Mammalian Cell Culture Processes Research Articles

Proteome analysis of antibody‐producing CHO cell lines with different metabolic profiles

Two-dimensional gel electrophoresis and tandem mass spectrometry were used to identify proteins associated with a metabolic shift during fed-batch cultures of two recombinant antibody-producing CHO cell lines. The first cell line underwent a marked change in lactate metabolism during culture, initially producing lactate and then consuming it, while the second cell line produced lactate for a similar duration but did not later consume it. The first cell line displayed a declining specific antibody productivity during culture, correlating to the 2-D gel results and the intracellular antibody concentration determined by HPLC. Several statistical analysis methods were compared during this work, including a fixed fold-change criterion and t-tests using standard deviations determined in several ways from the raw data and mathematically transformed data. Application of a variance-stabilizing transformation enabled the use of a global empirical standard deviation in the t-tests. Most of the protein spots changing in each cell line did not change significantly in the other cell line. A substantial fraction of the changing proteins were glycolytic enzymes; others included proteins related to antibody production, protein processing, and cell structure. Enolase, pyruvate kinase, BiP/GRP78, and protein disulfide isomerase were found in spots that changed over time in both cell lines, and some protein changes differed from previous reports. These data provide a foundation for future investigation of metabolism in industrially relevant mammalian cell culture processes, and suggest that along with differences between cell types, the proteins expressed in cultures with low lactate concentrations may depend on how those conditions were generated.

Proteomic profiling of recombinant cells from large-scale mammalian cell culture processes

Global expression profiling of mammalian cells used for the production of biopharmaceuticals will allow greater insights into the molecular mechanisms that result in a high producing cellular phenotype. These studies may give insights for genetic intervention to possibly create better host cell lines or even to provide clues to more rational strategies for cell line and process development. In this review I will focus on the contribution of proteomic technologies to a greater understanding of the biology of Chinese hamster ovary cells and other producing cell lines such as NS0 mouse cells.

Non-invasive flow cytometric monitoring of pH i in cell culture processes using EGFP

We have described a new method for monitoring of cell culture processes using green fluorescent protein (GFP) fluorescent intensity. GFP has been used as a non-invasive fluorescent reporter for various cellular processes. In this study, enhanced (EGFP) was found to be a very sensitive indicator of pH i in in vitro cell culture, and responded rapidly to extracellular pH (pH e) changes. EGFP transfected cells were evaluated for pH i changes by flow cytometry, by measuring EGFP fluorescent intensity, and compared to that of the pH-sensitive fluoroprobe, SNARF. EGFP intensity was found to reflect pH i values of cells at different pH e in the presence of nigericin and was affected by the addition of HCl and NaOH. Significant changes in pH i were detected at different stages of batch culture and when using different cell density and media composition. The EGFP assay can be used to minimise the perturbation of cells and processes under study, thus leading to accurate information about the physiological state of single cells in a population. The results establish the application of EGFP as a non-invasive indicator of pH i for monitoring of mammalian cell culture processes.

Validation of TSE Agent Removal by Immunoaffinity and Ion Exchange Chromatography Steps of the ReFacto Manufacturing Process.

ReFacto® (moroctocog alfa), a recombinant Factor VIII approved for treatment of hemophilia A is produced by a mammalian cell culture process that includes therapeutic-grade human serum albumin (HSA) in the cell culture medium. The plasma-derived HSA used in the cell culture process is sourced from countries whose blood supply does not pose a significant transmissible spongiform encephalopathy (TSE) risk, and is manufactured utilizing a fractionation process that has demonstrated the capability to significantly reduce the level of experimentally added TSE infectivity. While to date, no case of TSE has been identified with a history of exposure to fractionated HSA, Wyeth has conducted a study to demonstrate that the ReFacto manufacturing process has significant capacity to remove a TSE agent if it were present in the HSA. The ReFacto purification process consists of five chromatographic steps, two of which were validated for removal of a TSE agent: the immunoaffinity step (8A4 Sepharose chromatography) and anion exchange step (Q Sepharose chromatography). The GLP study was performed using appropriately qualified lab-scale systems, which model the performance of the production equipment. Brain homogenates from scrapie-infected hamsters (263K strain) were added to loads of the respective chromatography steps and levels of the scrapie agent in the product pools were determined using a validated Western blot quantitation method. Duplicate runs were performed to assess the consistency of the TSE agent reduction. The results show that both chromatography steps provide significant clearance of the TSE agent, with greater than a billion-fold (9 log) reduction in infectivity provided by the two process steps in combination. The consistency of the duplicate run results was very good (less than 0.7 log difference between runs). While the risk of TSE contamination of HSA is only theoretical, these data provide a high degree of assurance that in the event of an adventitious TSE contamination of the HSA used in the ReFacto cell culture process, the purification steps would remove the infectious agent to safe levels.

Modelling of Mammalian cells and cell culture processes.

