Continuous gradient elution chromatographic fractionation

The method of any of the preceding claims, wherein

• (i) EF1 and/or EF3 comprise the product of interest and impurities, wherein compared to the solution loaded in step 1, the concentration of the product of interest is increased by a factor of at least 2, preferably at least 5, more preferably at least 10, wherein
• a) the collection of EF1 is started at a first predetermined concentration XEF1-P of the product of interest in the eluate and stopped at a predetermined concentration YEF1-P of the product of interest in the eluate and/or the collection of EF3 is started at a first predetermined concentration XEF3-P of the product of interest in the eluate and stopped at a predetermined concentration YEF3-P of the product of interest in the eluate;
• and/or
• b) the collection of EF1 is started at a first predetermined concentration XEF1-I of the impurities in the eluate and stopped at a predetermined concentration YEF1-I of the impurities in the eluate and/or the collection of EF3 is started at a first predetermined concentration XEF3-I of the impurities in the eluate and stopped at a predetermined concentration YEF3-I of the impurities in the eluate;
• and/or
• (ii) EF2 comprises substantially pure product of interest, wherein
• a) the collection of EF2 is started at a predetermined concentration XEF2-P of product of interest in the eluate and stopped at a predetermined concentration YEF2-P of product of interest in the eluate;
• and/or
• b) the collection of EF2 is started at a predetermined concentration XEF2-I of impurities in the eluate and stopped at a predetermined concentration YEF2-I of impurities in the eluate.

The method of any of the preceding claims,

• wherein in steps 1 and 4 loading is stopped before any product of interest is eluted from the chromatography matrix with the flow-through;
• wherein steps 1 and 4 comprise binding the product of interest to the chromatography matrix;
• and/or
• wherein the chromatography matrices of step 1 and step 4 are of the same type.

A chromatography apparatus comprising

• one or more chromatography matrices with a first and a second end;
• a feed container;
• one or more a side fraction containers (SFC);
• conduit means connecting the second end of the one or more chromatography matrices with the one or more side fraction containers;
• conduit means connecting the one or more side fraction containers with the first end of the one or more chromatography matrices;
• conduit means connecting the feed container with the one or more side fraction containers;
• conduit means connecting the feed container with the first end of the one or more chromatography matrices;
• wherein the one or more side fraction containers have a volume of about 0.05 to about 8 volumes of the chromatography matrices.