HK1183890A - Apparatus and process of purification of proteins - Google Patents

Description

Apparatus and method for protein purification

Cross reference to related applications

This application claims priority from U.S. provisional patent application serial No. 61/345,634, filed on 18/5/2010, which is incorporated herein by reference in its entirety.

Background

The present invention relates generally to an apparatus and method for purifying proteins.

The economics of large scale protein purification are important, particularly for therapeutic antibodies, since antibodies constitute a large percentage of the therapeutic biology on the market. In addition to their therapeutic value, monoclonal antibodies, for example, are also important tools in the diagnostic field. Many monoclonal antibodies have been developed and used for diagnosis of many diseases, pregnancy and for drug testing.

Typical purification processes involve multiple chromatographic steps to meet purity, yield and throughput requirements. These steps typically involve trapping, intermediate purification or polishing, and final polishing. Affinity chromatography (protein a or G) or ion exchange chromatography is often used as the capture step. Traditionally, the capture step is followed by at least two other intermediate purification or polishing chromatography steps to ensure sufficient purity and virus clearance. The intermediate purification or polishing step is typically accomplished by affinity chromatography, ion exchange chromatography, or hydrophobic interaction, among others. In conventional methods, the final polishing step can be accomplished by ion exchange chromatography, hydrophobic interaction chromatography, or gel filtration chromatography. These steps remove process and product related impurities from the product stream and cell culture, including Host Cell Proteins (HCPs), DNA, leached protein a, aggregates, fragments, viruses, and other small molecule impurities.

Summary of The Invention

Briefly, the present invention is directed to an apparatus for purifying a protein from a sample containing the protein to be purified comprising a capture chromatography resin, a depth filter disposed relative to the capture chromatography resin such that the sample passes through the capture chromatography resin into the depth filter, and a mixed mode chromatography resin disposed relative to the depth filter such that the sample passes through the depth filter into the mixed mode chromatography resin.

In addition, the invention relates to a method of purifying a protein comprising providing a sample containing the protein, processing the sample through a capture chromatography resin to provide a first eluate comprising the protein, processing the first eluate through a depth filter after processing the sample through the capture chromatography resin to provide a filtered eluate comprising the protein, and processing the filtered eluate through a mixed mode chromatography resin after processing the first eluate through the depth filter to provide a second eluate comprising the protein.

The invention also relates to an apparatus and method for purifying a protein comprising providing a sample containing the protein, processing the sample through a capture chromatography resin to provide a first eluate comprising the protein, processing the first eluate through a depth filter to provide a filtered eluate comprising the protein, and processing the filtered eluate through a membrane adsorber or monolith to provide a second eluate comprising the protein.

Brief Description of Drawings

Figure 1 illustrates a schematic diagram of one embodiment of the process.

Figure 2 illustrates another schematic diagram of one embodiment of the process.

Figure 3 illustrates another schematic diagram of one embodiment of the process.

Figure 4 illustrates another schematic diagram of one embodiment of the process.

FIGS. 5a and 5b illustrate HCP clearance profiles for protein purification methods.

Fig. 6a and 6b illustrate the leached protein a clearance profile of the protein purification process.

FIGS. 7a and 7b illustrate aggregate clearance profiles for protein purification methods.

FIGS. 8a and 8b illustrate DNA clearance profiles for protein purification methods.

FIGS. 9a and 9b illustrate the step yields of the protein purification process.

Figure 10a illustrates HCP levels of protein purification process as a function of XOHC feed loading on depth filter under different buffer conditions.

Figure 10a illustrates HCP removal by protein a capture/depth filtration after pH inactivation at a 3000L production scale.

FIGS. 11a, 11b and 11c illustrate impurity clearance profiles obtained by a two-column protein purification method.

FIGS. 12a and 12b illustrate HCP clearance profiles for protein purification methods.

Figures 13a and 13b illustrate the leached protein a clearance profile of the purification process.

FIGS. 14a and 14b illustrate aggregate clearance profiles for protein purification methods.

FIGS. 15a and 15b illustrate DNA clearance profiles for protein purification methods.

FIGS. 16a and 16b illustrate the step yields of the protein purification process.

Detailed description of the embodiments

Reference now will be made in detail to embodiments of the invention, one or more examples of which are set forth below. The examples are provided as illustrations of the invention and not as limitations of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

In one embodiment, the invention includes a two-step chromatographic step protein purification system and method. The overall recovery using the system and method of the present invention is acceptable and the final product quality is comparable to the more traditional method. By eliminating specific steps in downstream processing, higher productivity is achieved while maintaining acceptable integrity and purity of the molecule. For example, minimizing the number of chromatography steps reduces the number of process components, buffers, tanks, and miscellaneous equipment commonly used in conventional protein purification processes.

Schematic diagrams of several embodiments of the two-step chromatographic step purification system of the present invention are provided in fig. 1-4. In one embodiment of the invention, a sample containing a protein is provided. Any protein-containing sample can be used in the present invention. Samples containing proteins may include, for example, cell cultures or mouse ascites fluid. The protein may be any protein or fragment thereof known in the art. In some embodiments, the protein is an antibody. In a specific embodiment, the protein is a monoclonal antibody or fragment thereof. In some cases, the protein may be a human monoclonal antibody. In other embodiments, the protein is an immunoglobulin G antibody. In still other embodiments, the protein is a fusion protein, such as an Fc-fusion protein.