Mammalian cell cultures represent the major source for a number of very high-value biopharmaceutical products, including monoclonal antibodies (MAbs), viral vaccines, and hormones. These products are produced in relatively small quantities due to the highly specialised culture conditions and their susceptibility to either reduced productivity or cell death as a result of slight deviations in the culture conditions. The use of mathematical relationships to characterise distinct parts of the physiological behaviour of mammalian cells and the systematic integration of this information into a coherent, predictive model, which can be used for simulation, optimisation, and control purposes would contribute to efforts to increase productivity and control product quality. Models can also aid in the understanding and elucidation of underlying mechanisms and highlight the lack of accuracy or descriptive ability in parts of the model where experimental and simulated data cannot be reconciled. This paper reviews developments in the modelling of mammalian cell cultures in the last decade and proposes a future direction - the incorporation of genomic, proteomic, and metabolomic data, taking advantage of recent developments in these disciplines and thus improving model fidelity. Furthermore, with mammalian cell technology dependent on experiments for information, model-based experiment design is formally introduced, which when applied can result in the acquisition of more informative data from fewer experiments. This represents only part of a broader framework for model building and validation, which consists of three distinct stages: theoretical model assessment, model discrimination, and model precision, which provides a systematic strategy from assessing the identifiability and distinguishability of a set of competing models to improving the parameter precision of a final validated model.

Gas sensor arrays for early detection of infection in mammalian cell culture

The detection of bacterial infections in a mammalian cell culture process is realised using a gas sensor array. In production-scale and laboratory-scale cultivations of a perfused recombinant CHO-cell culture producing human blood coagulation Factor VIII, we show that the gas sensor array identifies bacterial contamination earlier than conventional methods. The sensitivity of the instrument is verified by inoculation of a blank cell culture medium with defined bacterial cell counts.

Measurement and control of dissolved carbon dioxide in mammalian cell culture processes using an in situ fiber optic chemical sensor.

At high viable cell concentrations in large-scale mammalian cell culture processes, the accumulation of dissolved carbon dioxide (dCO(2), typically quantified as an equilibrium gas-phase concentration) becomes problematic as a result of low CO(2) removal rates at reduced surface-to-volume ratios. High dCO(2) concentrations have previously been shown to inhibit cell growth and product formation in mammalian cells and to alter the glycosylation pattern of recombinant proteins. Therefore, reliable monitoring and control of dCO(2) are important for successful large-scale operation. Off-line measurements by instruments such as blood gas analyzers (BGA) are constrained by the low frequency of data collection and cannot be used for on-line control. In a preliminary evaluation of the YSI 8500 in situ sensor, a response time (t(90%)) of 6 min, sensitivity of 0.5% CO(2) (3.6 mmHg), and linearity of measurement (R(2) = 0.9997) between the equivalent gas-phase partial pressure of 0-180 mmHg (0% and 25% CO(2)) were established. Measurements were found to be unaffected by culture pH and typical mammalian cell culture concentrations of glucose, glutamine, glutamate, lactate, and ammonium. The sensor withstood repeated sterilization and cleaning cycles. The reliability of this sensor was demonstrated in microcarrier-based Chinese hamster ovary (CHO) cell perfusion cultures at reactor scales of 30, 40, 340, and 2000 L and was successfully implemented in a dCO(2) control strategy using N(2) sparging.

A tool for modeling strategic decisions in cell culture manufacturing.

The development of a prototype tool for modeling manufacturing in a biopharmaceutical plant is discussed. A hierarchical approach to modeling a manufacturing process has been adopted to confer maximum user flexibility. The use of this framework for assessing the impact of manufacturing decisions on strategic technical and business indicators is demonstrated via a case study. In the case study, which takes the example of a mammalian cell culture process delivering a therapeutic for clinical trials, the dynamic modeling tool indicates how manufacturing options affect the demands on resources and the associated manufacturing costs. The example illustrates how the decision-support software can be used by biopharmaceutical companies to investigate the effects of working toward different strategic goals on the cost-effectiveness of the process, prior to committing to a particular option.

Monitoring of intracellular ribonucleotide pools is a powerful tool in the development and characterization of mammalian cell culture processes

Efficient cell culture process development for the industrial production of recombinant therapeutics is characterized by constraints which pertain to issues such as costs, competitiveness and the meeting of project timelines. These constraints require tools which can help the developer learn as much as possible as quickly as possible about the cell at hand and identify features of a particular culture which are amenable to improvement. Current on- and off-line monitoring parameters, however useful, provide only late indications (cell concentration, viability) and circumstantial evidence (lactate, ammonia, etc.) with regard to the physiologic status of cells at the time of sampling. The relative intracellular content of purine to pyrimidine nucleotide triphosphates as well as the ratio of UTP to UDP-N-acetylhexosamines have been previously described as sensitive indicators of a cell's metabolic status, growth potential, and overall physiological condition. The sensitivity of such nucleotide ratios and their usefulness in commercially relevant process development and characterization were tested at Boehringer Ingelheim Pharma KG in a large number of fermentations (>80) with a variety of culture modes, cells, and products in scales up to 10,000 litres. Monitoring of these intracellular parameters allows a timely and reliable assessment of cell state and growth potential, which is possible neither by classical cell number and viability measurements nor by a variety of fermentation data typically monitored. The view inside the cell afforded by nucleotide monitoring enables prediction of the behavior of a culture up to 2 days before any hint of physiological changes is given by cell number and viability estimation. In this paper, data relating the growth behavior of CHO and hybridoma cell lines to their nucleotide pools are shown. Two very different processes for the production of recombinant tPA in 10,000-litre bioreactors are compared and characterized with respect to their nucleotide profiles. Examples from industrial process development cases in which intracellular nucleotide information is used to advantage are also presented and discussed. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 357–367, 1999.

