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 - 6 . (canceled) 
     
     
         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) a sensor for determining the pH value in the cell cultivation vessel, and   e) a device for adding a mixed feed solution to the cell cultivation vessel, the device comprising separate inlets for the feed solutions, a chamber for mixing the feed solutions to obtain the mixed feed solution, and a single outlet for adding the mixed feed solution 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.   
     
     
         8 . Use of a device according to claim 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 within a cultivation vessel, wherein the cultivation vessel comprises a device for adding at least two feed solutions, wherein the device comprises separate inlets for the feed solutions, a chamber for mixing the feed solutions, and a single outlet for adding the mixed feed solution to the cell cultivation vessel;   mixing at least two feed solutions, wherein at least one feed solution is an acidic feed solution and has a pH value of less than pH 6.5, and at least one feed solution is an alkaline feed solution and has a pH value of more than pH 7.5, in the chamber to produce a mixed feed solution;   adding the mixed feed solution to the cultivation vessel containing the cultivation medium;   further cultivating the cell comprising the nucleic acid encoding the polypeptide in the fed-batch or continuous cultivation within the cultivation vessel, wherein the ratio of the volume of the mixed feed solution in the chamber to the volume of the cultivation medium in the cultivation vessel is from 0.8-1.2 ml chamber volume per 1 liter volume of the cultivation medium in the cultivation vessel; and   recovering the polypeptide from the cultivation medium or the cells.   
     
     
         10 . The method according to  claim 9 , wherein
 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 , wherein the mixing is immediately prior to the addition to the cultivation vessel. 
     
     
         12 . The method according to  claim 9 , wherein 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 , wherein 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 within a cultivation vessel, wherein the cultivation vessel comprises a device for adding at least two feed solutions, wherein the device comprises separate inlets for the feed solutions, a chamber for mixing the feed solutions, and a single outlet for adding the mixed feed solution to the cell cultivation vessel;   mixing at least two feed solutions, wherein at least one feed solution is an acidic feed solution and has a pH value of less than pH 6.5, and at least one feed solution is an alkaline feed solution and has a pH value of more than pH 7.5, in the chamber to produce a mixed feed solution;   adding the mixed feed solution to the cultivation vessel containing the cultivation medium;   further cultivating the cell comprising the nucleic acid encoding the polypeptide in the fed-batch or continuous cultivation within the cultivation vessel, wherein the ratio of the volume of the mixed feed solution in the chamber to the volume of the cultivation medium in the cultivation vessel is from 0.8-1.2 ml chamber volume per 1 liter volume of the cultivation medium in the cultivation vessel 0.8 ml/L to 1.2 ml/L; and   recovering the polypeptide from the cultivation medium or the cells,   wherein the feed solution mixture has a pH value upon addition to the cultivation vessel of from pH 4.0 to pH 6.0.