In one embodiment of the invention, the protein-containing sample may first be clarified using any method known in the art (see fig. 2, step 1). The clarification step seeks to remove cells, cell debris and some host cell impurities from the sample. In one embodiment, the sample may be clarified by one or more centrifugation steps (see fig. 3-4, step 1). Centrifugation of the sample may be performed as is known in the art. For example, canTo use about 1 x 10^-8Centrifugation of the samples was performed at a normalized loading of m/s and gravity of about 5,000 Xg to about 15,000 Xg.

In another embodiment, the sample may be clarified by microfiltration or ultrafiltration membranes. In some embodiments, the microfiltration or ultrafiltration membrane may be in a Tangential Flow Filtration (TFF) mode. Any TFF clarification method known in the art may be used in this embodiment. TFF refers to a membrane separation process in a cross-flow configuration driven by a pressure gradient, wherein the membrane fractionates the components of a liquid mixture according to particle and/or solute size and structure. In clarification, the membrane pore size is selected to allow some components to pass through the pores with the water while leaving cells and cell debris on the membrane surface. In one embodiment, TFF clarification can be performed with polysulfone membranes using, for example, a 0.1 μm or 750 kD molecular weight cut-off, 5-40 psig, and a temperature of about 4 ℃ to about 60 ℃.

In yet another embodiment, the sample may be clarified by one or more depth filtration steps (see fig. 3-4, step 1). Depth filtration refers to a method of removing particles from a solution using a series of filters arranged in sequence with decreasing pore size. The depth filter three-dimensional matrix creates a labyrinth-like path for the sample to flow through. The retention principle mechanism of depth filters depends on random adsorption and mechanical entrapment throughout the depth of the matrix. In various embodiments, the filter film or sheet can be cotton wool (wind cotton), polypropylene, rayon cellulose, glass fiber, sintered metal, porcelain, diatomaceous earth, or other known components. In certain embodiments, a composition comprising a depth filter membrane may be chemically treated to provide an electropositive charge, i.e., a cationic charge, to enable the filter to capture negatively charged particles, such as DNA, host cell proteins, or aggregates.

Any depth filtration system available to those skilled in the art may be used in this embodiment. In one particular embodiment, the depth filtration step may be carried out with Millistak +. Pod depth filter systems, XOHC media, available from Millipore Corporation. In another embodiment, the depth filtration step may be performed with a Zeta Plus ™ depth filter available from 3M Purification Inc.

In some embodiments, the depth filter media has a nominal pore size of about 0.1 microns to about 8 microns. In other embodiments, the depth filter media may have a pore size of about 2 microns to about 5 microns. In one particular embodiment, the depth filter media can have a pore size of about 0.01 microns to about 1 micron. In still other embodiments, the depth filter media may have a pore size greater than about 1 micron. In still other embodiments, the depth filter media may have a pore size of less than about 1 micron.

In some embodiments, the depth filtration clarification step may comprise the use of two or more depth filters arranged in series. In such an embodiment, for example, Millistak + mini DOHC and XOHC filters may be arranged in series and used in the clarification step of the invention.

Any combination of these or other clarification methods known in the art may be used as an optional clarification step in the present invention. For example, the clarification step may comprise centrifugation and depth filtration (see FIGS. 3-4, step 1).

In a particular embodiment, the present system comprises the use of a clarification step and further processing steps (see fig. 2, step 2). The further processing step may comprise a non-chromatographic purification step.

In one embodiment, the further processing step may comprise treatment with a detergent (see FIGS. 3-4, step 2). The detergent used may be any detergent known to be useful in protein purification processes. In one embodiment, the detergent may be applied to the sample at low levels and the sample is then incubated for a time sufficient to inactivate the enveloped mammalian virus. The amount of detergent to be applied may be in one embodiment from about 0 to about 1% (v/v). In another embodiment, the amount of detergent to be applied may be from about 0.05% to about 0.7% (v/v). In yet another embodiment, the amount of detergent to be applied may be about 0.5% (v/v). In one specific embodiment, the detergent may be polysorbate 80 (Tween 80) or Triton X-100. This step provides additional clearance of enveloped virus and improves the robustness of the overall process. This step may be referred to as a detergent virus inactivation step.

In one embodiment, after the optional clarification and further purification steps of the present invention, the sample may be subjected to a chromatographic capture step (see FIGS. 1-2). The capture step is designed to separate the protein from the clarified sample. Typically, the capture step reduces HCP, host cell DNA, and endogenous virus or virus-like particles in the sample. The chromatographic mechanism used in this embodiment may be any mechanism known in the art for use as a capture step. In one embodiment, the sample may be subjected to affinity chromatography, ion exchange chromatography or hydrophobic interaction chromatography as the capture step.

In one embodiment of the invention, affinity chromatography may be used as the capture step. Affinity chromatography utilizes specific binding interactions between molecules. The specific ligand is chemically immobilized or "coupled" to the solid support. Proteins in the sample having specific binding affinity for the ligand are bound as the sample passes through the resin. After washing away other sample components, the bound protein is then stripped from the immobilized ligand and eluted to purify it from the original sample.

In this embodiment of the invention, the affinity chromatography capture step may comprise an interaction between an antigen and an antibody, an enzyme and a substrate, or a receptor and a ligand. In a particular embodiment of the invention, the affinity chromatography capture step may comprise protein a chromatography, protein G chromatography, protein a/G chromatography or protein L chromatography.

