Feed mixing device and its use

Abstract

Herein is reported a feed mixing device for adding feed solutions with a non-physiologically pH value to a cell cultivation vessel comprising a chamber for mixing the feed solutions prior to their addition to the cell cultivation vessel as well as its use. With the feed mixing device as reported herein feed components can be provided in solution at a pH value at which they have good solubility and/or good stability whereby the pH value can be clearly different from the pH value of the cultivation medium, i.e. different from the physiological pH value. This allows performing the cultivation with more flexibility compared to a cultivation in which the pH value of the feed solution is limited to a small range around the pH value of the cultivation.

1 . A device for adding at least two feed solution each with a non-physiological pH value to a cell cultivation vessel comprising separate inlets for the feed solutions, a chamber for mixing the feed solutions, and a single outlet for adding the mixed feed solutions to the cell cultivation vessel, whereby the ratio of the volume of the chamber for mixing the feed solutions to the volume of cultivation medium in the cultivation vessel is of from 0.8 mL/L to 1.2 mL/L. 
     
     
         2 . The device according to  claim 1 , characterized in that the ratio is about 1 mL/L. 
     
     
         3 . The device according to  claim 1 , characterized in that the device is separated from the cultivation vessel and the outlet is to the inside of the cultivation vessel. 
     
     
         4 . The device according to  claim 1 , characterized in that the feed mixing device is outside of the cultivation vessel or inside the cultivation vessel. 
     
     
         5 . The device according to  claim 1 , characterized in that the chamber for mixing the feed solutions has a volume of from 0.5 mL to 1,000 mL. 
     
     
         6 . The device according to  claim 1 , characterized in that the device is made of sterilizable material. 
     
     
         7 . A cell cultivation apparatus comprising
 a) a cell cultivation vessel,   b) a gas supply,   c) at least two liquid storage units for feed solutions each with a non-physiological pH value,   d) at least a sensor for determining the pH value in the cell cultivation vessel, and   e) a device according to  claim 1 .   
     
     
         8 . Use of a device according to  claim 1  or of an apparatus according to  claim 7  in a fed-batch or continuous cultivation of a cell. 
     
     
         9 . A method for the production of a polypeptide comprising the steps of:
 cultivating a cell comprising a nucleic acid encoding the polypeptide in a fed-batch or continuous cultivation with a cultivation vessel comprising a device according to  claim 1  or an apparatus according to  claim 7 , wherein at least two feed solutions each with a non-physiologically pH value are added to the cultivation medium, and   recovering the polypeptide from the cultivation medium or the cells.   
     
     
         10 . The method according to  claim 9 , characterized in that
 any acidic feed solution has a pH value of pH 6.5 or lower, and/or any alkaline feed solution has a pH value of pH 8.0 or higher, and   the mixed feed solutions have a pH value of from pH 7 to pH 7.5.   
     
     
         11 . The method according to  claim 9 , characterized in that the mixing is immediately prior to the addition to the cultivation vessel. 
     
     
         12 . The method according to  claim 9 , characterized in that any acidic feed solution has a pH value of pH 4.5, or lower, and/or any alkaline feed solution has a pH value of pH 10.0 or higher. 
     
     
         13 . The method according to  claim 9 , characterized in that of from two to four separate feed solutions are added to the cultivation medium whereof at least one is an acidic feed solution and at least one is an alkaline feed solution. 
     
     
         14 . The method according to  claim 9 , characterized in that each of the alkaline or acidic feed solutions comprises at least a compound selected from amino acid, sugar, vitamin, trace element, lactate, and growth factor. 
     
     
         15 . A method for obtaining a polypeptide with a reduced G(0) glycoform and/or increased G(1) glycoform comprising the steps of:
 cultivating a cell comprising a nucleic acid encoding the polypeptide in a fed-batch or continuous cultivation with a cultivation vessel comprising a device according to  claim 1  or in an apparatus according to  claim 7 , wherein at least one acidic feed solution and at least one alkaline feed solution are added to the cultivation medium, and   recovering the polypeptide from the cultivation medium or the cells,   
       wherein the mixed feed solution has a pH value upon addition to the cultivation vessel of from pH 4.0 to pH 6.0.