Morbidelli, Massimo*
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Continuous manufacturing is currently being seriously considered in the biopharmaceutical industry as the possible new paradigm for producing therapeutic proteins, due to production cost and product quality related benefits. In this study, a monoclonal antibody producing CHO cell line was cultured in perfusion mode and connected to a continuous affinity capture step. The reliable and stable integration of the two systems was enabled by suitable control loops, regulating the continuous volumetric flow and adapting the operating conditions of the capture process. For the latter, an at-line HPLC measurement of the harvest concentration subsequent to the bioreactor was combined with a mechanistic model of the capture chromatographic unit. Thereby, optimal buffer consumption and productivity throughout the process was realized while always maintaining a yield above the target value of 99%. Stable operation was achieved at three consecutive viable cell density set points (20, 60, and 40 × 106 cells/mL), together with consistent product quality in terms of aggregates, fragments, charge isoforms, and N-linked glycosylation. In addition, different values for these product quality attributes such as N-linked glycosylation, charge variants, and aggregate content were measured at the different steady states. As expected, the amount of released DNA and HCP was significantly reduced by the capture step for all considered upstream operating conditions. This study is exemplary for the potential of enhancing product quality control and modulation by integrated continuous manufacturing. Biotechnol. Bioeng. 2017;114: 298-307. © 2016 Wiley Periodicals, Inc.
- MeSH
- bioreaktory MeSH
- buněčné kultury metody MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- křečci praví MeSH
- monoklonální protilátky analýza izolace a purifikace metabolismus MeSH
- perfuze metody MeSH
- protein - isoformy chemie MeSH
- proteinové agregáty MeSH
- rekombinantní proteiny analýza izolace a purifikace metabolismus normy MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A macroporous particulate support prepared previously by reactive gelation under shear and functionalized with poly(N-isopropylacrylamide), PNIPAM, brushes of variable length is applied for temperature-responsive chromatography, whereby temperature modulates hydrophobic interactions. Several different analytes, including small pharmaceuticals, peptides, proteins and monoclonal antibodies are employed. Contrary to the most commonly observed behavior in conventional chromatography, increasing retention is observed at elevated temperatures. Peak broadening is quantified using the peak standard deviation, which depends on both the polymer chain conformation and analyte adsorptivity. The favorable effect of grafted polymer thickness on retention becomes progressively less pronounced for thicker grafted PNIPAM layers. The effect of eluent composition on solute-sorbent interactions was investigated by introducing NaCl, methanol, dioxane and by varying the pH. Salt or organic solvent addition affects apart from the analytes solution properties, the hydrophobicity of the stationary phase itself. Frontal analyses performed at different temperatures to determine dynamic binding capacities, indicate small mass transfer resistances imposed by this novel packing material.
N-linked glycosylation is known to be a crucial factor for the therapeutic efficacy and safety of monoclonal antibodies (mAbs) and many other glycoproteins. The nontemplate process of glycosylation is influenced by external factors which have to be tightly controlled during the manufacturing process. In order to describe and predict mAb N-linked glycosylation patterns in a CHO-S cell fed-batch process, an existing dynamic mathematical model has been refined and coupled to an unstructured metabolic model. High-throughput cell culture experiments carried out in miniaturized bioreactors in combination with intracellular measurements of nucleotide sugars were used to tune the parameter configuration of the coupled models as a function of extracellular pH, manganese and galactose addition. The proposed modeling framework is able to predict the time evolution of N-linked glycosylation patterns during a fed-batch process as a function of time as well as the manipulated variables. A constant and varying mAb N-linked glycosylation pattern throughout the culture were chosen to demonstrate the predictive capability of the modeling framework, which is able to quantify the interconnected influence of media components and cell culture conditions. Such a model-based evaluation of feeding regimes using high-throughput tools and mathematical models gives rise to a more rational way to control and design cell culture processes with defined glycosylation patterns. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1135-1148, 2016.
- MeSH
- biologické modely * MeSH
- bioreaktory MeSH
- časové faktory MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- glykosylace MeSH
- koncentrace vodíkových iontů MeSH
- kultivované buňky MeSH
- monoklonální protilátky chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Long-term continuous protein production can be reached by perfusion operation. Through the continuous removal of waste metabolites and supply of nutrients, steady-state (SS) conditions are achieved after a certain transient period, where the conditions inside the reactor are not only uniform in space but also constant in time. Such stable conditions may have beneficial influences on the reduction of product heterogeneities. In this study, we investigated the impact of perfusion cultivation on the intracellular physiological state of a CHO cell line producing a monoclonal antibody (mAb) by global transcriptomics and proteomics. Despite stable viable cell density was maintained right from the beginning of the cultivation time, productivity decrease, and a transition phase for metabolites and product quality was observed before reaching SS conditions. These were traced back to three sources of transient behaviors being hydrodynamic flow rates, intracellular dynamics of gene expression as well as metabolism and cell line instability, superimposing each other. However, 99.4% of all transcripts and proteins reached SS during the first week or were at SS from the beginning. These results demonstrate that the stable extracellular conditions of perfusion lead to SS also of the cellular level.
