Method and apparatus for chromatographic purification

Abstract

The present invention relates to a method and an apparatus suitable for a continuous chromatography process which only needs three separation columns. The process is a two step procedure comprising two chromatographic steps. The first chromatographic step (capture) is performed alternating and sequentially on two separation columns, the second chromatographic step (polishing) is performed, also sequentially, on the third column.

1 . An apparatus comprising
 two separation units A1 and A2 both having the same chromatography matrix and a separation unit B having a chromatography matrix which differs from the chromatography matrix of separation units A1 and A2, all separation units having a fluid inlet and a fluid outlet, whereby there is at least fluid connection between the fluid outlet of separation unit A1 and the fluid inlet of separation unit B and fluid connection between the fluid outlet of separation unit A2 and the fluid inlet of separation unit B   at least one valve in the fluid connection between separation units A1 and A2 and separation unit B that allows to switch between fluid communication between the outlet of separation unit A1 and the fluid inlet of separation unit B and fluid communication between the outlet of separation unit A2 and the fluid inlet of separation unit B.   at least two buffer reservoirs and at least two pumps   a reservoir containing sample solution that is in fluid connection with the inlets of separation units A1 and A2   
     
     
         2 . Apparatus according to  claim 1 , characterized in that the separation units A1 and A2 have an affinity chromatography, a cation exchange, a mixed mode cation exchange or an anion exchange chromatography matrix. 
     
     
         3 . Apparatus according to  claim 1 , characterized in that the separation unit B has a cation exchange, a mixed mode anion exchange or an anion exchange chromatography matrix. 
     
     
         4 . Apparatus according to  claim 1 , characterized in that the separation units A1 and A2 have a an affinity chromatography matrix and the separation unit B has a cation exchange, a mixed mode anion exchange or an anion exchange chromatography matrix. 
     
     
         5 . Apparatus according to  claim 1 , characterized in that the separation units A1 and A2 have a cation exchange or a mixed mode cation exchange chromatography matrix and the separation unit B has an anion exchange or a mixed mode anion exchange chromatography matrix. 
     
     
         6 . Apparatus according to  claim 1 , characterized in that the apparatus further comprises a connecting line between the fluid outlet of separation unit A1 and the fluid inlet of separation unit A2 and a connecting line between the fluid outlet of separation unit A2 and the fluid inlet of separation unit A1. 
     
     
         7 . Apparatus according to  claim 1 , characterized in that the apparatus comprises a fluid inlet in the connecting line between the outlets of separation units A1 and A2 and the fluid inlet of separation unit B. 
     
     
         8 . Apparatus according to  claim 1 , characterized in that the apparatus comprises an additional reservoir with virus inactivation buffer that is at least in fluid connection with the inlet of one of the three separation units. 
     
     
         9 . Apparatus according to  claim 1 , characterized in that the apparatus comprises at least one additional reservoir with buffer for cleaning in place that is at least in fluid connection with the inlet of one of the three separation units. 
     
     
         10 . A method of purifying a target molecule from one or more impurities in a sample using an apparatus according to  claim 1 , the method comprising the steps of
 alternately loading the sample on the separation units A1 and A2 so that while the sample is loaded on separation unit A1 wherein the sample is at a first pH and conductivity enabling the target molecule to be bound to separation unit A1, separation unit A2 is at least part of that time in fluid communication with separation unit B so that the target molecule loaded on separation unit A2 is eluted onto separation unit B and separation unit A2 is re-equilibrated and while the sample is loaded on separation unit A2 wherein the sample is at a first pH and conductivity enabling the target molecule to be bound to separation unit A2, separation unit A1 is at least part of that time in fluid communication with separation unit B so that the target molecule loaded on separation unit A1 is eluted onto separation unit B and separation unit A1 is re-equilibrated   recovering the target molecule from the fluid outlet of separation unit B.   
     
     
         11 . Method according to  claim 10 , characterized in that the target molecule is an antibody. 
     
     
         12 . Method according to  claim 10 , characterized in that while loading the sample onto separation unit A1 the fluid outlet of separation unit A1 is at least part of that time in fluid communication with the fluid inlet of separation unit A2 to enable the capture of the starting to leach target molecule from the separation column A1 to be bound to separation column A2 and while loading the sample onto separation unit A2 the fluid outlet of separation unit A2 is at least part of that time in fluid communication with the fluid inlet of separation unit A1 to enable the capture of the starting to leach target molecule from the separation column A2 to be bound to separation column A1. 
     
     
         13 . Method according to  claim 10 , characterized in that a virus inactivation buffer is pumped through separation unit A1 after loading separation unit A1 with the target molecule and through separation unit A2 after loading separation unit A2 with the target molecule. 
     
     
         14 . Method according to  claim 10 , characterized in that, while washing the unbound sample from the separation unit A1, separation unit A2 is at least part of that time in fluid communication with the reservoir containing sample solution and separation unit A1 and while washing the unbound sample from the separation unit A2, separation unit A1 is at least part of that time in fluid communication with the reservoir containing sample solution and separation unit A2. 
     
     
         15 . Method according to  claim 10 , characterized in that the sample is a clarified sample.