In certain embodiments, protein a affinity chromatography may be used in the capture step of the invention (see fig. 3-4, step 3). Protein a affinity chromatography involves the use of protein a, a bacterial protein that demonstrates specific binding to the non-antigen binding portion of many types of immunoglobulins. The protein a resin used may be any protein a resin available to the person skilled in the art. In one embodiment, the protein A resin may be selected from the MabSelect ™ series of resins available from GE Healthcare Life Sciences. In another embodiment, the protein A resin may be ProSep Ultra Plus resin available from Millipore Corporation. Any column available in the art may be used in this step. In a specific embodiment, the column may be a MabSelect ™ column available from GE Healthcare Life Sciences or a ProSep Ultra Plus column available from Millipore Corporation.

If protein A affinity is used as a chromatography step, the column may have an inner diameter of about 5 cm and a column length of about 20 cm. In other embodiments, the column length may be from about 5 cm to about 100 cm. In yet another embodiment, the column length may be from about 10 cm to about 50 cm. In yet another embodiment, the column length may be about 5 centimeters or greater. In one embodiment, the column may have an inner diameter of about 0.5 cm to about 2 meters. In another embodiment, the column may have an inner diameter of about 1 cm to about 10 cm. In yet another embodiment, the column may have an inner diameter of about 0.5 cm or greater.

The particular method used for the chromatographic capture step, including passing the sample through the column, washing and elution, depends on the particular column and resin used and is generally supplied by the manufacturer or known in the art. The term "processing" as used herein may describe the process of a sample flowing through or past a chromatography column, resin, membrane, filter or other mechanism, and shall include continuous flow through each mechanism as well as flow that is paused or stopped between mechanisms.

Following the chromatographic capture step, the eluate may be subjected to viral inactivation (see FIGS. 2-4, step 4). In one embodiment, such a virus inactivation step may comprise low pH virus inactivation (see fig. 3-4, step 4). In one aspect, a high concentration glycine buffer at low elution pH can be used in the final eluate pool in the target range for low pH viral inactivation without further pH adjustment. Alternatively, an acetate or citrate buffer may be used for elution, and then the pool of eluate is titrated to the appropriate pH range for low pH viral inactivation. In one embodiment, the pH is from about 2.5 to about 4. In another embodiment, the pH is from about 3 to about 4.

In one embodiment, once the pH of the eluate pool is lowered, the pool is incubated for a period of about 15 to about 90 minutes. In one embodiment, the low pH viral inactivation step may be achieved by titration with 0.5M phosphoric acid to obtain a pH of about 3.5, followed by incubation of the sample for 1 hour.

After the low pH viral inactivation step, the inactivated eluate pool may be neutralized to a higher pH. In one embodiment, the neutralized, higher pH may be a pH of about 6 to about 10. In another embodiment, the neutralized, higher pH may be a pH of from about 8 to about 10. In yet another embodiment, the neutralized, higher pH may be a pH of from about 6 to about 10. In yet another embodiment, the neutralized, higher pH may be a pH of about 6 to about 8. In yet another embodiment, the neutralized, higher pH may be a pH of about 8.1.

In one embodiment, pH neutralization may be accomplished using 1M Tris pH 9.5 buffer or another buffer known in the art. The conductivity of the inactivated eluate pool may then be adjusted with purified water or deionized water. In one embodiment, the conductivity of the inactivated eluate pool can be adjusted to about 0.5 to about 50 mS/cm. In another embodiment, the conductivity of the inactivated eluate pool can be adjusted to about 6 to about 8 mS/cm.

In other embodiments, the virus inactivation step may be performed using other methods known in the art. For example, the virus inactivation step may, in various embodiments, include treatment with an acid, detergent, chemical, nucleic acid cross-linking agent, ultraviolet light, gamma radiation, heat treatment, or any other method known in the art for such use.

Following the optional virus inactivation step, the inactivated eluate pool may be subjected to depth filtration as described above (see FIGS. 1-4). This depth filtration step may be supplemented with depth filtration as a clarification step. In one embodiment, this step may include using two or more depth filters arranged in series. With proper sizing of the depth filter according to processing conditions known in the art, various impurities can be removed or reduced from the process stream prior to further processing.

In one embodiment, the depth filtration step may be followed by or combined with a sterile filtration step (see FIGS. 3-4, step 5). Any sterile filter known in the art may be used in this embodiment. In one embodiment, the sterile filter is a microfilter. In one aspect of the invention, the sterile filters may comprise Sartopore 2 bacteria grade filters. The sterilizing filter may for example have a 0.45 micron pre-filter before a 0.2 micron final filter. In another embodiment, the sterilizing filter may have a membrane porosity of about 0.1 microns to about 0.5 microns. In other embodiments, the sterilizing filter may have a membrane porosity of about 0.1 microns to about 0.3 microns. In one aspect, the sterilizing filter may have a membrane porosity of about 0.22 microns. In one embodiment, the sterilizing filter may be arranged in series with the depth filter.

After depth filtration and optional sterile filtration, the sample may then be subjected to an intermediate/final polishing step (see fig. 1-2). In one embodiment, the intermediate/final polishing step may comprise a mixed mode (also called multimodal) chromatography step (see fig. 3, step 6). In this step, the sample is cleared of residual HCP, DNA, leached protein a, and aggregates. The mixed mode chromatography step used in the present invention may employ any mixed mode chromatography known in the art. Mixed mode chromatography involves the use of solid phase chromatography supports in the form of resins, monoliths or membranes that use a variety of chemical mechanisms to adsorb proteins or other solutes. Examples useful in the present invention include, but are not limited to, chromatographic supports that utilize a combination of two or more of the following mechanisms: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, thiophilic interaction, hydrogen bonding, pi-pi bonding, and metal affinity. In particular embodiments, mixed mode chromatography is combined with: (1) anion exchange and hydrophobic interaction techniques; (2) cation exchange and hydrophobic interaction techniques; and/or (3) electrostatic and hydrophobic interaction techniques.

