SOP 6 Objectives

The objectives are:

To become familiar with the principles of ion exchange chromatography and apply these concepts to purify tPA from cell culture media using Cation Exchange.

To pack a chromatography column, condition it and use it for analysis.

To become familiar with the operation of the Millipore Amicon Vantage-L Biochromatography column, and collection of fractions for later confirmation by gel electrophoresis.

OVERVIEW:

Ion Exchange chromatography is a productive and cost effective methodology frequently used in downstream processing. Proteins must reversibly bind to the resin since it is necessary to remove the protein from the resin.

Poros 50 HS is a cation resin, which means that positively charged molecules will bind to the negatively charged resin. The extent of binding is dependent on the cationic strength of the protein of interest and can be manipulated by changing the pH and/or conductivity of the buffers used in the chromatography process. The main proteins in the media used to grow tPA are tPA, Bovine serum albumin (BSA) and Transferrin. Each protein has a specific isoelectric point called the pI. BSA has a pI of 4.9, tPA is 7.5 - 8.5 and transferrin is 5.9. We are able to selectively bind the tPA to the resin by controlling the pH and ionic strength of the Equilibration buffer. At a pH of 6.0, tPA is more cationic (positively charged) than either BSA or Transferrin. Therefore, the more positive charged tPA will bind to the resin and the others will flow through the column and out to waste. tPA is then removed from the column using a high concentration of salt, which competitively "bumps" the protein off the resin as the sodium ions bind.

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