Scale-Up analysis for a CHO cell culture process in large-scale bioreactors
ABSTRACT Bioprocess scale-up is a fundamental component of process development in the biotechnology industry. When scaling up a mammalian cell culture process, it is important to consider factors such as mixing time, oxygen transfer, and carbon dioxide removal. In this study, cell-free mixing studies were performed in production scale 5,000-L bioreactors to evaluate scale-up issues. Using the current bioreactor configuration, the 5,000-L bioreactor had a lower oxygen transfer coefficient, longer mixing time, and lower carbon dioxide removal rate than that was observed in bench scale 5- and 20-L bioreactors. The oxygen transfer threshold analysis indicates that the current 5,000-L configuration can only support a maximum viable cell density of 7 x 10(6) cells mL(-1). Moreover, experiments using a dual probe technique demonstrated that pH and dissolved oxygen gradients may exist in 5,000-L bioreactors using the current configuration. Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing-related engineering parameters in the 5,000-L bioreactors. These equations indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer and carbon dioxide removal. Furthermore, as the liquid volume increases in a production bioreactor operated in fed-batch mode, bulk mixing becomes a challenge. The mixing studies suggest that the engineering parameters related to bulk mixing and carbon dioxide removal in the 5,000-L bioreactors may need optimizing to mitigate the risk of different performance upon process scale-up.
- SourceAvailable from: Susan Mcdonnell
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- "Among the cell lines available for mAb production are myeloma, hybridoma, and Chinese hamster ovary (CHO) cell lines [3,4]. The use of CHO cells in large-scale production is common [3,5-7] because of their ability to express high recombinant protein levels [8,9], grow to high cell densities [10-13], and to grow in serum-free suspension culture [6,14-16]. CHO cells are also suitable for use with expression systems, such as dihydrofolate reductase (DHFR) and glutamine synthetase (GS) [17-19]. "
ABSTRACT: High recombinant protein productivity in mammalian cell lines is often associated with phenotypic changes in protein content, energy metabolism, and cell growth, but the key determinants that regulate productivity are still not clearly understood. The mammalian target of rapamycin (mTOR) signalling pathway has emerged as a central regulator for many cellular processes including cell growth, apoptosis, metabolism, and protein synthesis. This role of this pathway changes in response to diverse environmental cues and allows the upstream proteins that respond directly to extracellular signals (such as nutrient availability, energy status, and physical stresses) to communicate with downstream effectors which, in turn, regulate various essential cellular processes. In this study, we have performed a transcriptomic analysis using a pathway-focused polymerase chain reaction (PCR) array to compare the expression of 84 target genes related to the mTOR signalling in two recombinant CHO cell lines with a 17.4-fold difference in specific monoclonal antibody productivity (qp). Eight differentially expressed genes that exhibited more than a 1.5-fold change were identified. Pik3cd (encoding the Class 1A catalytic subunit of phosphatidylinositol 3-kinase [PI3K]) was the most differentially expressed gene having a 71.3-fold higher level of expression in the high producer cell line than in the low producer. The difference in the gene's transcription levels was confirmed at the protein level by examining expression of p110delta. Expression of p110delta correlated with specific productivity (qp) across six different CHO cell lines, with a range of expression levels from 3 to 51 pg/cell/day, suggesting that p110delta may be a key factor in regulating productivity in recombinant cell lines.BMC Biotechnology 02/2014; 14(1):15. DOI:10.1186/1472-6750-14-15 · 2.03 Impact Factor
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- "A practical explicit formula is (4) where H 2 O is the concentration of water in the liquid phase, He C Henry's constant for CO 2 , p the actual total pressure and y C the molar fraction of CO 2 in the gas phase. In literature (e.g., Xing et al. ), y C is most often assumed to be the carbon dioxide concentration in the air pressed into the sparger and that is the very problem. Due to mass transfer across the bubble interfacial area, A B , changes in the amount n C of CO 2 within a single representative gas bubble along its path in the continuous liquid phase can be described by the simple ordinary differential equation in n C (5) which can easily be derived from Eq. (1). "
ABSTRACT: Most discussions about stirred tank bioreactors for cell cultures focus on liquid-phase motions and neglect the importance of the gas phase for mixing, power input and especially CO(2) stripping. Particularly in large production reactors, CO(2) removal from the culture is known to be a major problem. Here, we show that stripping is mainly affected by the change of the gas composition during the movement of the gas phase through the bioreactor from the sparger system towards the headspace. A mathematical model for CO(2)-stripping and O(2)-mass transfer is presented taking gas-residence times into account. The gas phase is not moving through the reactor in form of a plug flow as often assumed. The model is validated by measurement data. Further measurement results are presented that show how the gas is partly recirculated by the impellers, thus increasing the gas-residence time. The gas-residence times can be measured easily with stimulus-response techniques. The results offer further insights on the gas-residence time distributions in stirred tank reactors.Biotechnology Journal 12/2011; 6(12):1547-56. DOI:10.1002/biot.201100153 · 3.49 Impact Factor
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- "Mammalian cell cultures are a predominant vehicle for the production of proteins at an industrial scale. Production methods range from transient, but large-scale, high efficiency transfections of cell cultures [1–3] to the establishment of stable cell lines that are subsequently grown in large-scale reactors [4, 5]. In the last case, identification of clones that couple high protein expression to good growth conditions is pivotal. "
ABSTRACT: The use of high stringency selection systems often results in the induction of very few recombinant mammalian cell lines, which limits the ability to isolate a cell line with favorable characteristics. The employment of for instance STAR elements in DNA constructs elevates the induced number of colonies and also the protein expression levels in these colonies. Here, we describe a method to systematically identify genomic DNA elements that are able to induce many stably transfected mammalian cell lines. We isolated genomic DNA fragments upstream from the human Rb1 and p73 gene loci and cloned them around an expression cassette that contains a very stringent selection marker. Due to the stringency of the selection marker, hardly any colony survives without flanking DNA elements. We tested fourteen ~3500 bp DNA stretches from the Rb1 and p73 loci. Only two ~3500 bp long DNA fragments, called Rb1E and Rb1F, induced many colonies in the context of the stringent selection system and these colonies displayed high protein expression levels. Functional analysis showed that the Rb1 DNA fragments contained no enhancer, promoter, or STAR activity. Our data show the potential of a methodology to identify novel gene expression augmenting DNA elements in an unbiased manner.Molecular Biotechnology 10/2010; 48(1):19-29. DOI:10.1007/s12033-010-9344-8 · 1.88 Impact Factor