Tangential flow filtration (TFF) is widely used in biopharmaceutical processing for protein purification – a common application for TFF is ultrafiltration for concentration/diafiltration of proteins. In this type of application, the product protein is retained (concentrated) within the feed side of the ultrafiltration membrane, while the buffer components and other small impurities (smaller than the membrane pore size) freely pass through the membrane into the permeate side. Several scholarly articles are available in literature which discuss the ultrafiltration application as well as its optimization strategies. Another category of application where TFF finds significant use is in the clarification of cell culture bioreactor and microbial fermenter feed solutions using microfiltration membranes. In some of these microfiltration TFF applications (e.g., mammalian cell culture clarification), the product (protein) freely passes through the microfiltration membrane and is recovered on the permeate side, while the contaminating impurities (cells, cell debris, colloids) are retained on the feed side of the membrane. In certain other microfiltration TFF applications (allantoic fluid clarification in egg-based flu process), the product (flu virus) may get concentrated on the feed side of the microfiltration membrane (similar to an ultrafiltration step), while the contaminating impurities (ovalbumin, etc.) may get removed into the permeate side…
Tag: <span>mammalian cell culture</span>
The evaluation of bacteriocin cytotoxicity is a critical first step in guaranteeing its safe use in food and therapeutic applications. In this study, the bacteriocins nisin, pediocin, and colicin ([Col] E1, E3, E6, E7, and K) were evaluated for cytotoxicity against cultured mammalian cells. Cellular membrane potential (delta psi) changes of bacteriocin-treated cells were used as an index of cytotoxicity. Simian virus 40-transfected human colon (SV40-HC) cells and African green monkey kidney epithelial cells (Vero) cells were cultured (35°C, 10% CO2, humidified air) as monolayers on tissue culture plates. Log phase cells (~104 cells/mL) were treated with partially purified, individual bacteriocin preparations at 170, 350, and 700 activity units (AU)/mL…
Raman spectroscopy offers an attractive solution for monitoring key process parameters and predictive modelling in cell culture processes using transgenic Chinese hamster ovary (CHO) cells. Frequent in-line measurements offer the potential for advanced control strategies. However, an erroneous value created by analytical signal noise is a significant issue that can affect process controls negatively. One such challenge is to differentiate the signal reflecting process changes, ranging from random to gross error, in a timely manner so the process control system can respond to these changes and maintain adequate control.
In the past 20 years, mammalian cell lines have been utilized to produce many viral veterinary vaccines. Cell lines such as baby hamster kidney (BHK)-21, Vero, and Madin Darby canine kidney (MDCK) are widely used because they help facilitate shorter manufacturing lead times and tighter process controls. As compared to other biotech products, viral vaccine manufacturing processes present some specific constraints linked to the cell substrates used. With the global veterinary vaccine market value predicted to be almost $7 billion per year by 2021[2], to remain competitively priced as well as profitable, bioprocess scientists are under pressure to develop methods for faster and more cost-efficient cell culture production. This has led to a shift from the use of expensive, two-dimensional T-flask and roller bottles to single-use, stirred tank bioreactors with microcarriers, or the adaptation of attachment-dependent cell lines such as BHK-21 for suspension culture. This requires time-consuming optimization and scale-up development experiments, which are real drawbacks. However, utilizing automated, single-use mini bioreactors as a scale-down model can enable more efficient use of time and optimization of media, feed, and culture conditions to de-risk upstream process development. In this article, single-use, mini bioreactors are evaluated to determine if they are geometrically comparable to benchtop bioreactors (both glass and single-use vessels) and pilot-scale, single-use bioreactors for effectively modelling mammalian cell culture at 2 L and 50 L scale…
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...
Viable cell density (VCD), the quantitative assessment of living cells, is commonly determined by laborious and inaccurate off-line cell counting methods. Single-function in situ probes have been developed using various technologies including optical density, radio frequency and dielectric permittivity. Optical density measurements predict total biomass but are sensitive to cell debris accumulation and inherent media turbidity. Near-infrared (NIR) has the advantage of being able to measure many key analytes in the cell culture simultaneously. NIR has been used to acquire real-time measurements of glucose, glutamine, glutamate, and media nutrients such as amino acids and metabolites (e.g., lactate and ammonia). NIR probes can be sterilized in place, and real-time measurements can be acquired throughout the mammalian cell culture processes…
Many laboratories have utilized cell-free systems or prokaryotic systems designed to produce biological molecules with single polypeptide chains, limited folding requirements, and without glycosylation. The yeast systems are used to generate glycoproteins; however, their glycosylation profiles are vastly different from those of mammalian cells. Without significant glycoengineering, the yeast-produced recombinant glycoproteins may be unsuitable as therapeutic molecules. As such, the use of mammalian cells is still the preferred method to produce complex biological molecules…