Tag: <span>stirred-tank bioreactor</span>

A Comparative Bioreactor Vessel Study: Conventional Reusable Glass and Single-Use Disposables for the Production of Alkaline Phosphatase

Single-use, stirred-tank bioreactor systems have been used in large-scale production for a number of years. Bench-scale, stirred-tank bioreactors have not been commercially-available for single-use until recently. The New Brunswick™ CelliGen® BLU pitched-blade bioreactor was introduced in 2009, and the CelliGen BLU packed-bed bioreactor, in 2012. Little information is currently available on the utility of these bioreactors for bench-scale production of recombinant products. Thus, we designed this study to perform multiple comparisons with these single-use bioreactors and their traditional glass vessel counterparts. The data comparisons included: (1) CelliGen BLU pitched-blade vs. glass pitched-blade; and (2) CelliGen BLU pitched blade in batch mode vs. CelliGen BLU packed-bed in perfusion mode. Chinese hamster ovary (CHO) cells were used to measure alkaline phosphatase (ALKP) production in each bioreactor. The final measured concentration of ALKP, after eight days of batch-mode culture in the single-use, pitched-blade bioreactor, was 1.6 U/mL compared to 2.1 U/mL in the reusable bioreactor. After six perfusion harvests in the single-use, packed-bed bioreactor, the combined ALKP production was 16.2 U/mL compared to 17.4 U/mL in the reusable bioreactor in batch mode. Multiple batch culture runs in the pitched-blade bioreactor would be required to match the output of a single run in the packed-bed bioreactor in perfusion mode. Results demonstrate that there are no significant differences between the reusable and single-use systems for bench-scale production of recombinant proteins. Our results also suggest that the CelliGen BLU packed-bed bioreactor, when operated in perfusion mode, is superior to the CelliGen BLU pitched-blade bioreactor when operated in batch mode, confirming our studies from 2012…

Read MoreA Comparative Bioreactor Vessel Study: Conventional Reusable Glass and Single-Use Disposables for the Production of Alkaline Phosphatase

Biologics Production

alkaline phosphatase alkp production batch mode batch production bench-scale bioproduction biologics celligen blu cho cells glass bioreactors glass vessels perfusion mode single-use single-use bioreactor stirred-tank bioreactor

Study of a Single-Use Stirred Tank Bioreactor for Manufacturing a Therapeutic Protein in a High Cell Density Microbial Fermentation

Stirred tank single-use bioreactors (SUBs) have been widely adopted for production of biopharmaceuticals such as monoclonal antibodies in mammalian cell culture. However, they are seldom used for commercial production of biologics with microbial fermentation. SUBs offer time-saving advantages because they do not require significant downtime for cleaning and sterilization, so finding a SUB that can perform well with high cell density microbial fermentation processes has the potential to increase the number of production runs. Therefore, for this study, a His-tagged protease inhibitor was chosen as a model protein to demonstrate that the Sartorius Biostat STR® MO, a SUB recently developed for microbial fermentation, is suited for recombinant protein production by high cell density Escherichia coli fermentation processes.

At 50 L scale, the SUB achieved good process control and allowed an oxygen uptake rate (OUR) of up to 240 mmoles/L/h. The fermentation runs produced up to 5.8 g/L of the soluble recombinant protein and a dry cell weight of >60 g/L at the end of fermentation. Additionally, the SUB showed a similar fermentation profile when compared with data from parallel runs in 15 L sterilise-in-place (SIP) vessels using identical media and process parameters. This study indicates that with a minimum investment of capital resources, stirred tank SUBs could be used in pilot-scale manufacturing with high cell density microbial fermentations to potentially shorten the timelines and costs of advancing therapeutic proteins to clinic.

Read MoreStudy of a Single-Use Stirred Tank Bioreactor for Manufacturing a Therapeutic Protein in a High Cell Density Microbial Fermentation

Manufacturing

fermentation microbial fermentation recombinant proteins single-use bioreactor stirred-tank bioreactor therapeutic proteins

Mouse Hybridoma Cell Culture in a Protein-Free Medium Using a Bio-Mimicking Fish-Tail Disc Stirred Bioreactor

Because the Lambda MINIFOR bioreactor provides good mixing of cell culture, nutrients, and gases without any damaging hydrodynamic forces by using a bio-mimicking “fish-tail“ disc stirrer, it can be successfully applied for the cultivation of bacteria and yeast, insect, plant, and mammalian cells. However, reports on its application in mouse hybridoma cell culture using protein-free media is non-existent in the scientific literature. Therefore, this study describes preliminary findings of the Lambda MINIFOR bioreactor suitability in mouse hybridoma cell culture and antibody production using the SP2/O-Ag14-CB.Hep-1 mouse hybridoma cell and the PFHM-II protein-free medium as models. Results verified 2.45 × 106 viable cells/mL as the highest cell concentration, 86% as maximum cell viability, 0.0156/h as the exponential growth rate, 44 h as cell population doubling time, a stable phenotype measured by limiting dilution after 2.5 months, no antibiotic and antifoam requirements, 71.4% of IgG SDS-PAGE purity in the cell culture harvested supernatant, 38.68 ± 22.29 µg/mL, 39.23 ± 10.66 pg/cell/day, up to 99.5% of purity (sample measured by SDS-PAGE and SE-HPLC) after an IgG capture step based on protein A-Sepharose, a low pH incubation, and size-exclusion chromatography, no molecule aggregation, specificity for the CKTCTT epitope (located in the HBsAg “a” determinant), an IgG affinity constant equal to 1.11 × 1010 M-1, and < 78 pg mouse DNA/mg of IgG. In conclusion, this study corroborated a cumulative CB.Hep-1 mAb production of 1.77 g/15 days and validated the usefulness of the Lambda MINIFOR bioreactor in mouse hybridoma cell culture in protein-free media for research applications...

Read MoreMouse Hybridoma Cell Culture in a Protein-Free Medium Using a Bio-Mimicking Fish-Tail Disc Stirred Bioreactor

Biologics Production

fish-tail disc stirrer hybridoma mammalian cell culture protein-free media stirred-tank bioreactor

Cell Culture Bioreactor Optimization: Growing Recombinant HEK293 Cells to Produce a G-Protein-Coupled Receptor (rGPCR) Membrane Protein

Heterologous expression of membrane proteins remains a bottleneck for structural characterization by x-ray crystallography. Such proteins represent approximately 30% of the proteome and are not sufficiently represented in the Protein Data Bank (PDB). G-protein-coupled receptors (GPCRs) are an area of particular interest as it is estimated that one third of current FDA approved drugs act through this class of receptors. We have been studying rhodopsin with an interest in determining the conformational change that leads to signal transduction in this class of receptors. Although there has been some success in expressing select members of the large GPCR family in bacterial systems, the best characterized expression systems have generally been in mammalian tissue culture…

Read MoreCell Culture Bioreactor Optimization: Growing Recombinant HEK293 Cells to Produce a G-Protein-Coupled Receptor (rGPCR) Membrane Protein

Biologics Production

cell culture bioreactor G-protein-coupled receptors membrane proteins protein expression recombinant proteins stirred-tank bioreactor