• Fast Ion Exchange – Ion exchange fractionation of ascites fluid
• Fast Affinity – Affinity purification of an anti-melanoma antibody
• High Throughput Hydrophobic Interaction – Hydrophobic interaction chromatography using axial and radial flow columns
• Fast Desalting – Fast desalting of a protein solution
• Low Cost Reverse Phase – Reverse phase of nucleotides
• Direct Purification – Direct purification of crude cell lysates (when speed counts)
• Tandem Radial Flow – Tandem radial flow column chromatography of a refolded recombinant protein sample

Fast Ion Exchange

Ion exchange fractionation of ascites fluid
	Sample: 150 mL ascites fluid Column: 1500mL Superflo® Column Packing: DEAE Cellulose Flow Rate: 150 mL/min Start Buffer: 10mM phosphate, pH 8.5 Step Gradient: 60 mM NaCl in start buffer
Radial flow columns are routinely used in initial capture steps after fermentation and cell disruption. The radial flow design of Superflo® columns helps overcome typical problems encountered with most ion exchange media. These packings, especially cellulose, are inexpensive and offer high capacities. However, on axial columns they characteristically yield low flow rates, high backpressures, and swell and shrink with buffer changes. With the radial flow design, fast flow rates are easily achieved with only a peristaltic pump. You save time and can use inexpensive packings and equipment. Saxena, V. and Well, A.E., Biochromatography, Vol 2(2), p.90, 1987.

Fast Affinity

Affinity purification of an anti-melanoma antibody on Protein A Sepharose®* in a 1500mL Superflo® column
Flow Rate:    104 mL/min loading    107 mL/min wash    92 mL/min elution Yield:    2600 mg estimated recovery    3126 mg actual recovery Purity:    95% (HPLC gel filtration)    IgG2A (double immunodiffusion)
Dilute cell culture fluids can be purified without prior concentration using affinity chromatography and Superflo® columns. High purification and recovery levels are possible with columns due to the high flow rates and therefore reduced residence times in the column. There is lower nonspecific binding and less proteolysis than in conventional columns. Saxena, V., et al, American Laboratory News, October, 1987, p.112.

High Throughput Hydrophobic Interaction

Fast Desalting

Fast desalting of a protein solution
	Sample:    40g BSA in 20L    1M NaCl Flow Rate:    1.8 L/min loading at 3 psi    5.0 L/min run at 16 psi Elution Buffer:    Tap water
The low bed of Superflo® columns makes them ideal for fast desalting applications. Using dextran, polyacrylamide, or agarose packings, desalting can be performed at several times the speed previously possible. In this example, a 20 L sample was fully desalted in 45 minutes in a 100 L Superflo® Column.

Low Cost Reverse Phase

Reverse phase of nucleotides
Sample: 5mg CMP, 5mg UMP, 5mg ATP Column: Superflo® 250 ml Stainless Steel Packing: Nu-Gel® C-18, 40-60 microns Flow Rate: 30 ml/min Solvent A: 100% methanol Solvent B: 0.05M tetrabutyl ammonium phosphate, 0.02M KH2PO4,5% methanol Linear Gradient: 0 to 100% B over 60 minutes
Reverse phase separations are generally associated with high pressures, high equipment costs, and difficult packing procedures. However, with radial flow, preparative HPLC separations can be achieved at low pressures (10-20 psi). Capital equipment costs are low and the separations can easily be scaled up for large process applications.

Direct Purification

Direct purification from crude cell lysates (when speed counts)

The Superflo® column system provides a rapid, low-pressure method for directly processing total crude cell lysates. Crude lysate is pumped directly onto a column packed with fast flow ion exchanger. The large porosity of the Superflo® column bed support and the interstitial space of the agarose beads permits fast, low-pressure flow of the lysate without clogging by cell debris.

Tandem Radial Flow

Tandem radial flow column chromatography of a refolded recombinant protein sample
	Peak I: Q-Sepharose®* flow-through Peak II: Q-Sepharose®* and S-Sepharose®* flow-through Elution Buffer: 10mM PO4, 1nM EDTA, 500mM NACl Peak III: S-Sepharose®* peak
In the example above, tandem radial flow anion and cation exchange columns were used to partially purify and concentrate a dilute recombinant protein. The sample was first passed through a column packed with Q-Sepharose®* to remove contaminating proteins and endotoxins. A column packed with S-Sepharose®* was connected to the outlet of the first column to further purify the product. The tandem arrangement enabled the rapid processing of multiple preparations. Data courtesy of Creative Biomolecules, Inc.