In one embodiment, the mixed mode chromatography step may be accomplished using columns and resins, such as Capto # adhesive columns and resins available from GE Healthcare Life Sciences. The Capto adhesive columns are multi-mode media for intermediate purification and polishing of the captured monoclonal antibodies. In a particular embodiment, the mixed mode chromatography step may be performed in flow-through mode. In other embodiments, the mixed mode chromatography step may be performed in bind-elute mode.

In other embodiments, the mixed mode chromatography step may be accomplished using one or more of the following systems: capto MMC (available from GE Healthcare Life Sciences), HEA HyperCel cassettes (available from page Corporation), PPA HyperCel cassettes (available from page Corporation), MBI HyperCel cassettes (available from page Corporation), MEP HyperCel cassettes (available from page Corporation), Blue tristryl M (available from page Corporation), CFT kinetic fluorapatate (available from Bio-Rad Laboratories, Inc.), CHT nuclear hydroxamate (available from Bio-Rad Laboratories, Inc.), and/or ABx (available from j.t. Baker). The particular method used for the mixed mode chromatography step may depend on the particular column and resin used and is generally supplied by the manufacturer or known in the art.

Each column used in the process can be large enough to provide maximum throughput and maximum economies of scale. For example, in certain embodiments, each column can define an internal volume of about 1 liter to about 1500 liters, about 1 liter to about 1000 liters, about 1 liter to about 500 liters, or about 1 liter to about 250 liters. In some embodiments, the mixed mode column may have an inner diameter of about 1 cm and a column length of about 7 cm. In other embodiments, the mixed mode column may have an inner diameter of about 0.1 cm to about 10 cm, about 0.5 cm to about 5 cm, about 0.5 cm to about 1.5 cm, or may be about 1 cm. In one embodiment, the mixed mode column may have a column length of from about 1 to about 50 centimeters, from about 1 to about 20 centimeters, from about 5 to about 10 centimeters, or may be about 7 centimeters.

In some embodiments, the systems of the invention are capable of handling high titer concentrations, such as about 5 g/L, about 6 g/L, about 7 g/L, about 8 g/L, about 9 g/L, about 10 g/L, about 12.5 g/L, about 15 g/L, about 20 g/L, about 25 g/L, about 1 g/L to about 5 g/L, about 5 g/L to about 10 g/L, about 5 g/L to about 12.5 g/L, about 5 g/L to about 15 g/L, about 5 g/L to about 20 g/L, or about 5 g/L to about 55 g/L, or a concentration of about 5 g/L to about 100 g/L. For example, some systems are capable of handling high antibody concentrations while processing from about 200L to about 2000L of culture per hour, from about 400L of culture to about 2000L per hour, from about 600L to about 1500L of culture per hour, from about 800L to about 1200L of culture per hour, or greater than about 1500L of culture per hour.

In one embodiment of the invention as shown in fig. 3, the capture column and mixed mode column are single use chromatography columns. In one aspect of this embodiment, a third chromatography column is not used; however, if additional chromatography steps are required for further processing, those steps are also included herein.

In one embodiment, the intermediate/final polishing step may be accomplished by one or more membrane adsorbers or monoliths (see fig. 4, step 6) rather than a mixed-mode column. Membrane adsorbers are thin synthetic microporous or macroporous membranes derivatized with functional groups similar to those on equivalent resins. On their surface, the membrane adsorbers carry functional groups, ligands, interwoven fibers, or reactants that are capable of interacting with at least one substance in contact with a fluid phase passing through the membrane under the influence of gravity. The membranes are typically stacked 5 to 15 layers deep in a relatively small cartridge to create a much smaller footprint than a column with similar output. The membrane adsorber used herein may be a membrane ion exchanger, a mixed mode, a ligand membrane, and/or a hydrophobic membrane.

In one embodiment, the membrane adsorbers used may be ChromaSorb membrane adsorbers available from Millipore Corporation. A ChromaSorb membrane adsorber is a membrane-based anion exchanger designed for MAb and protein purification that is intended to remove trace impurities, including HCPs, DNA, endotoxins, and viruses. Other membrane adsorbers that may be used include Sartobind Q (available from Sartorium BBI Systems GmbH), Sartobind S (available from Sartorium BBI Systems GmbH), Sartobind C (available from Sartorium BBI Systems GmbH), Sartobind D (available from Sartorium BBI Systems GmbH), Pall Mustang (available from Pall Corporation), or any other membrane adsorber known in the art.

As mentioned above, monoliths may also be used in the intermediate/final polishing step of the present invention (see fig. 4, step 6). Monoliths are monolithic porous structures of uninterrupted and interconnected channels of a certain controlled size. The sample is transported through the monolith by convection to cause rapid mass transfer between the mobile and stationary phases. Thus, the chromatographic properties are flow independent (non-flow dependent). Monoliths exhibit low back pressure even at high flow rates, thereby significantly reducing purification time. In one embodiment, the monolith may be an ion-exchange or mixed mode ligand-based monolith. In one aspect, the monoliths used may comprise UNO monoliths (available from Bio-Rad Laboratories, Inc.) or prosrift or IonSwift monoliths (available from Dionex Corporation).