Monitoring of intracellular ribonucleotide pools is a powerful tool in the development and characterization of mammalian cell culture processes.

Efficient cell culture process development for the industrial production of recombinant therapeutics is characterized by constraints which pertain to issues such as costs, competitiveness and the meeting of project timelines. These constraints require tools which can help the developer learn as much as possible as quickly as possible about the cell at hand and identify features of a particular culture which are amenable to improvement. Current on- and off-line monitoring parameters, however useful, provide only late indications (cell concentration, viability) and circumstantial evidence (lactate, ammonia, etc.) with regard to the physiologic status of cells at the time of sampling. The relative intracellular content of purine to pyrimidine nucleotide triphosphates as well as the ratio of UTP to UDP-N-acetylhexosamines have been previously described as sensitive indicators of a cell's metabolic status, growth potential, and overall physiological condition. The sensitivity of such nucleotide ratios and their usefulness in commercially relevant process development and characterization were tested at Boehringer Ingelheim Pharma KG in a large number of fermentations (>80) with a variety of culture modes, cells, and products in scales up to 10,000 litres. Monitoring of these intracellular parameters allows a timely and reliable assessment of cell state and growth potential, which is possible neither by classical cell number and viability measurements nor by a variety of fermentation data typically monitored. The view inside the cell afforded by nucleotide monitoring enables prediction of the behavior of a culture up to 2 days before any hint of physiological changes is given by cell number and viability estimation. In this paper, data relating the growth behavior of CHO and hybridoma cell lines to their nucleotide pools are shown. Two very different processes for the production of recombinant tPA in 10,000-litre bioreactors are compared and characterized with respect to their nucleotide profiles. Examples from industrial process development cases in which intracellular nucleotide information is used to advantage are also presented and discussed.

Regulated multicistronic expression technology for mammalian metabolic engineering

Contemporary basic research is rapidly revealing increasingly complex molecular regulatory networks which are often interconnected via key signal integrators. These connections among regulatory and catalytic networks often frustrate bioengineers as promising metabolic engineering strategies are bypassed by compensatory metabolic responses or cause unexpected, undesired outcomes such as apoptosis, product protein degradation or inappropriate post- translational modification. Therefore, for metabolic engineering to achieve greater success in mammalian cell culture processes and to become important for future applications such as gene therapy and tissue engineering, this technology must be enhanced to allow simultaneous, in cases conditional, reshaping of metabolic pathways to access difficult-to-attain cell states. Recent advances in this new territory of multigene metabolic engineering are intimately linked to the development of multicistronic expression technology which allows the simultaneous, and in some cases, regulated expression of several genes in mammalian cells. Here we review recent achievements in multicistronic expression technology in view of multigene metabolic engineering.

Fermenter balancing for semi-continuous, multi-tank mammalian cell culture processes

Abstract Process design and scheduling for optimal economic return from semi-continuous, multi-stage mammalian cell culture processes are considered. Heuristics are identified for the optimization of the production phase duration for processes that display linear accumulation of a monoclonal antibody. These heuristics are used to gauge the validity of the commonly accepted process design assumption that harvest titre be maximized irrespective of the fermenter configuration. The heuristics are further tested against performance predictions for non-linear cell culture data. We show that, in general, the choice of production schedules that result in a balanced utilization of fermentation vessels leads to higher economic return and reduced wastage of expensive culture media and consumables.

Mammalian cell culture processes.

Over the past year, mammalian cell culture research has been aimed at investigating the influence of culture conditions on viability, productivity and the consistency of post-translational modifications. Studies of the effect of medium conditions and the development of kinetic models are being made in relation to current efforts to develop fed-batch strategies that will optimize recombinant protein production processes. Recent advances have included novel biosensor and bioreactor developments. New technologies have also been applied to investigate high cell density bioreactor and culture conditions.

Performance characteristics of Mammalian Cell Culture process operating continuously with protein-free medium

In this communication we briefly describe a continuous industrial process for the production of Tissue Plasminogen Activator (tPA), operating with a standard, basal medium (DME/F12). Process data of two culture runs has been chosen to present a performance profile over a production period of more than 3 mo. Developmental aspects of glucose utilization (as a process parameter) are briefly discussed.