For large volumes of dilute cell culture supernatants

Several in vitro cell cultures techniques are currently used to produce monoclonal antibodies from hybridomas on a laboratory scale. The hydrophobic surface of tissue culture flasks (T flasks) and cell culture dishes is ideal for stationary culture of hybridomas clones. T flasks vary in size from 12.5 cm 2 to 300 cm2 with working volumes between 5 and 400 ml. 225 cm2 flasks with a maximum working volume of 370 ml are commonly used for monoclonal antibody production. Cells are seeded in the T flask and handled as a batch culture in a humidified CO2 incubator. Fetal calf serum (5-15 %) is frequently added to media to provide essential growth factors and hormones. However, undefined serum supplements have a particularly large effect upon the purity of monoclonal antibodies with concentrations less than 100 µg/ml derived from low cell density cultures.

Monoclonal antibody concentrations, derived from T flasks, are typically between 5 and 50 µg/ml (Altshuler,G et al (1986) Biotech. & Bioeng. 28, 646) after a 7-10 day incubation period. Even more expensive production methods using roller bottles produce monoclonal antibodies in the range of 10-200 µg/ml over a longer 12-14 day period (Kuhlmann, I. et al (1989) ATLA 17, 73). Researchers often use T flasks and roller bottles because production costs are low and low monoclonal antibody concentrations are required. As the antibody represents only a small fraction of the total protein content of the supernatant, these antibodies need to be concentrated during an affinity-based purification step. The ProVac Protein A and G kits are designed to concentrate and purify antibodies from large volumes of dilute cell culture supernatants.

Features
• High speed concentration and purification of large volumes of dilute cell culture supernatant
• Uses universal 50 ml collection tubes
• Works under low or high vacuum (N.B. Capture efficiency of ProVac is enhanced at lower vacuum!)
• Simple to set-up and easy-to-use
• Ultrafiltration concentrators to de-salt and concentrate the purified monoclonal antibody
• Can be performed at room temperature or in the cold room

Benefits
• ProVac uses vacuum as the driving force. It permits large volumes of dilute samples to be continuously loaded on to the columns without the need of a centrifuge.
• Residence times between the target protein and the resin matrix can be more closely controlled under vacuum. Higher recoveries are obtained with slower sample loading flow rates.

Equipment Required
• Test tube rack to accommodate 50 ml collection tubes.
• Vacuum pump with tubing of appropriate internal diameter.
• Pre-filtration units to ensure sample is clarified to 0.2 µm pore size prior to purification.