In yet another embodiment, the intermediate/final polishing step may be accomplished by an additional depth filtration step rather than a membrane adsorber, monolith, or mixed mode column. In such an embodiment, the depth filtration for intermediate/final polishing can be a CUNO VR depth filter. In such embodiments, the depth filter may serve intermediate/final polishing as well as virus removal purposes.

After the intermediate/final polishing or mixed mode chromatography step, the eluate pool may be subjected to a viral or nanofiltration step (see fig. 2-4, step 7). In one embodiment, this filtration step may be accomplished by a nanofilter or a viral filter. As shown in fig. 2-4, step 8, a virus or nanofiltration step may optionally be followed by UF/DF to achieve target drug concentrations and buffer conditions prior to bottling. The virus filtration and UF/DF steps can be combined or exchanged with any method known in the art to provide a purified protein that can be bottled (fig. 2-4, step 9).

It can be seen that the process of the present invention provides consistently high product quality and process yield. Furthermore, the method of the present invention can reduce the total downstream batch processing time by about 40% to 50% and significantly reduce the production cost compared to conventional protein purification methods.

In one embodiment, the entire purification process can be completed in less time than usual, e.g., the entire process can be completed in less than 5 days. For example, steps 1 and 2, or steps 3 and 4, or steps 5,6, and 7 (as shown by the dashed lines in fig. 3-4), respectively, may be completed in one day or less. This is about half of the purification time required for a typical three-column process.

The following examples describe various embodiments of the invention. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered as exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples.

Examples 1

Purification experiments were performed and the yields and purities compared to a standard three-column method. The clarified harvest for MAb a (referred to herein as "CH") and protein a eluate for MAb B (referred to herein as "PAE 1") were used in this study. Two runs of each protein sample were performed (example 1 and example 2).

Step (ii) of

The samples were centrifuged and filtered using Millistak +. Pod depth filter systems, XOHC media, available from Millipore Corporation. After filtration, 0.5% (v/v) final concentration of Tween 80 was added to the clarified harvest and the mixture cooled with ice bags. 5 cm (internal diameter (i.d.)) x 20 cm (column length) ProSep Ultra Plus columns for capture. After equilibration, the column was loaded with CH to 45 g/L of MAb A at 400 cm/hr, followed by washing with equilibration and intermediate salt buffers, and then elution with pH 3.5 acetate buffer. The column was regenerated with 0.15M phosphoric acid before the next run. The eluate pool was then mixed and titrated with 0.5M phosphoric acid to pH 3.5, incubated for 1 hour, and then neutralized to pH 8.1 using 1M Tris, pH 9.5 buffer. The conductivity of the cell was adjusted to 6-8 mS/cm using Milli-Q water.

Two sets of conditions for the subsequent steps were evaluated. In example 1, the pH-inactivated protein A pool was passed 23 cm²Millistak +. mini XOHC filters 60L/m²The loading was filtered, followed by 13 cm of filtration available from Sartorius Stedim Biotech²0.45/0.22 μm Sartopore 2 film filters. In a second example, two Millistak +. mini XOHC filters were connected in series and 100L/m per device²The protein a eluate pool was loaded. Each filtrate was then passed through: (1) 1 cm (i.d.) x 7 cm of Capto adhesive posts; or (2) in a standard three-column process comprising 0.66 cm (i.d.) x 21.3 cm Q Sepharose Fast Flow (QSFF) columns (available from GE Healthcare Life Sciences) followed by bind-elute purification on 0.66 cm (i.d.) x 15.2 cm phenyl Sepharose HP columns (available from GE Healthcare Life Sciences). The detailed fine purification conditions are summarized in table 1. All steps were performed at room temperature.

TABLE 1 Experimental conditions for the polishing chromatography steps

Similar experiments were performed to purify PAE1 for MAb B. Instead of starting from a clarified harvest, a protein a eluate pool sample was used in this case. XOHC depth filter is loaded to 60L/m in two operations²And the Capto adhesive columns were loaded to 200 to 250 g/L. Key impurities such as HCP, leached protein a, aggregates/fragments and DNA were measured for each step, as well as step yield.

Results

FIGS. 5-8 show the HCP, leached protein A, aggregates and DNA levels after each unit operation of the three-column method (designated protein A-QSFF-phenyl) vs two-column method of the invention (designated protein A-Capto attachment). It can be seen that the protein A eluate pool (labeled protein A eluate) contains approximately 1700 to 2000 ng/mg HCP, 15 to 26 ng/mg leached protein A and 2.7% to 3.5% of high molecular weight species (in this case no DNA detected). After low pH inactivation, the protein a eluate was filtered through an XOHC depth filter at two different loading levels.

In example 1, in which two XOHC filters were assembled in series and each filter was loaded to 100L/m²(thus the average loading based on total filter area is 50L/m²) Almost all HCP was removed, with residual HCP levels of about 1.8 to about 2.4 ng/mg (shown as XOHC filtrate in the figure). In addition, about 65% of the leached protein a and about 54% of the aggregates were removed. Host cell DNA was also removed from the product pool to a level below the lower limit of detection. In example 2, only one XOHC filter was used and loaded to 60L/m². This results in slightly higher impurity levels: about 56 ng/mg HCP, about 7.2 to 8.6 ng/mg protein A, about 1.8% to 2.0% aggregates and about 30 to 40 pg/mg DNA. Despite the different impurity levels, both XOHC filtrates were purified to yield acceptable XOHC filtrates when processed through subsequent chromatographic steps, either by standard Q plus phenyl columns (standard three column process) or by Capto adhesive columns (two column process) (shown in the figure as flow-through)Quality of final product. The Capto adhesive flow cells contained less than 4 ng/mg HCP, which was within typical acceptable limits (< 10 ng/mg). This step appears to provide more efficient clearance of protein a than both Q and phenyl columns, with residual protein a levels below 1 ng/mg. Furthermore, the final product aggregate levels from both processes were comparable, less than 1%, with DNA below the limit of quantitation. Fig. 8a and 8b summarize the product yields for each purification step. Similar to most other unit operations, the two-column process yields 90% step yield, similar to the combined yield of Q and phenyl operations, thereby making the overall process yields of these two processes comparable.

