Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-02-09
2004-10-12
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069500, C435S069600, C435S070100, C435S070210, C435S404000, C435S408000, C436S548000, C530S413000, C530S417000
Reexamination Certificate
active
06803209
ABSTRACT:
TECHNICAL DESCRIPTION
A serum containing preparation is provided that is depleted in or devoid of interfering proteins, the proteins being otherwise present in the serum and methods for preparing the same. Methods of purifying cell culture product is further provided.
BACKGROUND TO THE INVENTION
Preparation of proteins using eukaryotic cell culture techniques is a well established methodology. Purification of proteins manufactured in eukaryotic cell cultures that rely on serum enriched media is problematic in part because the serum contains related proteins. These proteins are bovine proteins if the serum is derived from a cow or calf or bovine fetus. Alternatively, the proteins may be rabbit, horse or other animal proteins where these animals are the source of the serum. It is desirable to avoid a step or several steps of purification of the desired protein away from related contaminating proteins derived from the serum.
One approach to this problem is to avoid the use of serum in the medium in which the cells are cultured. This is often problematic because cells rely on the rich source of nutrients provided by serum. Alternatively, the serum may be treated prior to sterilization and use by means of affinity chromatography techniques to remove the contaminating proteins. The problem here is that serum is very viscous and the capacity of the affinity chromatography column is small. Therefore affinity chromatography of serum is costly and time consuming.
As the requirement for therapeutic molecules and research reagents increases, the need for rapid methods for producing large amount of serum containing medium that is devoid of interfering proteins increases.
An example of a group of proteins that is of interest in bio-diagnostics, therapeutics and as research reagents and is prepared in cell culture are the antibodies and the Fc chimeric proteins that have the Fc part of an antibody fused with another protein. It is desirable to purify this group of proteins as a product of tissue culture. One of the most commonly used procedures for purification of antibodies is based on immunoaffinity purification using protein A or G. The original protocols use high porosity cross-linked carbohydrate supports for example, Sepharose. Since these supports are compressible, the purification using these supports done at slow flow rates. These slow flow rates are problematic and to speed up the rate of purification, newer chromatography technologies have recently been developed which permit much higher flow rates in chromatographic processes, for example, Perfusion Chromatography (PE Biosystems), HyperDiffusion Chromatography (BioSepra, Inc.) etc. Perfusion chromatography involves the flow of liquid through a non-compressible porous chromatographic particle POROS® Media, PE Biosystems) and the maximum flow rate is limited by the maximum pressure the chromatography system can withstand. It has become extremely desirable to produce antibody molecules and the Fc-fusion proteins in cell culture media that are devoid of bovine antibodies that may interfere with the purification of the desired protein molecules.
SUMMARY
The embodiments of the invention provide a novel culture medium and method of purifying proteins.
In a preferred embodiment, a cell culture medium is provided that includes a mixture of a serum supplement and a culture medium, wherein the mixture is deficient in a compound normally present in the serum supplement.
In a preferred embodiment, a method of preparing a culture medium containing serum, suitable for production in cells of a first protein in a class of proteins is provided where the medium being deficient in a second protein in a related class, the second protein normally present in the serum and capable of interfering with either the growth of the cells or the purification of the first compound; the steps of the method comprising; selecting the culture medium containing serum; subjecting the mixture to a chromatography step so as to provide an eluant, the eluant being deficient in the interfering second protein; and utilizing the eluant as a culture medium for production of the first protein by cells.
In a preferred embodiment, a method for obtaining a purified cell culture product; is provided that includes the steps of selecting a serum supplement and a nutrient medium suitable for cell culture; combining the serum supplement with the nutrient medium to form a mixture; subjecting the mixture to an affinity chromatography step so as to remove a compound capable of interfering with the preparation of the cell culture product, the chromatography step providing a flow through, and obtaining the purified cell culture product from cells grown or maintained in the flow through.
In further embodiments of the invention, affinity chromatography includes protein A and protein G column and more particularly perfusion column. Cell culture products include but are not limited to monoclonal antibodies, Fc-fusion proteins (Fc-Chimeric proteins), MHC proteins, growth factors, cytokines, hormones and serum albumin. Proteins to be removed from serum include but are not limited to antibodies, proteins or protein fragments or peptides capable of binding MHC, growth factors, cytokines serum albumin and pathogenic material.
REFERENCES:
patent: 5019270 (1991-05-01), Afeyan et al.
Kriegsman & Kriegsman
Saunders David
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