DESC:The following specification particularly describes the invention and the manner in which it is to be performed:

FRACTIONATION SCHEME IN ION EXCHANGE CHROMATOGRAPHY OF AN ANTIBODY

FIELD OF THE INVENTION
The invention relates to protein purification methods. In particular, the invention relates to methods using ion exchange chromatography for removing contaminants from an antibody composition.

BACKGROUND OF THE INVENTION
Monoclonal antibodies (mAbs) are effective targeted therapeutic agents. The high specificity of the antibodies makes them ideal to reach their intended target and hence is useful to treat a wide variety of diseases.
The commercial production of recombinant human monoclonal antibody therapeutics demands robust processes, i.e., the purification scheme needs to reliably and predictably produce antibody composition intended for use in humans. A process should be designed to remove the product related contaminants such as high molecular weight aggregates, product variants such as charged variants (acidic, deamidated/oxidized, basic), sequence variants and other species, as well as process related contaminants such as leached Protein-A, host cell protein, DNA, adventitious and endogenous viruses, endotoxin, extractable from resins and filters, process buffers and agents such as detergents that may have been employed for virus reduction. In designing a purification scheme and other conditions for each of the chromatographic steps, along with removal of contaminants, an important consideration is recovery from each step of the purification scheme and from the overall purification scheme. Hence, for a commercially viable process, the purification scheme needs to be designed to ensure adequate removal of contaminants from an antibody composition while maintaining the yield of the same.
Chromatographic techniques exploit the physical and chemical differences between the antibodies and the contaminant for the separation. Majority of purification schemes for mAbs involve a Protein-A based chromatography, which results in a high degree of purity and recovery in a single step. One or two additional chromatography steps are employed as polishing steps, generally selected from cation and anion exchange chromatography, although hydrophobic interaction chromatography, mixed mode chromatography or hydroxyapatite chromatography may be chosen as well.
Ion exchange chromatography is ideal for reducing contaminants such as high molecular weight aggregates, charge-variants of the antibody (e.g. deamidated/oxidized variants), residual DNA and host cell protein, leached Protein-A and viral particles. Depending on the nature of the contaminants, the cation or anion exchange chromatography or both, may be employed in the purification scheme. Maximizing the potential of the ion exchange chromatography in separating the contaminants, requires optimization/elucidation of numerous factors such as mode of chromatography (i.e. bind/elute or flow through mode), loading amount, type of matrix or ligand to be used, binding/loading and elution buffer etc. In particular, the elution scheme and the ensuing discarding or collection of eluent/flow-through fractions (i.e., fractionation/pooling scheme) is critical for ensuring robust product quality and in optimized recovery of the antibody composition.
Generally, easily measured output parameters such as absorbance and/or volume are used for fractionation/pooling scheme. However, the volume based scheme suffers from disadvantage of lacking the robustness since it is sensitive to even slight variation in factors such as dynamic binding capacity, pH of the buffer used, protein concentration in the load etc. The absorbance (measured at UV 280 nm) based scheme is well known to lead to signal saturation problems especially during process scale up. Hence, there exists a need for optimization of an appropriate fractionation/pooling scheme for balancing the purity and yield ratio in a/any commercial mAb production process.