The use of high efficiency depth filters, such as Millistak +. Pod XOHC depth filter systems, along with the positive charge function in the two column process enhanced the robustness of impurity removal without significantly affecting product yield. Figure 10a shows HCP levels in filtrates from protein a eluate pools passed through an XOHC depth filter under different feed loading conditions. Higher pH and lower loading levels resulted in better HCP clearance. The second pass of the filtrate through another XOHC filter also resulted in almost complete clearance of HCP without further column purification. Similar trends were also observed in examples 1 and 2 as shown in fig. 5-8. Thus, proper sizing of the depth filter prior to the mixed mode intermediate/polishing step ensures robust removal of product-related and process-related impurities and consistent production of quality material throughout the process.

Figure 10b illustrates the application of an XOHC depth filter to post-protein a capture/pH inactivation material at a 3000L production scale. The feed was adjusted to pH 7.9 and conductivity 5.4 mS/cm and at 49L/M²Depth filter area loading. Samples taken during filtration showed greater than 500-fold clearance of residual HCP from the feedstock prior to filtration through the Q membrane device.

To evaluate the general applicability of this two-column method to different MAb molecules, the inventors also evaluated PAE1 for MAb B under the above processing conditions. As shown in fig. 11a and 11b, the overall process yield and final product purity were similar to the levels obtained for CH of MAb a and comparable to that observed in the standard three-column process for this molecule. Therefore, this method has the potential to be a platform technology for large-scale purification of monoclonal antibodies.

By using a high efficiency protein a resin and integrating depth filtration with mixed mode flow-through operation, the two-column process of the present invention can provide yields and product purity comparable to standard three-column processes. A separate detergent inactivation step used prior to protein a capture may provide additional viral clearance for this method. In addition, the process results in no need to use ammonium sulfate salts, reduced hardware, tank inventory, column packing, cleaning and validation, significant reduction in batch processing time and ultimately improved process economics.

Examples 2

In this example, a MabSelect ­ protein a eluate (herein designated "PAE 2") of MAb a was pH inactivated, neutralized to pH 8 with 1M Tris, pH 9.5 buffer, then filtered through CUNO 60/90 ZA and a deplipidated depth filter train, each followed by a Sartopore 20.45/0.22 μ M sterile filter. The filtrate was then adjusted to pH 9.5 with 5M NaOH and diluted with water to a conductivity range of 6-7 mS/cm. This filtrate contained approximately 3% aggregates, 15 ng/mg HCP and <1 ng/mg protein A. To better assess protein A clearance, samples were loaded with an additional 20 ng/mg MabSelect ™ protein A prior to loading into 5 mL Capto adhesive columns. The operation was carried out twice at room temperature and the specific conditions are summarized in table 2. The elution pool was analyzed for yield, HCP, protein a and aggregate/fragment levels.

TABLE 2 Experimental conditions for the bind-elute operation of PAE2 for MAb A on Capto @ adhesive columns

Table 3 summarizes the purification performance of the process of the invention in the bind-elute mode using Capto adhered columns of PAE 2. The impurity levels were comparable to those obtained by the standard three-column method. Although the yield is slightly lower in this two-column process than in the standard three-column process, the performance of this two-column process is within acceptable ranges and can be further optimized, thereby increasing the step yield without compromising product purity.

TABLE 3 summary of Capto adhesive columns for PAE2 binding-elution purification Performance of MAb A

Examples 3

Another set of purification experiments was performed with a method consisting of protein a capture, low pH inactivation, XOHC depth filtration and anion exchange membrane for final polishing.

The CH for MAb a was reused in this study, and two runs were performed at different loading levels of the XOHC depth filter (example 1 and example 2). The protein a capture, pH inactivation and XOHC filtration steps were run in the same manner as shown in example 1. However, the phenyl column was removed from this process and the QSFF columns were exchanged for 0.08 ml ChromaSorb film devices (Millipore Corporation) that were also run in flow-through mode. The Chromasorb device was wetted and washed according to the manufacturer's protocol, equilibrated with 25 mM Tris buffer, pH 8, containing 50 mM NaCl, and then charged with a 3 kg/L load and a flow rate of 1 ml/min. After loading, the device was washed with the same flow rate of equilibration buffer. The flow-through fractions were pooled from 200 mAU (UV280) at load to 200 mAU at wash. Key impurities, such as HCP, leached protein a, aggregates/fragments and DNA were measured after each step. This method also compares yield and purity with a standard three-column method (as detailed in example 1).

