Agency for Science, Technology and Research

Bio-rad Laboratories, Inc.

Peter Stanley Gagnon

Wei Zhang

Hui Theng Gan

Vincent Vagenende

Yan Ling Phyllicia Toh

Tze Yang Lee

This application describes compositions, kits, and methods for removing contaminants from a protein preparation, for example a cell culture harvest comprising a recombinant protein. The methods described herein can enhance the performance of a later applied chromatographic purification step. The method generally involves contacting a protein preparation comprising a desired protein with a combination of allantoin, a cationic polymer, and a fatty acid, thereby causing the formation of solids and removal of the solids using a suitable process. Further treatment by contacting the treated sample with a chemically reactive surface further enhances results achieved by the first purification step.

Method of enhancing the performance of chromatography methods for purification of proteins

Methods are disclosed for use of apatite chromatography, particularly without reliance upon phosphate gradients, for purification or separation of at least one intact non-aggregated antibody, or at least one immunoreactive antibody fragment, from an impure preparation. Integration of such methods into multi-step procedures with other fractionation methods are additionally disclosed.

Enhanced purification of antibodies and antibody fragments by apatite chromatography

Provided herein is a novel method of purifying an IgG antibody from a preparation by use of an electropositive membrane having a defined porosity.

Method of purifying an antibody

A method of reducing the aggregate content in a preparation having a target protein includes contacting the preparation with an aryl anion to form a mixture and contacting the mixture with at least one electropositive solid to remove excess aryl anion.

Methods for reducing aggregate content of protein preparations by treatment with aryl anions

Methods are provided for enhancing reduction of virus and viral DNA levels in protein preparations.

Virus reduction method

Methods are provided for extracting DNA-compaction proteins from biological samples.

Protein extraction methods

A method of purifying a desired protein from a preparation includes providing the preparation in a form having less than about 5% of the chromatin residing in the original production medium, contacting the preparation with a nonionic organic polymer in an amount sufficient to cause the desired protein to precipitate or adsorb on a nonionic hydrophilic surface, and adjusting a salt concentration before or during the contacting step, the adjusting step providing a sufficient salt concentration to produce a conductivity greater than physiological conductivity.

Protein purification in the presence of nonionic organic polymers at elevated conductivity

Methods for reduction of aggregate levels in antibody and other protein preparations through treatment with low concentrations of electropositive organic additives (e.g., ethacridine, chlorhexidine, or polyethylenimine) in combination with ureides (e.g., urea, uric acid, or allantoin) or organic modulators (e.g., nonionic organic polymers, surfactants, organic solvent or ureides). Some aspects of the invention relate to methods for reducing the level of aggregates in conjunction with clarification of cell culture harvest. It further relates to the integration of these capabilities with other purification methods to achieve the desired level of final purification.

Methods for reducing levels of protein-contaminant complexes and aggregates in protein preparations by treatment with electropositive organic additives

A method of purifying a target protein includes contacting a cell culture harvest or a protein preparation including at least one target protein with at least one fatty acid having 8 to 10 carbon atoms to form a mixture, contacting the mixture with one or more solids to form a mixture, the one or more solids comprise a cationic functional group, a metal binding functional group, or both, the metal binding functional group including a nitrogen-containing moiety selected from (1) a polyamine, (2) an imine, (3) an N-heterocycle, (4) an amino acid, (5) an N-hydroxyamide, (6), an arylamine, and combinations thereof, and separating solid materials after contacting the mixture with the one or more solids to provide a solution comprising the target protein.

Protein purification process

A method of purifying a target antibody includes contacting a cell culture harvest or a protein preparation including at least one target antibody with at least one fatty acid having 7 to 10 carbon atoms to form a mixture, contacting this mixture with allantoin, and then separating solid materials to provide a solution comprising the target antibody. Solid materials can be removed by filtration, sedimentation or centrifugation, and the fatty acids can be enanthic, caprylic, pelargonic, nonenoic or capric acid. The invention is also directed to kits used to facilitate this method of antibody purification.

Antibody purification process

A method of purifying a desired protein from a preparation includes (a) providing the preparation in a form having less than about 5% of chromatin residing in an original production medium, (b) contacting the preparation with a nonionic organic polymer and a salt, a concentration of nonionic organic polymer being sufficient to precipitate the desired protein or cause its accretion on a hydrophilic surface, or maintain it in a precipitated state or accreted on the hydrophilic surface, the salt concentration being sufficient to produce greater than physiological conductivity, and (c) contacting the preparation with at least one electropositive surface, optionally in the presence of a salt concentration sufficient to produce greater than physiological conductivity, the desired protein does not substantially adsorb to the at least one electropositive surface while not preventing adsorption of acidic contaminants to the at least one electropositive surface.

Protein purification in the presence of nonionic organic polymers and electropositive surfaces

A method of reducing aggregate content in a preparation having a target protein includes contacting the preparation with an alkyl cation to form a mixture, and contacting the mixture with at least one functionalized solid to remove excess alkyl cation.

Methods for reducing chromatin content in protein preparations by treatment with alkyl cations

Methods for the improved purification of antibodies and other proteins from protein preparations including the steps of conditioning the protein preparation by contacting it with multivalent organic ions, then applying the conditioned preparation to an adsorptive chromatography medium.

Chromatographic purification of antibodies from chromatin-deficient cell culture harvests

A method of reducing the aggregate content in a protein preparation having a target protein includes contacting the protein preparation with a thio-heterocyclic cation to form a mixture, contacting the mixture with at least one functionalized solid to remove excess thio-heterocyclic cations; and optionally contacting the mixture, simultaneously or sequentially, with at least one further functionalized solid to further reduce aggregate content of the protein preparation.

Methods for reducing aggregate levels in protein preparations by treatment with thio-heterocyclic cations

A method for the purification of a desired protein from a protein preparation includes conditioning the protein preparation by treatment with soluble organic multivalent ions, immobilized organic multivalent ions, or both, optionally in the presence of supersaturated allantoin, thereby removing at least 90% of chromatin, then (1) precipitating the desired protein with a nonionic organic polymer in the presence of non-protein-precipitating salts at greater than physiological concentration to provide a precipitate of the desired protein; or (2) precipitating the desired protein with a nonionic organic polymer in the absence of non-precipitating salts at greater than physiological concentration to provide a precipitate and subsequently washing the precipitate with a nonionic organic polymer in the presence of non-protein-precipitating salts at greater than physiological concentration.

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Singapore, Singapore

Peter Stanley Gagnon