FIELD OF INVENTION

This invention relates to a method of separation of charge variants using ion-exchange chromatography operated in displacement mode.

BACKGROUND OF INVENTION

Recombinant monoclonal antibodies (MAbs) are an important class of therapeutic proteins and constitute a significant proportion of pharmaceutical therapeutics due to their high degree of specificity ability to initiate immune response against the target antigens and long serum persistence thus reducing the need for frequent administration.

Composition of MAbs exhibit significant heterogeneity due to the presence of large number of charged variants. These variants may be the result of glycosylation deamidation oxidation reduction and other post-translational modifications as well as factors such as the temperature pH at which cells expressing the antibody are cultured.

Presence of charged variants can have a significant impact on the potency immunogenicity or pharmacokinetics of the therapeutic molecule. These charge variants are therefore critical to the product’s quality and shelf life of a therapeutic antibody. In addition the advent of concept of “Quality by Design” in industry requires a thorough understanding of the relationship between these critical quality attributes and process that impact the product’s life cycle. Hence there is an increased focus on the development of methods for the efficient separation of charged variants to ensure the safety and efficacy of a therapeutic antibody.

Ion-exchange chromatography is one of the most widely used methods for the separation and purification of antibody charge variants. Ion-exchange chromatography can be operated by two general modes - elution mode or displacement mode.

In elution chromatography a solution of the sample to be purified is applied to a stationary phase using a mobile phase. The mobile phase is chosen such that the sample is neither strongly nor weakly adsorbed but rather binds reversibly. The bound sample components are then eluted from the column using buffers of different composition and typically accompanied by linear or step changes in the salt concentration and/or pH.

Pabst et.al (Biotechnol Prog. 2008 Sep-Oct; 24(5):1096-106) discloses a method of separation of protein charge variants with induced pH gradients using anion exchange chromatographic columns in elution mode. In Analytical Chemistry 2009 81(21): 8846-8857 Farnan and Moreno discloses a method of monoclonal antibody charge variant separations using a pH gradient in ion-exchange chromatography operated in elution mode.

However significant drawbacks exist in the use of elution chromatography. These include operational complexity due to gradient solvent pumping low throughput and low column loadings especially at the preparative scale.

Displacement chromatography is a technique that has been successfully used for the purification of peptides monoclonal antibodies and provides greater advantages over elution chromatography in separation and concentration of sample components at significantly higher concentration and in permitting very high column loading.

Displacement chromatography involves sorption of a sample mixture near the inlet of the column and employs a displacer compound in the mobile phase. The displacer compound has a much higher affinity for the stationary phase than do any of the components in the mobile phase. As the mobile phase is applied to the column the displacer forms a sharp-edged zone at the head of the column pushing other components downstream. Each sample component then acts as a displacer for the lower-affinity solutes and the solutes sort into a series of contiguous bands ("displacement train") that move downstream at the rate set by the displacer. The solutes appear at the bottom of the column as contiguous zones each zone consisting essentially of one uniformly purified component.

U.S. Patent No. 5 028 696 describes a method of separating charged molecules using an ion-exchange chromatography and a displacer molecule. U.S. Patent No. 5 478 924 discloses a method of purifying a protein comprising loading on a displacement chromatography wherein a cationic displacer is used for displacing the molecules.

In Journal of Chromatography 499 (1990) 47-54 Torres and Peterson discloses a method of purification of mAbs by complex-displacement chromatography wherein the displacer was bound to the protein and displacer/protein complexes were then eluted from the resin.

International patent application no. WO 2009135656 discloses a method for the purification of antibodies using ion exchange chromatography in displacement mode wherein a polycationic organic compound (Expell SP1TM) is used as a displacer.

Khawli et.al in mAbs (2010) 2:6 613-624 describes a procedure for separation and characterization of charge variants of monoclonal antibody using displacement chromatography wherein Sachem SP1TM is used as a displacer.

Recently Zhang et.al. in Journal of Chromatography 1218 (2011) 5079-5086 disclose a cation-exchange displacement chromatography for the isolation and characterization of antibody charge variants wherein the displacer buffer included Expell SP1TM as a displacer.

