Hoffmann-la Roche Inc.

F. Hoffmann-la Roche Ag

Alexander Jockwer

Michael Pohlscheidt

Oskar Braunersreuther

Torsten Klabuhn

Joerg Thiele

Christian Klinger

Detlef Eisenkraetzer

Betina Da Silva Ribeiro

Barbara Bezdek-Sueess

Thomas Noll

Jochen Gaetgens

Markus Emmler

Thomas Troebs

Jens Hoffmann

Sylvia Baumann

Barbara Bezdek-Suess

Separating apparatus, comprising a sedimentation settler and a collection vessel disposed underneath and being in fluid communication with the sedimentation settler, the collection vessel forming a receiving chamber having an outlet at or adjacent to the chamber bottom and having an inlet opening, wherein the collection vessel is arranged such the flow direction of the fluid in the area underneath the sedimentation settler is substantially in line with the direction of the channels of the sedimentation settler.

Separation apparatus and use thereof

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.

Feed mixing device and its use

A dissolved COchange in aqueous solutions affects directly the intracellular pH (pHi) value as it does so by influencing therefore important cellular processes. The enzyme carbonic anhydrase II (CAII) catalyzes the equilibrium of COin aqueous solutions and because it alters the speed at which this equilibrium is reached it was identified as a strong candidate for metabolic engineering. The cell line stably expressing hCAII presented a better initial re-alkalinization of cytoplasm after induced COacid load. The most alkaline pHi value associated to the lowest pHi variations was observed for that cell line in long term increased COlevels. In general, the increased COprofile triggered the quicker progress of G0G1-cell cycle phase for both transfected and control cell lines.

COprofile cultivation

A method for the recombinant production of a polypeptide in a eukaryotic host cell modified in the citrate cycle is described, wherein the method comprises cultivating the eukaryotic host cell in a suitable medium under conditions which allow the expression of the polypeptide, wherein the content of dissolved CO(pCO) in the medium is maintained at a constant value in the range of 10% to 20%.

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Eurasburg, Germany

Alexander Jockwer