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. 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.

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 × 106 cells/ml, was integrated with the downstream process, consisting of a capture step with the recently developed Protein A ligand, ZCa ; a solvent/detergent-based virus inactivation; and two ion-exchange chromatography steps. The use of a mild pH in the downstream process makes this ICB suitable for the purification of acid-sensitive monoclonal antibodies. Integration and automation of the downstream process were achieved using the Orbit software, and the same equipment and control system were used in initial small-scale trials and the pilot-scale downstream process. High recovery yields of around 90% and a productivity close to 1 g purified antibody/L/day were achieved, with a stable glycosylation pattern and efficient removal of impurities, such as host cell proteins and DNA. Finally, negligible levels of antibody aggregates were detected owing to the mild conditions used throughout the process. The present work paves the way for future industrial-scale integrated continuous biomanufacturing of all types of antibodies, regardless of acid stability.

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. 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.

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. 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.

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

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.

Hydrocyclones as cell retention device for CHO perfusion processes in single-use bioreactors.

In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50-L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low-viability late fed-batch culture, and viability was restored. The successive runs allowed optimization of the HC-bag configuration, and cultivations with 20-25 days duration at cell concentrations up to 50 × 106 cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor.

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. 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.

Rapid development of clone-specific, high-performing perfusion media from established feed supplements.

Perfusion cultivation of recombinant CHO cells is of substantial interest to the biopharmaceutical industry. This is due to increased space-time-yields (STYs) and a short residence time of the recombinant protein in the bioreactor. Economic processes rely on cultivation media supporting rapid growth in the exponential phase and high protein production in the stationary phase at minimal media consumption rates. To develop clone-specific, high-performing perfusion media we present a straightforward and rapid two-step approach combining commercially available basal media and feed supplements using design-of-experiment. First, the best performing feed supplements are selected in batch cultures. Then, the mixing ratio of selected feed supplements is optimized in small-scale semicontinuous perfusion cultures. The final media formulation is supported by statistical response surface modeling of a set of cultivation experiments with blended media formulations. Two best performing novel media blends were finally applied to perfusion bioreactor verification runs to reach 200 × 106 c/ml within 2 weeks at minimum cell-specific perfusion rates as low as 10-30 pL/c/d. Obtained STYs of 0.4-1.2 g/L/d represent a 10-fold increase compared to batch cultures. This general workflow is universally applicable to any perfusion platform combining a specific cell line, basal medium, and established feed solutions.

Repurposing fed-batch media and feeds for highly productive CHO perfusion processes.

Perfusion is a cell culture mode that is gaining popularity for the manufacture of monoclonal antibodies and their derivatives. The cell culture media supporting perfusion culture need to support higher cell densities than those used in fed-batch culture. Therefore, when switching from a fed-batch to a perfusion mode, a new medium need to be developed which supports high cell densities, high productivity, and favorable product quality. We have developed a method for deriving perfusion culture media based on existing fed-batch media and feeds. We show that we can obtain culture media that successfully support perfusion cultures in a single-use rocking bioreactor system at cell-specific perfusion rates below 25 pL-1 cell-1 day-1 . High productivities and favorable product quality are also achievable.

Bioprocessing of Recombinant CHO-K1, CHO-DG44, and CHO-S: CHO Expression Hosts Favor Either mAb Production or Biomass Synthesis.

Chinese hamster ovary (CHO) cells comprise a variety of lineages including CHO-DXB11, CHO-K1, CHO-DG44, and CHO-S. Despite all CHO cell lines sharing a common ancestor, extensive mutagenesis, and clonal selection has resulted in substantial genetic heterogeneity among them. Data from sequencing show that different genes are missing in individual CHO cell lines and each cell line harbors a unique set of mutations with relevance to the bioprocess. However, not much literature is available about the influence of genetic differences of CHO on the performance of bioprocess operations. In this study, the host cell-specific differences among three widely used CHO cell lines (CHO-K1, CHO-S, and CHO-DG44) and recombinantly expressed the same monoclonal antibody (mAb) in an isogenic format by using bacterial artificial chromosomes (BACs) as transfer vector in all cell lines is examined. Cell-specific growth and product formation are studied in batch, fed-batch, and semi-continuous perfusion cultures. Further, two different cell culture media are used to investigate their effects. The authors find CHO cell line-specific preferences for mAb production or biomass synthesis that are determined by the host cell line. Additionally, quality attributes of the expressed mAb are influenced by the host cell line and media.

Label-free independent quantitation of viable and non-viable cells using a multivariable multi-resonant sensor.

Biological cells are utilized for diverse biotechnological and bioengineering purposes ranging from the production of biopharmaceuticals, to cell therapy, "human-on-a-chip" drug and toxicology assays, and drug-resistance tests. In these and other applications, it is critical to quantify the levels of not only viable but also non-viable cells. While traditional off-line cell-staining methods are available for counting of non-viable cells, many applications cannot periodically remove cells for their off-line analysis because of the risk of contamination or workflow logistics. Here we show in-situ label-free quantitation of viable and non-viable cells with multivariable multi-resonant sensors. We used Chinese hamster ovary (CHO) cells in suspension culture in single-use bioreactors as a representative example. The resonant sensor design strategy permitted enhanced sensor sensitivity versus conventional non-resonant measurements and probed the spectral dispersion of viable and non-viable cells with multiple resonances. These capabilities of label-free in-situ analysis of cell viability can be attractive in diverse cell applications such as cell suspensions, adhered cells, and their 3D assemblages.