SUMMARY OF THE INVENTION
The present invention discloses a method for purifying a monoclonal antibody from the contaminants, the method comprising ion exchange chromatography, wherein the ion exchange chromatography uses conductivity based fractionation scheme for chromatography eluent collection. As the eluent containing the desired antibody composition is collected based on conductivity values, the method is easily scalable to high-volume production batches. Thus, the method of conductivity based eluent collection is robust compared to volume/absorbance based fractionation, and offers strict control over quality and recovery of the antibody composition.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an illustration of a chromatogram as obtained from following chromatographic procedure in example 1, when the path length of U.V. cell is 2 mm.
Figure 2 is an illustration of a chromatogram as obtained from following chromatographic procedure in example 1, when the path length of U.V. cell is 5 mm.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The phrase "ion exchange material" refers to a solid phase which is negatively charged (i.e. a cation exchange resin) or positively charged (i.e. an anion exchange resin). The charge may be provided by attaching one or more charged ligands to the solid phase, e.g. by covalent linking. Alternatively, or in addition, the charge may be an inherent property of the solid phase (e.g. as is the case-for silica, which has an overall negative charge).
The term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, the current flows by ion transport. Therefore, with an increasing amount of ions present in the aqueous solution, the solution will have a higher conductivity. The unit of measurement for conductivity is mS/cm, and can be measured using a conductivity meter, e.g., by Orion. The conductivity of a solution may be altered by changing the concentration of ions therein. For example, the concentration of a buffering agent and/or concentration of a salt (e.g. NaCl or KCl) in the solution may be altered in order to achieve the desired conductivity.
A "contaminant" is a material that is different from the desired polypeptide product. The contaminant may be a variant of the desired polypeptide (e.g. a deamidated variant or an aminoaspartate variant of the desired polypeptide) or another non-product related polypeptide e.g. host cell protein, host cells nucleic acid, endotoxin etc. A contaminant can also be process related for example Protein-A-leachates.
An "acidic variant" is a variant of a polypeptide of interest which is more acidic (e.g. as determined by cation exchange chromatography) than the polypeptide of interest. An example of an acidic variant is a deamidated variant.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention discloses a method for purifying the monoclonal antibody from the contaminants, the method comprising loading the sample containing the monoclonal antibody and the contaminants onto an ion-exchange column, followed by optionally washing (one or more washes) the column, and thereafter eluting the monoclonal antibody from the ion-exchange column, wherein the elution solution (eluent) collection/fractionation is conductivity based and the eluent comprises the monoclonal antibody purified from the contaminants.
In any of the above mentioned embodiments, the ion exchange chromatography is a cation exchange chromatography.
In any of the above mentioned embodiments, the ion exchange chromatography is preceded by at least one chromatography. In particular, the ion exchange chromatography is preceded by at least one chromatography, one of which is an affinity chromatography. More particularly, the ion exchange chromatography is preceded by at least one chromatography, one of which is a Protein-A chromatography.
In any of the above mentioned embodiments, the ion exchange chromatography is followed by at least one chromatography. In particular, the ion exchange chromatography is followed by at least one chromatography, one of which is an ion exchange chromatography. More particularly, the ion exchange chromatography is followed by at least one chromatography, one of which is an anion exchange chromatography.
In any of the above mentioned embodiments, the method employs use of one or more steps such as viral inactivation, filtration and diafiltration. These steps may be interspersed between the chromatographic steps or the after all the chromatographic steps.
In any of the above mentioned embodiments, the percentage of acidic variants is less than 40%.
In any of the above mentioned embodiments, the method results in increase in protein recovery.
In any of the above mentioned embodiments, the monoclonal antibody is an anti-HER2 antibody.
In any of the above mentioned embodiments, the monoclonal antibody is trastuzumab.
EXAMPLE
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.
Protein-A chromatography column (ProSep®-vA) 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. Post elution the Protein-A eluent was subjected to low pH viral inactivation followed by pH neutralization. The neutralized Protein-A eluent (NTEL) was there after loaded onto CEX chromatography.
The cation exchange resin (POROS® XS 50) was pre-equilibrated with 5 CV of equilibration buffer (15 mM Phosphate buffer, pH 7.3, conductivity of 3.0 mS/cm). NTEL having antibody concentration in the range of 25-40 mg per ml of resin was loaded onto CEX chromatography. Thereafter, 5 CV of post load wash (PLW) comprising of phosphate buffer pH 7.3, conductivity of 25 ± 3 mS/cm was passed through the column. The product was eluted from the column with a linear conductivity gradient of 15 mM phosphate buffer pH 7.3, conductivity of 25 ± 3 mS/cm. The eluent collection was evaluated using different criteria, as elucidated in Examples 1, 2 and 3.
For all the following strategies, the variation in pH, load factor, dynamic binding capacity results in change in the slope of the chromatogram. Based on design of experiments and the analysis of offline chromatograms, an empirical correlation was built between the input variables pH, load factor and DBC and fractionation scheme corresponding to % X. % X refers to fraction that needs to be discarded, named fraction 1 following which fraction 2 needs to be collected.
Example 1: UV based fractionation
The UV absorbance corresponding to % X was determined by integrating the chromatogram offline. The absorbance range observed for targeting %X was ~500 mAu to 3000 mAu at a path length of 2mm of U.V. cell (Figure 1). The scale up of this process to large scale system required converting it with a factor of 2.5 considering the path length of 5 mm. This resulted in a range ~1250 mAu to ~7500 mAu. This led to saturation of UV signal when path length of U.V cell was 5mm, as can be seen from Figure 2.
Example 2: Volume based fractionation
The volume of the eluent to be discarded corresponding to %X was determined by integrating the chromatogram offline. The volume to be discarded for targeting %X was determined to be 1.75 CV. The recovery of the process was calculated using the formula:
(Total protein in load/ Total protein in the eluate)*100
Although, this resulted in optimum quality of the eluate but the recovery of the process was compromised, i.e. acidic variants less than 40%. The volume based fractionation led to a variation in the recovery in the range of 45%-60%.
Example 3: Conductivity based fractionation
The conductivity corresponding to % X was determined by integrating the chromatogram offline was determined to be 4-6 mS/cm. The recovery of the process was calculated using the formula:
(Total protein in load/ Total protein in the eluate)*100
Thus, during the course of elution the fraction changed from 1 to 2 as soon as the desired conductivity condition occurred. The first fraction was discarded. The conductivity based fractionation led to a variation in the recovery in the range of 55-66%.
,CLAIMS:CLAIMS
We claim:
1. A method for purifying an antibody from a composition comprising the antibody and at least one contaminant by ion exchange chromatography, the method comprising steps of:
a. loading the composition comprising the antibody and at least one contaminant onto an ion-exchange material,
b. optionally washing the material using a wash buffer solution, and
c. eluting the antibody from the ion-exchange material,
wherein the collection/fractionation of the elution solution (eluent) is conductivity based.
2. The method as claimed in claim 1, wherein the ion exchange chromatography is cation exchange chromatography.
3. The method as claimed in claim 1, wherein the ion exchange chromatography is preceded by an affinity chromatography step and/or a viral inactivation step.
4. The method as claimed in claim 1, wherein the ion exchange chromatography is followed by an anion exchange chromatography step and/or filtration step.
5. The method as claimed in claim 1, wherein the method is used to recover up to 66% of the antibody in the eluent.
6. The method as claimed in claim 1, wherein the antibody is an anti-HER2 antibody.
7. The method as claimed in claim 1, wherein the antibody is trastuzumab.