- MeSH
- buněčné kultury metody MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- glykosylace MeSH
- monoklonální protilátky analýza genetika MeSH
- perfuze metody MeSH
- proteom analýza genetika MeSH
- proteomika metody MeSH
- transkriptom * MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The pharmaceutical production of recombinant proteins, such as monoclonal antibodies, is rather complex and requires proper development work. Accordingly, it is essential to develop appropriate scale-down models, which can mimic the corresponding production scale. In this work, we investigated the impact of the bioreactor scale on intracellular micro-heterogeneities of a CHO cell line producing monoclonal antibodies in fed-batch mode, using a 10 mL micro-bioreactor (ambr™) scale-down model and the corresponding 300 L pilot-scale bioreactor. For each scale, we measured the time evolution of the proteome, which enabled us to compare the impact of the bioreactor scale on the intracellular processes. Nearly absolute accordance between the scales was verified by data mining methods, such as hierarchical clustering and in-detail analysis on a single protein base. The time response of principal enzymes related to N-glycosylation was discussed, emphasizing major dissimilarities between the glycan fractions adorning the heavy chain and the corresponding protein abundance. The enzyme expression displayed mainly a constant profile, whereas the resulting glycan pattern changed over time. It is concluded that the enzymatic activity is influenced by the changing environmental conditions present in the fed-batch processes leading to the observed time-dependent variation.
- MeSH
- biologické modely * MeSH
- bioreaktory * MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- glykosylace MeSH
- křečci praví MeSH
- monoklonální protilátky metabolismus MeSH
- proliferace buněk MeSH
- proteomika metody MeSH
- rekombinantní proteiny metabolismus MeSH
- shluková analýza MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
N-linked glycosylation is of key importance for the efficacy of many biotherapeutic proteins such as monoclonal antibodies (mAbs). Media components and cell culture conditions have been shown to significantly affect N-linked glycosylation during the production of glycoproteins using mammalian cell fed-batch cultures. These parameters inevitably change in modern industrial processes with concentrated feed additions and cell densities beyond 2 × 107 cells/mL. In order to control the time-dependent changes of protein glycosylation, an automated microbioreactor system was used to investigate the effects of culture pH, ammonia, galactose, and manganese chloride supplementation on nucleotide sugars as well as mAb N-linked glycosylation in a time-dependent way. Two different strategies comprising of a single shift of culture conditions as well as multiple media supplementations along the culture duration were applied to obtain changing and constant glycosylation profiles. The different feeding approaches enabled constant glycosylation patterns throughout the entire culture duration at different levels. By modulating the time evolution of the mAb glycan pattern, not only the endpoint but also the ratios between different glycosylation structures could be modified. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1123-1134, 2016.
Although several scaling bioreactor models of mammalian cell cultures are suggested and described in the literature, they mostly lack a significant validation at pilot or manufacturing scale. The aim of this study is to validate an oscillating hydrodynamic stress loop system developed earlier by our group for the evaluation of the maximum operating range for stirring, based on a maximum tolerable hydrodynamic stress. A 300-L pilot-scale bioreactor for cultivation of a Sp2/0 cell line was used for this purpose. Prior to cultivations, a stress-sensitive particulate system was applied to determine the stress values generated by stirring and sparging. Pilot-scale data, collected from 7- to 28-Pa maximum stress conditions, were compared with data from classical 3-L cultivations and cultivations from the oscillating stress loop system. Results for the growth behavior, analyzed metabolites, productivity, and product quality showed a dependency on the different environmental stress conditions but not on reactor size. Pilot-scale conditions were very similar to those generated in the oscillating stress loop model confirming its predictive capability, including conditions at the edge of failure.
The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a 13 C6 glucose containing feed media during constant operation at 20 × 106 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13 C6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, τST (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time τR (1 day) and characteristic time for glucose uptake τGlc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of 13 C6 glucose in the bioreactor and thus the intracellular 13 C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1630-1639, 2017.
- MeSH
- bioreaktory * MeSH
- buněčné kultury metody MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- glukosa metabolismus MeSH
- hydrodynamika MeSH
- izotopové značení metody MeSH
- křečci praví MeSH
- metabolismus * MeSH
- perfuze MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 106 cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 106 cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:879-890, 2017.
- MeSH
- bioreaktory * MeSH
- buněčné kultury * MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- kultivované buňky MeSH
- metabolom * MeSH
- monoklonální protilátky biosyntéza MeSH
- multivariační analýza MeSH
- perfuze * MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- tandemová hmotnostní spektrometrie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can conclude that the modulation of glycosylation in a sequential steady state approach in combination with mechanistic model represents an efficient and rational strategy to develop continuous processes with desired N-linked glycosylation patterns. Biotechnol. Bioeng. 2017;114: 1978-1990. © 2017 Wiley Periodicals, Inc.
- MeSH
- analýza selhání vybavení MeSH
- biologické modely * MeSH
- bioreaktory * MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- design s pomocí počítače MeSH
- design vybavení MeSH
- glykosylace MeSH
- monoklonální protilátky izolace a purifikace metabolismus MeSH
- perfuze přístrojové vybavení metody MeSH
- počítačová simulace MeSH
- polysacharidy metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
