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.

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.

Optimization of medium with perfusion microbioreactors for high density CHO cell cultures at very low renewal rate aided by design of experiments.

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.

Integrated continuous biomanufacturing on pilot scale for acid-sensitive monoclonal antibodies.

In this study, we demonstrated the first, to our knowledge, integrated continuous bioprocess (ICB) designed for the production of acid-sensitive monoclonal antibodies, prone to aggregate at low pH, on pilot scale. A high cell density perfusion culture, stably maintained at 100 × 10  cells/ml, was integrated with the downstream process, consisting of a capture step with the recently developed Protein A ligand, Z ; a solvent/detergent-based virus inactivation; and two ion-exchange chromatography steps. The use of a mild pH in the downstream process makes this ICB suitable for the purification of acid-sensitive monoclonal antibodies.

Capillary and microchip electrophoresis method development for amino acid monitoring during biopharmaceutical cultivation.

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.

On convergence for hybrid models of gene regulatory networks under polytopic uncertainties: a Lyapunov approach.

Hybrid models of genetic regulatory networks allow for a simpler analysis with respect to fully detailed quantitative models, still maintaining the main dynamical features of interest. In this paper we consider a piecewise affine model of a genetic regulatory network, in which the parameters describing the production function are affected by polytopic uncertainties. In the first part of the paper, after recalling how the problem of finding a Lyapunov function is solved in the nominal case, we present the considered polytopic uncertain system and then, after describing how to deal with sliding mode solutions, we prove a result of existence of a parameter dependent Lyapunov function subject to the solution of a feasibility linear matrix inequalities problem.

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

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.

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

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.

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

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.

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

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.

An integrated continuous downstream process with real-time control: A case study with periodic countercurrent chromatography and continuous virus inactivation.

Integrated continuous downstream processes with process analytical technology offer a promising opportunity to reduce production costs and increase process flexibility and adaptability. In this case study, an integrated continuous process was used to purify a recombinant protein on laboratory scale in a two-system setup that can be used as a general downstream setup offering multiproduct and multipurpose manufacturing capabilities. The process consisted of continuous solvent/detergent virus inactivation followed by periodic countercurrent chromatography in the capture step, and a final chromatographic polishing step.

Optimal loading flow rate trajectory in monoclonal antibody capture chromatography.

In this paper, we determined the optimal flow rate trajectory during the loading phase of a mAb capture column. For this purpose, a multi-objective function was used, consisting of productivity and resin utilization. Several general types of trajectories were considered, and the optimal Pareto points were obtained for all of them.

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

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.

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

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.

Optimization study on periodic counter-current chromatography integrated in a monoclonal antibody downstream process.

An optimization study of an integrated periodic counter-current chromatography (PCC) process in a monoclonal antibody (mAb) downstream process at lab scale, is presented in this paper. The optimization was based on a mechanistic model of the breakthrough curve in the protein-A capture step. Productivity and resin utilization were the objective functions, while yield during the loading of the capture column was set as a constraint.

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

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.

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

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.

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

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.

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

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.

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.

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.