These highly macroporous media provide fast linear velocities at low backpressure. UNOsphere media are designed for high-efficiency capture of biomolecules from crude feedstreams and are fully supported for regulatory submission.
Purification of a Monoclonal Antibody Using a Combination of UNOsphere Rapid S and UNOsphere Q Media Ion Exchange Chromatography After Protein A Capture
Introduction Antibody-based drugs represent a growing segment of the pharmaceutical industry and one of the most promising classes of therapeutic drugs. The demand for protein-based drugs is expected to grow steadily for the foreseeable future. However, production of monoclonal antibody (mAb)–based drugs remains very costly, and solutions to lower production costs are needed. Downstream purification steps are becoming a target for cost reduction. The use of ion exchange chromatography to directly capture MAbs from cell culture streams or as a polishing step after an affinity separation may be a way to reduce costs.
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Binding and backpressure properties of UNOsphere Q support: A 5 mg/ml sample of BSA in 10 mMTris, pH8.5, was loaded onto a 1.1 x 20 cm column. Red, backpressure; blue, 10% breakthrough capacity.
Purification of plasmid DNA on a UNOsphere Q column. Clarified bacterial lysate (10 ml, adjusted to pH 8.0) was loaded onto a 0.5 x 11 cm column (2.1 ml) in buffer A (10 mM sodium phosphate, 0.3 M NaCl, 1 mM EDTA, pH 8.0). The sample was eluted with a 0–40% gradient of buffer B (10 mM sodium phosphate, 1.0 M NaCl, 1 mM EDTA, pH 8.0) at a flow rate of 2 ml/min (600 cm/hr). The column was washed with 10 column volumes of 40% buffer B, followed by 100% buffer B for 5 column volumes. The effluent was monitored at 254 nm. Each fraction was 5 ml. Blue, A254; red, conductivity; black, theoretical gradient.
Analysis of plasmid DNA purified on UNOsphere Q support. Fractions were separated on a 0.8% agarose gel. Lane 1, 1 kb marker (catalog #170-8204); lane 2, crude lysate; lane 3, flowthrough (fractions 2–6); lane 4, fractions 9–18; lane 5, fractions 19–30; lane 6, fractions 31–35; lane 7, fraction 36.
Restriction enzyme analysis of plasmid DNA purified on UNOsphere Q support. Lane 1, 1 kb marker; lane 2, undigested clarified lysate; lane 3, EcoRI-digested clarified lysate; lane 4, undigested fractions 31–35; lane 5, digested fractions 31–35; lane 6, undigested fraction 36; lane 7, digested fraction 36.
* 10% breakthrough capacity determined with 2.0 mg/ml BSA in a 1.1 x 10 cm column. ** UNOsphere Q support packed into a 20 cm bed height and run at 1,200 cm/hr generates less than 2 bar backpressure.
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