The methods described in the prior art require the use of a mobile phase with a displacer compound such as polycationic organic compound Expell SP1TM to displace charge variant(s). However since the displacer molecule itself has a very high affinity for the stationary phase removal of the displacer from the column poses a significant challenge. Furthermore the method is very difficult to optimize in terms of choice of displacer displacer concentration and in obtaining displacer compounds at an economical cost. This has effectively reduced the use of displacement chromatography as a mainstream chromatographic technique despite its clear advantage over elution chromatography.

The present invention provides a method for the separation of charge variants by “self-displacement ion-exchange chromatography” whereby displacement chromatography is performed without the use of a displacer compound. The method as described in the present invention exploits the differential binding capacity of protein charged variants when the protein charge variants are loaded onto the chromatographic column in an amount that equals or exceeds the dynamic binding capacity of the column. As a result variants with higher affinity for competitively bind to the resin and effectively displace variants with lower affinity as enriched components in the flow-through. The higher affinity variants bound to the column may then be eluted using a mobile phase of varying salt concentrations / and or pH.

Thus the principle object of the present invention is to provide a method wherein the chromatographic conditions are optimized to utilize this principle of self-displacement chromatography for the separation of charge variants. Separation of variants is achieved in the absence of any displacer compound in the mobile phase. The invention thus avoids the complications and expense in using a displacer compound but retains the advantageous characteristics of displacement chromatography.

SUMMARY OF THE INVENTION

The present invention discloses a method of separation of antibody charge variants using ion-exchange chromatography operated in displacement mode. Continued loading of the mobile phase comprising antibody charge variants results in column overload so that the higher affinity antibody variants competes for the binding sites on the stationary phase and thereby displaces low affinity charge variants from the chromatographic column. The invention thus provides a method for the separation of antibody charge variants by ion-exchange chromatography operated in displacement mode but that avoids the use of a displacer compound in the mobile phase.

The higher affinity charge variants bound to the column may then be eluted using a gradient elution i.e. by means of an elution buffer having varying salt concentrations and/or pH.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an illustration of a chromatogram from the procedure as described in example 2. DBC is the dynamic binding capacity of the resin and the arrow indicates the breakthrough point. The amount of antibody loaded onto the POROS HS 50 cation exchange resin is 60 mg/ml of the resin and equaled the DBC of the resin. The line marked “Cond” represents the increase in conductivity in mS/cm. Region marked D represents the displaced acidic variants. Peak A represents the mid peak and peak B represents the basic variants of the antibody.

Figure 2 is an illustration of a chromatogram from the procedure as described in example 2. DBC is the dynamic binding capacity of the resin and the arrow indicates the breakthrough point. The amount of antibody loaded onto the POROS HS 50 cation exchange resin is greater than 60 mg/ml of the resin and exceeded the DBC of the resin. The line marked “Cond” represents the increase in conductivity in mS/cm. Region marked D represents the displaced acidic variants. Peak A represents the mid peak and peak B represents the basic variants of the antibody.

Figure 3 is an illustration of a chromatogram from the procedure as described in example 3. The amount of antibody loaded onto the POROS XS cation exchange resin is greater than 100 mg/ml of the resin and exceeded the DBC of the resin. The line marked “Cond” represents the increase in conductivity in mS/cm. Region marked D represents the displaced acidic variants. Peak A represents the mid peak and peak B represents the basic variants of the antibody.

Figure 4 is an illustration of the HPLC profiles of the antibody mixture on cation exchange load and eluate pool from the procedure as described in example 2. The profile could be primarily divided into three main regions. Region 1 corresponds to the acidic variants of the antibody. Region 2 corresponds to the mid peak. Region 3 corresponds to the basic variants of the antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a method of separation of antibody charge variants by ion-exchange chromatography operated in displacement mode.

The invention provides a method of separation of antibody charge variants comprising loading onto an ion-exchange resin a mobile phase comprising the antibody charge variants in an amount that equals or exceeds the dynamic binding capacity of the resin resulting in displacement and enrichment of low affinity variants in the flow-through. The invention thus avoids the use of a displacer compound in the mobile phase.