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. 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.

Differential gene expression of a feed-spiked super-producing CHO cell line.

Feed supplements are concentrated cell culture media that contain a variety of nutrients, which can be added during a bioprocess. During fed-batch cultivation, feed media are typically added to a growing cell culture to maximize cell and product concentrations. In this study, only a single shot of feed medium was added on day 0 to a basal cell culture medium and compared to non-supplemented basal medium (feed-spiked at day 0 versus control experiments) by cultivation of a recombinant mAb expressing CHO cell line in batch mode under controlled conditions in a bioreactor. Since the feed-spike at day 0 was based on existing medium components without introducing additional supplements, a desirable process with decreased complexity was generated. Unlike cells in basal medium, feed-spiked cultures reached almost 2× higher peak cell concentrations (10 × 106 c/mL vs. 18 × 106 c/mL) and 3× higher antibody concentrations (0.8 g/L vs. 2.4 g/L). Batch process time and the integral over the viable cell count were similar for both process types. Constantly high cell-specific production rates in feed-spiked cultures (70 pg/cell/day) compared to continuously declining rates in basal medium (from 70 to 10 pg/cell/day) were responsible for an overall 70% higher cell-specific production rate and the higher product concentrations. To associate gene expression patterns to different process proceedings, transcriptome analysis was performed using microarrays. Several transcripts that are involved with glutamine de novo synthesis and citric acid cycle were significantly upregulated on several days in feed-spiked cultures. The top identified gene ontology (GO) terms related well to cell cycle and primary metabolism, cellular division as well as nucleobase formation or regulation, which indicated a more active proliferative state for feed-spiked cultures. KEGG biochemical pathway analysis and Gene set enrichment analysis (GSEA) further confirmed these findings from a complementary perspective. Moreover, several interesting gene targets, which have not yet been associated with recombinant protein expression, were identified that related to a higher proliferative state, growth, protein synthesis, cell-size control, metabolism, cell survival as well as genes that are associated with the control of the mammalian target of rapamycin (mTOR) in feed-spiked cultures. Analysis of critical product quality attributes (i.e. glycosylation, charge variants and size distribution) showed that feed-spiking did not change antibody quality.

Biphasic cultivation strategy to avoid Epo-Fc aggregation and optimize protein expression.

In biphasic cultivations, the culture conditions are initially kept at an optimum for rapid cell growth and biomass accumulation. In the second phase, the culture is shifted to conditions ensuring maximum specific protein production and the protein quality required. The influence of specific culture parameters is cell line dependent and their impact on product quality needs to be investigated. In this study, a biphasic cultivation strategy for a Chinese hamster ovary (CHO) cell line expressing an erythropoietin fusion protein (Epo-Fc) was developed. Cultures were run in batch mode and after an initial growth phase, cultivation temperature and pH were shifted. Applying a DoE (Design of Experiments) approach, a fractional factorial design was used to systematically evaluate the influence of cultivation temperature and pH as well as their synergistic effect on cell growth as well as on recombinant protein production and aggregation. All three responses were influenced by the cultivation temperature. Additionally, an interaction between pH and temperature was found to be related to protein aggregation. Compared with the initial standard conditions of 37°C and pH 7.05, a parameter shift to low temperature and acidic pH resulted in a decrease in the aggregate fraction from 75% to less than 1%. Furthermore, the synergistic effect of temperature and pH substantially lowered the cell-specific rates of glucose and glutamine consumption as well as lactate and ammonium production. The optimized culture conditions also led to an increase of the cell-specific rates of recombinant Epo-Fc production, thus resulting in a more economic bioprocess.

Influence of bioreactor scale and complex medium on probing control of glucose feeding in cultivations of recombinant strains of Escherichia coli.

The objective of this work was to evaluate the performance of a feedback glucose control strategy (the probing strategy) in production relevant bioreactors with complex and mineral media. Experimental results from fed-batch cultivations with two recombinant Escherichia coli constructs expressing two different human therapeutic proteins were used to assess the performance and limitations of the glucose probing technique. Even though the performance of the probing strategy was affected by scale and complex media, this methodology rapidly identified a glucose feed protocol similar to an experimentally derived feed regime. This methodology may serve as a powerful tool for industrial process development and in optimization of glucose feed regimes when transferring process technology from one bioreactor system to another.

Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli.

Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process. Analysis of the concentration of DNA in the medium revealed that the release of DNA coincided with the accumulation of formate. It was found that increasing concentrations of DNA correlated in almost linearly increasing concentrations of formate. Formate accumulation is caused by mixed acid fermentation, although no oxygen limitation was measured with the DO electrode. It is proposed that extracellular DNA restrained mass transfer between the bulk medium and the cell. To investigate if the DNA accumulation caused formate production, DNA was removed by continuous feeding of a DNA binding polymer to the medium. The addition of the polymer decreased the content of free DNA in the broth and the formate was reassimilated. Furthermore, additional DNA early in the process resulted in early formate accumulation.