The Combined Use of Expanded-Bed Adsorption and Gradient Elution for Capture and Partial Purification of Mutant Diphtheria Toxin (CRM 9) fromCorynebacterium diphtheriae

Expanded-bed adsorption (EBA) is a new approach for performing the initial recovery or capture of proteins from various crude feedstocks. The essence of the method is direct adsorption of the desired protein from the unclarified suspension by passing it through a stable expanded bed of the adsorbent. This type of operation replaces centrifugation, clarification, dialysis, and concentration with one simple unit operation. The recovery is done by pumping the feedstock upward on the expanded column and eluting it downward in a step mode from the packed bed. One of the unique properties of the expanded bed is its behavior as a true plug-flow column, which makes it possible to use gradient elution and to achieve better purification in addition to the other benefits. In this work the “traditional” recovery and purification process of the extracellular mutant diphtheria toxin (CRM 9) was replaced with an expanded bed adsorption process in which the protein was eluted using a linear salt gradient in an upward mode instead of the standard downward step elution. This combined procedure is a simpler, shorter process that yielded a purified protein preparation (that only had to pass through a gel filtration column instead of through the next ion-exchange step). Twenty grams of protein suitable for clinical use was prepared using this method.