In an embodiment the invention provides a method of separation of antibody charge variants comprising

a) loading onto an ion-exchange resin a mobile phase comprising the antibody charge variants in an amount that equals or exceeds the dynamic binding capacity of the resin resulting in displacement and enrichment of low affinity charge variants in the flow-through and

b) eluting from the resin the bound charge variants by varying the salt concentrations and/or pH of the mobile phase

wherein the mobile phase in step a) is devoid of a displacer compound.

In an embodiment the invention provides a method of separation of antibody charge variants comprising loading onto a cation-exchange resin a mobile phase comprising the antibody charge variants in an amount that equals or exceeds the dynamic binding capacity of the column resulting in displacement and enrichment of acidic variants in the flow-through and wherein the mobile phase is devoid of a displacer compound.

In another embodiment the invention provides a method of separation of antibody charge variants comprising

a) loading onto a cation-exchange resin a mobile phase comprising the antibody charge variants in an amount that equals or exceeds the dynamic binding capacity of the column resulting in displacement and enrichment of acidic variants in the flow-through and

b) eluting from the resin the mid peak and basic variants by varying the salt concentrations and/or pH of the mobile phase

wherein the mobile phase in step a) is devoid of a displacer compound.

Likewise separation of antibody charge variants may also be achieved using an anion-exchange chromatographic column with an appropriate mobile phase and resin thereof wherein the basic variants are displaced and obtained in the flow-through.

Ion-exchange chromatography step mentioned in the embodiments may be preceded by a protein-A affinity chromatography.

Cation exchange chromatographic column mentioned in the embodiments include any weak or strong cation exchange chromatographic resin. Commercially available cation exchange resins include but are not limited to those having a sulfonate based group e.g. MonoS MiniS Source 15S and 30S SP Sepharose Fast Flow SP Sepharose High Performance from GE Healthcare Toyopearl SP-650S and SP-650M from Tosoh S-Ceramic Hyper D from Pall Corporation or a carboxymethyl based group e.g. CM Sepharose Fast Flow from GE Healthcare Macro-Prep CM from BioRad CM-Ceramic Hyper D from Pall Corporation Toyopearl CM-650S CM-650M and CM-650C from Tosoh. In embodiments of the invention a strong cation exchange resin such as POROS HS® or a POROS XS® (Applied Biosystems) is used; they are made of cross-linked poly(styrene-divinylbenzene) flow-through particles and the particles are surface coated with a polyhydroxylated polymer functionalized with sulfopropyl group.

The chromatographic steps mentioned in the embodiments may include one or more tangential flow filtration concentration diafiltration or ultrafiltration steps.

The embodiments mentioned herein may include one or more viral inactivation steps or sterile filtration or nano filtration steps.

The embodiments mentioned herein may include one or more neutralization steps.

The term ‘charge variants’ as used here in refers to a group of low-pI mid-pI and high-pI variants and are described as “acidic variants” “mid peak” and “basic variants” respectively based on their differential elution from an analytical ion-exchange HPLC. Though not to be construed as limiting the scope of the said term examples of analytical ion exchange HPLC may be found in web references http://www.dionex.com/en-us/products/columns/bio/protein/mabpac-scx/lp-86977.html and http://www.crawfordscientific.com/downloads/Application-Notes/Techniques/LC/5990-6844EN.pdf

The term ‘mobile phase’ as used herein refers to any solution that percolates through or along the chromatographic resin. The mobile phase comprising the antibody charged variants includes cell culture harvests supernatant filtrate etc. Such a mobile phase may contain aggregates host cell proteins host cell DNA viruses etc. The mobile phase comprising the charged variants to be separated may also include partially purified solution that has been subjected to a chromatography step or an equivalent thereof and to which appropriate buffer and salts may be added

The ‘dynamic binding capacity’ (DBC) or ‘break-through capacity’ of a chromatographic resin is the amount of target protein the resin will bind under actual flow conditions before significant breakthrough of unbound protein occurs.

The break-through capacity of a resin can be obtained experimentally by passing a solution of a particular solute through the column and by directly monitoring UV absorbance. The point at which some protein passing through the resin is first detected in the eluate (as is no longer able to find an available binding site in the packed resin) is the break through point and is indicated by an increase in the UV absorbance of the process stream leaving the column. The DBC or break-through capacity may be expressed in millimoles or milligrams taken up per gram of dry ion exchanger or per cm3 of bed volume.