Fig. 12-15 illustrate impurity profiles for each unit operation in a one-column vs three-column process. As discussed above, when a relatively low feed loading is applied to the XOHC depth filter (case 1), HCP, aggregates, leached protein a and DNA are more effectively reduced, resulting in very low residual impurity levels. Upon further processing of such POD filtrate via the Q-membrane, all impurities are further removed to acceptable levels. For example, the Q membrane filtrate of example 1 contains approximately 0.7 ng/mg HCP, 1.5 ng/mg protein A, 1.4% aggregates, and DNA below the limit of quantitation. Although the aggregate level is slightly higher than that observed in the phenyl eluate, it can be further minimized by optimizing the process conditions of the Q membrane, including pH, conductivity, and loading levels. Alternatively, by enlarging the depth filter size prior to the Q-membrane step, the impurity levels can be reduced from those observed here. As shown in fig. 16, the step yield of the Q membrane flow-through is comparable to that of the Q column; thus, the elimination of the phenyl column not only reduces the overall processing time, but also improves the overall purification yield as compared to the two-column process.

All references cited in this specification, including but not limited to all papers, publications, patents, patent applications, reports, texts, reports, manuscripts, manuals, books, netbooks, journals, journal articles and/or periodicals, are hereby incorporated by reference into this specification in their entirety. The discussion of references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the citation.

These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. Further, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention so further described in such appended claims. Therefore, the spirit and scope of the appended claims should not be limited to the description of the versions contained therein.

Claims (60)

1. An apparatus for purifying a protein from a sample containing the protein to be purified, comprising:

a. a capture chromatography resin;

b. a depth filter disposed relative to the capture chromatography resin such that the sample passes through the capture chromatography resin into and through the depth filter; and

c. a mixed mode chromatography resin disposed relative to the depth filter such that the sample passes through the depth filter into and through the mixed mode chromatography resin.

2. The device of claim 1, wherein the capture chromatography resin is selected from the group consisting of an affinity resin, an ion exchange resin, and a hydrophobic interaction resin.

3. The device of claim 1, wherein the capture chromatography resin is selected from the group consisting of a protein a resin, a protein G resin, a protein a/G resin, and a protein L resin.

4. The device of claim 1, wherein the capture chromatography resin and/or mixed mode chromatography resin is contained within a chromatography column.

5. The device of claim 1, further comprising one or more clarification devices for clarifying proteins arranged to receive the sample prior to its passage into the capture chromatography resin.

6. The apparatus of claim 5, wherein the clarification device is selected from one or more of a centrifuge, a microfilter, an ultrafilter, and a depth filter.

7. The device of claim 1, further comprising a second depth filter arranged to receive the sample from the first depth filter prior to processing the sample via the mixed mode chromatography resin.

8. The device of claim 1, further comprising a sterile filter arranged to receive the sample from the depth filter prior to processing the sample via the mixed mode chromatography resin.

9. The device of claim 1, wherein the mixed mode chromatography resin comprises a chromatography resin employing one or more chromatography mechanisms selected from the group consisting of: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, and metal affinity.

10. The device of claim 1, wherein the mixed mode chromatography resin comprises a chromatography resin that employs a combination of anion exchange and hydrophobic interaction chromatography mechanisms.

11. An apparatus for purifying a protein from a sample containing the protein to be purified, comprising:

a. a capture chromatography resin;

b. a depth filter disposed relative to the capture chromatography resin such that the sample passes through the capture chromatography resin into and through the depth filter; and

c. a membrane adsorber arranged relative to the depth filter such that the sample passes through the depth filter into and through the membrane adsorber.

12. The device of claim 11, wherein the capture chromatography resin is selected from the group consisting of a protein a resin, a protein G resin, a protein a/G resin, and a protein L resin.

13. The device of claim 11, further comprising one or more clarification devices for clarifying proteins arranged to receive the sample prior to its passage into the capture chromatography resin.

14. The apparatus of claim 13, wherein the clarification device is selected from one or more of a centrifuge, a microfilter, an ultrafilter, and a depth filter.

15. The device of claim 11, further comprising a second depth filter arranged to receive the sample from the first depth filter prior to processing the sample via the membrane adsorber.

16. The device of claim 11, further comprising a sterile filter arranged to receive the sample from the depth filter prior to processing the sample via the membrane adsorber.

17. The device of claim 11, wherein the membrane adsorber is selected from the group consisting of a membrane ion exchanger, a mixed-mode ligand membrane, and a hydrophobic membrane.

18. The device of claim 11, further comprising a pre-bottled filter positioned relative to the membrane adsorber such that the sample enters and passes through the filter through the membrane adsorber.

19. The device of claim 18, wherein the pre-bottled filter is selected from the group consisting of a viral filter, a nano-filter, an ultra-filter, and a percolator.

20. An apparatus for purifying a protein from a sample containing the protein to be purified, comprising:

a. a capture chromatography resin;

b. a depth filter disposed relative to the capture chromatography resin such that the sample passes through the capture chromatography resin into and through the depth filter; and

c. a monolith disposed relative to the depth filter such that the sample enters and passes through the monolith through the depth filter.

21. A method of purifying a protein, comprising:

a. providing a sample containing a protein;

b. processing the sample via a capture chromatography resin to provide a first eluate comprising the protein;

c. processing the first eluate through a depth filter after processing the sample through the capture chromatography resin to provide a filtered eluate comprising the protein; and

d. after processing the first eluate through the depth filter, the eluate is filtered through mixed mode chromatography resin processing to provide a second eluate comprising the protein.

22. The method of claim 21, wherein the capture chromatography resin is selected from the group consisting of an affinity resin, an ion exchange resin, and a hydrophobic interaction resin.

