However, the lactic acid present in the conditioned medium could inhibit ESC growth and induce spontaneous differentiation when its concentration exceeded 1.5 g/l. In addition, the 3D static culture could be limited by oxygen, which was depleted in the long-term culture when cell density in the matrix was high. However, these problems can be alleviated in dynamic culture with improved oxygen transfer and continuous media perfusion. The matrix pore size also had profound effects on ESCs. The smaller-pore (30 -60 m) matrix gave a higher proliferation rate and Oct-4 and stage specific embryonic antigen-1 expressions. Overall, the 3D culturing method is superior to the 2D culture method and can provide an economical way to mass-produce undifferentiated ESCs in uncoated matrices and conditioned media. STEM CELLS 2007;25:447-454
As the number of therapeutic proteins produced by mammalian cell cultures in the pharmaceutical industry continues to increase, the need to improve productivity and ensure consistent product quality during process development activities becomes more significant. Rational medium design is known to improve cell culture performance, but an understanding of nutrient consumption and metabolite accumulation within the medium is required. To this end, we have developed a technique for using 1D (1)H NMR to quantitate nonprotein feed components and metabolites in mammalian cell cultures. We refer to the methodology as "Fermentanomics" to differentiate it from standard metabolomics. The method was found to generate spectra with excellent water suppression, signal-to-noise, and resolution. More importantly, nutrient consumption and metabolite accumulation was readily observed. In total, 50 media components have been identified and quantitated. The application of Fermentanomics to the optimization of a proprietary CHO basal medium yielded valuable insight regarding the nutrient levels needed to maintain productivity. While the focus here is on the extracellular milieu of CHO cell cultures, this methodology is generally applicable to quantitating intracellular concentrations and can be extended to other mammalian cell lines, as well as platforms such as yeasts, fungi, and Escherichia coli.
Background: Sarilumab is the first fully human monoclonal antibody (mAb) directed against the interleukin-6 receptor alpha (IL-6Rα). Sarilumab was developed using VelocImmune ® mice immunized with the human IL-6 (hIL-6) receptor. VelocImmune mice are genetically-engineered to express human antibody variable domain genes in the same robust fashion that the replaced mouse genes are typically expressed. Sarilumab is currently being explored as a new therapeutic modality for the treatment of rheumatoid arthritis. Objectives: To evaluate the kinetic binding parameters and in vitro functional activity of two monoclonal antibodies directed against IL-6Rα: the fully human mAb sarilumab and the humanized mAb tocilizumab. Methods: Kinetic binding parameters were measured using Surface Plasmon Resonance (SPR) technology. The ability to block hIL-6 induced activation of the human IL-6Rα was investigated using several bioassays; a human hepatocellular carcinoma cell line HepG2, transfected with a STAT3-luciferase reporter plasmid, as well as a proliferation assay using the human B-lymphoma cell line, DS-1. Results: Sarilumab bound with high affinity to recombinant monomeric human and monkey IL-6 receptor with a K D value of 61.9 pM and 71.9 pM, respectively. The binding affinity of sarilumab to the dimeric human IL-6 receptor Fc-fusion was 12.8 pM. Cross-reactivity to mouse IL-6 receptor was not observed using SPR, indicating that sarilumab is specific to human and monkey IL-6 receptor. In contrast, tocilizumab bound to monomeric and dimeric forms of the human IL-6 receptor with a 15-22 fold weaker affinity than that of sarilumab as determined by SPR. In the HepG2 cell luciferase reporter assay, sarilumab effectively blocked luciferase activity induced by 50 pM hIL-6 with an IC 50 of 146 pM and was ~4 fold more potent than tocilizumab. Similarly, in the DS-1 cell proliferation assay, sarilumab effectively blocked growth induced by 1.0 pM hIL-6 with an IC 50 of 226 pM and was several fold more potent than tocilizumab. Conclusions: Based on these in vitro assay data, sarilumab has both a higher relative binding affinity for IL-6Rα, blocks IL-6Rα activation, and inhibits IL-6-induced cellular responses such as cell proliferation at lower concentrations than tocilizumab. Acknowledgements: VelocImmune ® is a registered trademark of Regeneron Pharmaceuticals, Inc.
Cell culture process conditions including media components and bioreactor operation conditions have a profound impact on recombinant protein quality attributes. Considerable changes in the distribution of galactosylated glycoforms (G0F, G1F, and G2F) were observed across multiple CHO derived recombinant proteins in development at Eli Lilly and Company when switching to a new chemically defined (CD) media platform condition. In the new CD platform, significantly lower G0F percentages and higher G1F and G2F were observed. These changes were of interest as glycosylation heterogeneity can impact the effectiveness of a protein. A systematic investigation was done to understand the root cause of the change and control strategy for galactosylated glycoforms distribution. It was found that changes in asparagine concentration could result in a corresponding change in G0F, G1F, and G2F distribution. A follow-up study examined a wider range of asparagine concentration and it was found that G0F, G1F, and G2F percentage could be titrated by adjusting asparagine concentration. The observed changes in heterogeneity from changing asparagine concentration are due to resulting changes in ammonium metabolism. Further study ascertained that different integrated ammonium level during the cell culture process could control G0F, G1F, and G2F percentage distribution. A mechanism hypothesis is proposed that integrated ammonium level impacts intracellular pH, which further regulates β-1, 4 galactosyltransferase activity.
Background : Embryonic stem cells (ESCs) have unlimited proliferation potential and can differentiate into all cell and tissue types, and thus are ideal sources for cell therapy and drug screening. Current supplies of ESCs are limited by the available cell sources and inefficient culture methods that grow ESCs on surfaces coated with expensive extracellular matrix (ECM) proteins and in media containing expensive growth factors. Objective : To meet the demand for ESCs, it is necessary to develop an economical process for their mass production. Methods : We review the latest development in in vitro ESC culture and introduce a two-stage perfusion bioreactor system that uses 3-D fibrous matrices and conditioned media for production of ESCs. Results/conclusion : The two-stage process can produce billions of ESCs in a small bioreactor without using ECM proteins and growth factors, and is promising for further scale-up for clinical applications.
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