Flow-through protein purification process

Medicilon's protein scientists have been working on protein expression and purification for many years. We can start your project even you have nothing in hand but the name of your protein. In Medicilon's laboratories, protein purification is performed in scales from micrograms and milligrams. All Protein Purification Services start with the analysis of physico-chemical and biological properties of a target protein resulting in the development of tailored procedures for its extraction, purification and characterization. email:marketing@medicilon.com.cn Web:www.medicilon.com

A process for isolating a protein of interest from a sample includes the steps of providing a sample comprising the protein of interest; and subjecting the sample to chromatography in flow-through mode, wherein the sample is applied to a chromatography material under conditions that highly bind the protein of interest but where the amount of protein of interest exceeds the binding capacity of the chromatography material such that a substantial amount of the protein of interest does not adsorb to the material; and collecting at least a portion of the sample that does not adsorb to the material, to thereby isolate the protein of interest.

The present invention relates to the field of protein purification. In the large-scale production of pharmaceutically important proteins, which production typically uses prokaryotic or eukaryotic cell lines engineered to express the protein of interest, biologies manufacturers face formidable challenges in the separation of the desired protein from cellular byproducts and other impurities created in the production process. These challenges include compliance with strict regulatory standards, which typically require that protein-based pharmaceutical products be substantially free from impurities, such as product related contaminants, e.g., aggregates, fragments and variants of the recombinant protein, and process related contaminants, e.g., host cell proteins (HCPs), media components, viruses, DNA, and endotoxins. Reported Developments

While various protein purification schemes are available to the biopharmaceutical industry, these schemes typically include one or more "bind-and-elute" steps and/or affinity purification steps in order to reach a pharmaceutically acceptable degree of purity. The bind-and-elute processes adsorb the protein of interest to a chromatography medium, which is then washed to remove impurities, followed by elution of the protein of interest from the chromatography medium by altering solvent conditions. Because these methods employ multiple steps and washes, they result in high costs.

An example of these processes is disclosed in US Pat. No. 7,323,553, which describes a method for purifying antibodies from a mixture containing host cell proteins using non-affinity chromatography purification steps, including cation exchange chromatography in a bind-and-elute mode, followed by a high-performance tangential- flow filtration (HPTFF) step. Another example is disclosed in WO 2007/108955, which describes the purification of antibodies or antibody-like proteins, from a cell culture supernatant, using cation exchange in a bind-and-elute mode followed by anion exchange chromatography.

The present invention relates to the flow-through protein purification system, eliminating the expensive and time-consuming bind-and-elute protein purification process.

Summary of the Invention

The present invention is based, at least in part, on the surprising discovery that significant purification of proteins from mixtures containing multiple contaminants can be obtained by using a chromatographic medium under conditions where the protein of interest binds to the medium, but where the amount of protein of interest contacted with the medium exceeds the capacity of the medium. This discovery permits the design of a downstream flow-through process, eliminating the typical bind-and-elute process, for the purification of proteins. The processes described herein can, as compared to prior systems, result in a shortening of processing time and lower costs due to, e.g., reduced buffer consumption, faster purification time, reduced cleaning validation cost, and/or lower capital expenditure (e.g., due to smaller footprint facilities with simple equipment).

More particularly, the present invention relates to a process for purifying a protein of interest, comprising:

(a) providing a sample comprising an amount of protein of interest and impurities;

(b) contacting said sample with a cation exchange, anion exchange or hydrophobic interaction material under chromatography conditions, which absorb said protein of interest, wherein said amount of protein of interest in said sample exceeds the capacity of said material to adsorb more than an insubstantial portion of said amount of protein of interest, and wherein a substantial amount of said impurities adsorb to said material; and

(c) separating said material to which impurities have been adsorbed from the sample containing the substantial portion of said protein of interest that has not adsorbed to said material, to result in a purified sample.

In a preferred aspect of the present invention, the process is conducted in a flow- through mode.

In a particularly preferred aspect of the present invention, the material is a cation exchange material and wherein said chromatography conditions of step (b) provide for said protein of interest to exhibit a net positive charge. In another aspect of the present invention, the pH and conductivity of said sample are selected to (i) maximize the purification yield of said protein of interest, and (ii) reduce the impurities retained in said purified sample. In some embodiments, the protein of interest has a pi between 6.5 and 9.5, and the pH and conductivity values of the chromatography conditions are set based on such pi.

In another preferred aspect of the present invention, the material is hydrophobic interaction chromatography material, and wherein said chromatography conditions of step (b) provide for said sample to contain concentration of lyotropic salt selected to maximize binding of said protein of interest. A preferred sample contacted with said hydrophobic interaction chromatography material contains the protein of interest together with aggregate impurities from about 1% to about 20%, and of host cell protein impurities from about 10 ppm to about 1000 ppm. In another preferred aspect of the present invention, the material is anion exchange material, and wherein said chromatography conditions of step (b) provide for said protein of interest to exhibit a net positive charge.

In a further preferred aspect of the present invention, the purified sample obtained from the cation exchange chromatography (CEX) is subjected to anion exchange chromatography (AEX) in a flow-through mode to form a second purified sample.

In a most preferred aspect of the present invention, the second purified sample is subjected to hydrophobic interaction chromatography (HIC) in a flow-through mode to form a further purified sample.

In some embodiments of the present invention, the insubstantial portion of said amount of protein of interest that binds to said material is less than about 20%, preferably less than about 15%, more preferably less than about 10%, and most preferably less than about 5% of the total amount of the protein of interest in the sample.

In other embodiments, the present invention process includes at least two additional processing steps prior to step (b) above, such as (i) filtering the sample, and/or (ii) removing cells from the sample by sedimentation, flocculation, enhanced cell settling, and/or centrifugation, and/or (iii) precipitating the protein of interest with polyethylene glycol (PEG). In further embodiments, the process includes inactivating a virus present in the sample or a portion thereof, as well as filtering the sample or a portion thereof to remove viruses.

In some embodiments of the present invention, at least 50%, preferably at least 60%, more preferably at least about 75%, and most preferably at least about 90% of the protein of interest originally present in the sample is collected at step (c).

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below.