23. The method of claim 21, wherein the capture chromatography resin is selected from the group consisting of a protein a resin, a protein G resin, a protein a/G resin, and a protein L resin.

24. The method of claim 21, wherein the protein is selected from the group consisting of a protein fragment, an antibody, a monoclonal antibody, an immunoglobulin, and a fusion protein.

25. The method of claim 21, wherein the sample is a cell culture.

26. The method of claim 21, wherein the sample is clarified prior to processing through the capture chromatography resin.

27. The method of claim 26, wherein the sample is clarified by a clarification method selected from the group consisting of centrifugation, microfiltration, ultrafiltration, depth filtration, sterile filtration, and treatment with detergents.

28. The method of claim 21 wherein the first eluate is subjected to viral inactivation after processing through the capture chromatography resin but before processing through the depth filter.

29. The method of claim 28, wherein said viral inactivation comprises a method selected from the group consisting of treatment with an acid, detergent, chemical, nucleic acid cross-linking agent, ultraviolet light, gamma radiation, and heat.

30. The method of claim 28 wherein viral inactivation comprises reducing the pH of the first eluate to a pH of about 3 to about 4.

31. The method of claim 30 wherein the first eluate is incubated for about 30 to about 90 minutes during virus inactivation.

32. The method of claim 21 wherein the filtered eluate is processed through the depth filter a second time.

33. The method of claim 32 wherein the filtered eluate is processed twice through the same depth filter.

34. The method of claim 32 wherein the filtered eluate is processed through two separate depth filters.

35. The method of claim 21, wherein the mixed mode chromatography resin comprises a chromatography resin employing one or more chromatography techniques selected from the group consisting of: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, and metal affinity.

36. The method of claim 21, wherein the mixed mode chromatography resin comprises a chromatography resin employing a combination of anion exchange and hydrophobic interaction chromatography techniques.

37. The method of claim 21, wherein the second eluate is subjected to further filtration after processing through the mixed-mode chromatography resin.

38. The method of claim 37, wherein said further filtration comprises one or more methods selected from the group consisting of viral filtration, nanofiltration, ultrafiltration and diafiltration.

39. The method of claim 21, wherein the filtered eluate is processed through the mixed-mode chromatography resin in flow-through mode.

40. The method of claim 21, wherein the filtered eluate is processed through the mixed mode chromatography resin in bind-elute mode.

41. A method of purifying a protein, comprising:

a. providing a sample containing the protein;

b. processing the sample via a capture chromatography resin to provide a first eluate comprising the protein;

c. processing the first eluate through a depth filter after processing the sample through the capture chromatography resin to provide a filtered eluate comprising the protein; and

d. after processing the first eluate through the depth filter, the eluate is filtered through a membrane adsorber process to provide a second eluate comprising the protein.

42. The method of claim 41, wherein the capture chromatography resin is selected from the group consisting of an affinity resin, an ion exchange resin, and a hydrophobic interaction resin.

43. The method of claim 41, wherein the capture chromatography resin is selected from the group consisting of a protein A resin, a protein G resin, a protein A/G resin, and a protein L resin.

44. The method of claim 41, wherein the protein is selected from the group consisting of a protein fragment, an antibody, a monoclonal antibody, an immunoglobulin, and a fusion protein.

45. The method of claim 41, wherein the sample is a cell culture.

46. The method of claim 41, wherein the sample is clarified prior to processing through the capture chromatography resin.

47. The method of claim 46, wherein the sample is clarified by a clarification method selected from the group consisting of centrifugation, microfiltration, ultrafiltration, depth filtration, sterile filtration, and treatment with detergents.

48. The method of claim 41 wherein the first eluate is subjected to viral inactivation prior to processing through the depth filter.

49. The method of claim 48, wherein said viral inactivation comprises a method selected from the group consisting of treatment with an acid, a detergent, a chemical, a nucleic acid cross-linking agent, ultraviolet light, gamma radiation, and heat.

50. The method of claim 48, wherein viral inactivation comprises reducing the pH of the first eluate to a pH of about 3 to about 4.

51. The method of claim 49, wherein the first eluate is incubated for about 30 to about 90 minutes during virus inactivation.

52. The method of claim 41 wherein the filtered eluate is processed through the depth filter a second time.

53. The method of claim 41 wherein the filtered eluate is processed twice through the same depth filter.

54. The method of claim 41 wherein the filtered eluate is processed through two separate depth filters.

55. The process of claim 41, wherein the membrane adsorber is selected from the group consisting of a membrane ion exchanger, a mixed-mode ligand membrane, and a hydrophobic membrane.

56. The process of claim 41 wherein the second eluent is processed through the membrane adsorber a second time.

57. The method of claim 41 wherein the second eluate is subjected to further filtration after processing through a membrane adsorber.

58. The method of claim 57, wherein the further filtration comprises one or more of the methods selected from the group consisting of viral filtration, nanofiltration, ultrafiltration and diafiltration.

59. The method of claim 41, wherein:

a. processing the first eluate through a depth filter after processing the sample through the capture chromatography resin; and

b. the first eluate is processed through the depth filter and then filtered through the membrane adsorber.

60. A method of purifying a protein, comprising:

a. providing a sample containing the protein;

b. processing the sample via a capture chromatography resin to provide a first eluate comprising the protein;

c. processing the first eluate through a depth filter after processing the sample through the capture chromatography resin to provide a filtered eluate comprising the protein; and

d. after processing the first eluate through the depth filter, the eluate is filtered through a monolith process to provide a second eluate comprising the protein.