion in CHO cell culture.

J Biotechnol 2016 06 6;228:37-49. Epub 2016 Apr 6.

Cell Technology Group, Department of Industrial Biotechnology/Bioprocess Design, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). The authors of the paper wish to retract the paper due to the discovery of a calculation error in the processing of the raw data. The discovered error concerns the calculation of the specific uptake/secretion rates for several metabolites in one of the experimental conditions, i.e. glutamine omission (called Q0). In other words, in Figure 2, the variations of the metabolic fluxes for the condition Q0 are not correct. When this error is corrected, the resulting mathematical model changes (in particular for the results associated with Q0 conditions), several figures and tables are modified, and the interpretation of the fluxes in Q0 has to be slightly modified. Therefore the authors wish to retract the article. However, the error does not affect the modelling approach or the methodology presented in the article. Therefore, a revised version with the correct data has since been published: http://www.sciencedirect.com/science/article/pii/S0168165617302663. We apologize to the scientific community for the need to retract the article and the inconvenience caused.
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http://dx.doi.org/10.1016/j.jbiotec.2016.03.015DOI Listing
June 2016

Observation of Chinese Hamster Ovary Cells retained inside the non-woven fiber matrix of the CellTank bioreactor.

Data Brief 2015 Dec 20;5:586-8. Epub 2015 Oct 20.

School of Biotechnology, Department Industrial Biotechnology/Bioprocess Design, Cell Technology Group (CETEG), Royal Institute of Technology, KTH, SE-10691 Stockholm, Sweden.

This data article shows how the recombinant Chinese Hamster Ovary (CHO) cells are located in the interstices of the matrix fibers of a CellTank bioreactor after completion of a perfusion culture, supporting the article entitled "Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor" by Zhang et al. [1]. It provides a visualization of the cell distribution in the non-woven fiber matrix in a deeper view.
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http://dx.doi.org/10.1016/j.dib.2015.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773380PMC
December 2015

Robustness analysis of elementary flux modes generated by column generation.

Math Biosci 2016 Mar 31;273:45-56. Epub 2015 Dec 31.

Department of Mathematics, Optimization and Systems Theory, KTH Royal Institute of Technology, Stockholm SE-100 44, Sweden.

Elementary flux modes (EFMs) are vectors defined from a metabolic reaction network, giving the connections between substrates and products. EFMs-based metabolic flux analysis (MFA) estimates the flux over each EFM from external flux measurements through least-squares data fitting. The measurements used in the data fitting are subject to errors. A robust optimization problem includes information on errors and gives a way to examine the sensitivity of the solution of the EFMs-based MFA to these errors. In general, formulating a robust optimization problem may make the problem significantly harder. We show that in the case of the EFMs-based MFA, when the errors are only in measurements and bounded by an interval, the robust problem can be stated as a convex quadratic programming (QP) problem. We have previously shown how the data fitting problem may be solved in a column-generation framework. In this paper, we show how column generation may be applied also to the robust problem, thereby avoiding explicit enumeration of EFMs. Furthermore, the option to indicate intervals on metabolites that are not measured is introduced in this column generation framework. The robustness of the data is evaluated in a case-study, which indicates that the solutions of our non-robust problems are in fact near-optimal also when robustness is considered, implying that the errors in measurement do not have a large impact on the optimal solution. Furthermore, we showed that the addition of intervals on unmeasured metabolites resulted in a change in the optimal solution.
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http://dx.doi.org/10.1016/j.mbs.2015.12.009DOI Listing
March 2016

Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor.

J Biotechnol 2015 Nov 23;213:28-41. Epub 2015 Jul 23.

School of Biotechnology, Dept. Industrial Biotechnology/Bioprocess Design, Cell Technology Group (CETEG), Royal Institute of Technology, KTH, SE-10691 Stockholm, Sweden. Electronic address:

Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 × 10(6)viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 °C to 29 °C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 °C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.
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http://dx.doi.org/10.1016/j.jbiotec.2015.07.006DOI Listing
November 2015

On dynamically generating relevant elementary flux modes in a metabolic network using optimization.

J Math Biol 2015 Oct 17;71(4):903-20. Epub 2014 Oct 17.

Department of Mathematics, Optimization and Systems Theory, KTH Royal Institute of Technology, SE-100 44, Stockholm, Sweden,

Elementary flux modes (EFMs) are pathways through a metabolic reaction network that connect external substrates to products. Using EFMs, a metabolic network can be transformed into its macroscopic counterpart, in which the internal metabolites have been eliminated and only external metabolites remain. In EFMs-based metabolic flux analysis (MFA) experimentally determined external fluxes are used to estimate the flux of each EFM. It is in general prohibitive to enumerate all EFMs for complex networks, since the number of EFMs increases rapidly with network complexity. In this work we present an optimization-based method that dynamically generates a subset of EFMs and solves the EFMs-based MFA problem simultaneously. The obtained subset contains EFMs that contribute to the optimal solution of the EFMs-based MFA problem. The usefulness of our method was examined in a case-study using data from a Chinese hamster ovary cell culture and two networks of varied complexity. It was demonstrated that the EFMs-based MFA problem could be solved at a low computational cost, even for the more complex network. Additionally, only a fraction of the total number of EFMs was needed to compute the optimal solution.
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http://dx.doi.org/10.1007/s00285-014-0844-1DOI Listing
October 2015

Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor™. Part I. Effect of the cell density on the process.

Biotechnol Prog 2013 May-Jun;29(3):754-67. Epub 2013 May 21.

School of Biotechnology, Cell Technology Group, KTH (Royal Institute of Technology), SE-106 91, Stockholm, Sweden.

High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor™ using external hollow fiber filter as cell separation device. Both "classical" tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF- and ATF-based cultures was shown at 20-35 × 10(6) cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by-product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9-1.3 × 10(8) cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 × 10(8) cells/mL, achieved for the first time in a wave-induced bioreactor, and 1.32 × 10(8) cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO2 level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 × 10(8) cell/mL based on the analysis of the theoretical distance between the cells for the present cell line.
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http://dx.doi.org/10.1002/btpr.1704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3752962PMC
January 2014

Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE Bioreactor™-part II: Applications for antibody production and cryopreservation.

Biotechnol Prog 2013 May-Jun;29(3):768-77. Epub 2013 May 21.

School of Biotechnology, Cell Technology Group, KTH (Royal Institute of Technology), Stockholm, SE-10691, Sweden.

A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor™ using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed-batch were compared. Cell densities higher than 10(8) cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell-specific productivity was comparable at cell densities up to 1.3 × 10(8) cells/mL in perfusion and was comparable or lower in fed-batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed-batch and 28× more in a 1-month perfusion at 10(8) cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 × 10(8) and 10(8) cells/mL was performed using cells from a perfusion run at 10(8) cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing.
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http://dx.doi.org/10.1002/btpr.1703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3752935PMC
January 2014

Study of a recombinant CHO cell line producing a monoclonal antibody by ATF or TFF external filter perfusion in a WAVE Bioreactor™.

BMC Proc 2011 22;5 Suppl 8:P105. Epub 2011 Nov 22.

School of Biotechnology, Animal Cell Technology Group, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden.

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http://dx.doi.org/10.1186/1753-6561-5-S8-P105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285023PMC
April 2015
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