The term ‘displacer compound’ or ‘displacer’ as used herein refers to a non-proteinaceous compound in the mobile phase that has a higher affinity for the stationary phase of the chromatographic column than the protein charge variants whose differential separation is sought.

The term “flow-through” as used herein denotes a solution that is obtained from the chromatographic column during loading or post loading washes of the column with the mobile phase. Flow-through solution generally contain substances that are not bound (or loosely bound) to the stationary phase of the column. The flow-through solution may be collected in various fractions and pooled together or may be collected as a single fraction.

The invention is more fully understood by reference to the following examples. These examples should not however be construed as limiting the scope of the invention.

EXAMPLES:

Example 1: Protein A chromatography

An anti-HER2 antibody was cloned and expressed in a Chinese Hamster Ovary cell line and the cell culture broth containing the expressed antibody was harvested clarified and subjected to protein A affinity chromatography as described below.

The clarified cell culture broth was loaded onto a protein A chromatography column (Prosep vA ultra VL44x250 205 mL) that was pre-equilibrated with Tris NaCl pH 7.5 buffer. The column was then washed with a high salt buffer and the bound antibody was eluted using a low pH acetate buffer.

Example 2: Cation exchange chromatography I

To begin with the DBC or break through capacity was determined for the cation exchange chromatographic resins (POROS HS and XS) based on the breakthrough point or breakthrough curve analysis.

The break-through capacity of a resin can be obtained experimentally by passing a solution of a particular solute through the column and by directly monitoring UV absorbance. The point at which some MAb passing through the resin is first detected in the eluate (as is no longer able to find an available binding site in the packed resin) is the break through point and is indicated by an increase in the UV absorbance of the process stream leaving the column.

DBC was calculated from the volume of protein solution that has been applied up to a specific break through point (usually 5 or 10%). The volume applied at 5% breakthrough (V5%) was defined from the fraction with 5% of the peak area in the start material.
DBC at 5% = (V (5%) - Vo) Co/Vc
where Co = antibody concentration (mg/ml)
Vc = geometric total volume (ml)
Vo = void volume (ml).

Based on the above the DBC for POROS HS and POROS XS was estimated to be 60 mg and 100 mg per mL of the resin respectively.

The eluate obtained from the protein A chromatography procedure described in Example 1 was neutralized with Tris Cl buffer (pH 9.0) to a pH of 6.0 and loaded onto the cation exchange resin (POROS HS 50 VL 11 ? 20) pre-equilibrated with 5 CV of equilibration buffer (Citrate buffer pH 6.0 at a conductivity of 3.0 mS/cm). The mobile phase with a load concentration of =60 mg of antibody per ml of the cation exchange resin was loaded onto the resin. Flow through fractions enriched with acidic variants of the antibody was displaced from the cation exchange column during loading or on washing the column with the equilibration buffer. The mid peak was eluted using a citrate buffer of pH 6.0 at a conductivity of 5-6 mS/cm. Basic variants were eluted using citrate buffer of pH 6.0 and with a step-increase in conductivity from 5-6 mS/cm to 8 mS/cm.

Example 3: Cation-exchange chromatography II

Alternatively the eluate obtained from the protein A chromatography procedure described in Example 1 was neutralized with Tris Cl buffer (pH 9.0) to a pH of 6 and loaded onto the cation exchange resin (POROS XS VL 11 ? 20) pre-equilibrated with 5 CV of equilibration buffer (Citrate buffer pH 6.0 at a conductivity of 3.0 mS/cm). The mobile phase with a load concentration of > 100 mg of antibody per ml of the cation exchange resin was loaded onto the column. Flow through fractions enriched with acidic variants of the antibody was displaced from the cation exchange column during loading. The mid peak was eluted using a citrate buffer of pH 6.0 at a conductivity of 5-6 mS/cm. Basic variants were eluted using citrate buffer of pH 6.0 and with a step-increase in conductivity from 5-6 mS/cm to 8 mS/cm.