The present invention relates to the field of antibody purification in biotechnological production. In particular, the present invention provides an improved method for purifying an antibody from a harvested cell culture fluid comprising the antibody and removing one or more impurities using Protein A affinity chromatography.

Background of the invention

Monoclonal antibodies (mAbs) as a class of therapeutic molecules are finding an increasing demand in the biotechnology industry for the treatment of diseases like cancer and multiple sclerosis. A key challenge associated to successful commercialization of mAbs is that from the various physical and chemical instabilities that are inherent to these molecules. (A Singla, et al. “Aggregation Kinetics for lgG1-Based Monoclonal Antibody Therapeutics” Feb 2016, doi: 10.1208/s12248-016-9887-0.)

The large-scale, economic purification of proteins is an increasingly important problem for the biotechnology industry. Generally, proteins are produced by cell culture, using either eukaryotic or prokaryotic cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cells typically used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors, feed medium and etc. Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge.

Purification of pharmaceutical grade mAb protein from cell culture media includes harvest/clarification followed by purification using a series of column chromatography steps in combination with filtration, ultrafiltration and diafiltration. After purification, the desired antibody preparation is suitably formulated and stored in appropriate conditions. However, many times, these steps do not provide the antibody with the desired level of purity and quality that are required for their pharmaceutical use. Sometimes, process-related and product-related impurities are observed to co-elute with the desired antibody during column purification. Therefore, it is important to reduce or remove such impurities from the desired preparation. Once a clarified solution containing the protein of interest has been obtained, its separation from the other host cell related, product related and process related impurities being generated through the upstream process is usually attempted using a combination of different chromatography techniques. These techniques separate mixtures of proteins on the basis of their charge, degree of hydrophobicity, or size. Several different chromatography resins are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved. The essence of each of these separation methods is that proteins can be caused either to move at different rates down a long column, achieving a physical separation that increases as they pass further down the column, or to adhere selectively according to its charge or specificity etc. to the separation medium, being then differentially eluted by different solvents. In some cases, the desired protein is separated from impurities when the impurities specifically adhere to the column, and the protein of interest does not, that is, the protein of interest is present in the “flow through”. Therefore, it is important to reduce or remove such impurities from the desired preparation.

Industrial-scale production of biopharmaceuticals is a challenging task; preserving the product by limiting stresses that may cause degradation, combined with maximising yield and minimising resources consumed are key elements of bioprocess development. One form of protein degradation is aggregation. In the biopharmaceutical industry, aggregates are classed as impurities and must be cleared to acceptable levels if a protein formulation is to be used therapeutically. Aggregates are thought to pose a risk of unwanted immunogenicity and may affect product potency and reproducibility.

Out of all probable instabilities, protein aggregation of mAbs has been a major problem that has been associated with a change in the protein structure and is a hurdle in various upstream and downstream processes. It can stimulate immune response causing protein misfolding having deleterious and harmful effects inside a cell. Also, the extra cost incurred to remove aggregated mAbs from the rest of the batch is huge. (A Singla, et al. “Aggregation Kinetics for lgG1-Based Monoclonal Antibody Therapeutics” Feb 2016, doi: 10.1208/s 12248- 016-9887-0.)

Aggregates may be formed when subjected to different environmental conditions such as medium type, temperature, pH and salt concentration during upstream, downstream, and storage processes. Aggregates from such stages should be minimized and the product should have a similar profile with the reference product in the final purification stage. Usually, they are removed by anion or cation exchange chromatography steps. Moreover, optimization studies are required to remove aggregates at these stages, and these studies may result in losses from the binding capacity or efficiency of the column or an additional chromatography stage may be needed to further purification.

Current methods for the purification of proteins, such as antibodies, include two or more chromatographic steps. For example, the first step in the protein purification protocol can involve an affinity chromatography step that utilizes a specific interaction between the protein of interest and an immobilized capture reagent. Protein A adsorbents are particularly useful for affinity capture of proteins such as antibodies that contain an Fc region. However, there are numerous drawbacks to using Protein A affinity chromatography for protein purification. In some instances, leakage of the Protein A capture agent results in contamination of the eluted proteins product, while in other instances, affinity capture does not separate protein variants, such as charge variants and aggregated forms of the protein, from the protein of interest. Additionally, varying levels of turbidity and/or precipitates can be formed in the Protein A elution pool following low pH inactivation and pH neutralization. This turbidity and/or precipitation can lead to significant product losses in the neutralized Protein A elution pool. Accordingly, there is a need for purification methods that reduce product losses and enhance the product purity in the elution pool.

Protein A is a cell surface protein found in Staphylococcus aureus. It has the property of binding the Fc region of a mammalian antibody.

Protein A affinity chromatography is the most frequently used affinity chromatography method in biomanufacturing. It is the standard technique for capturing recombinant monoclonal antibodies, which relies on the reversible and specific binding between the immobilized protein A ligand and antibodies.

WO2014207763 patent application discloses an invention provides an improved method for the purification of monoclonal antibody from cell culture. Process of purification of the desired monoclonal antibody comprises affinity, hydrophobic interaction and optionally ion exchange column chromatography.

However, the state of the art is insufficient for a specific method that removes impurities using a single chromatography step instead of a chromatography series.

According to the literature, in order to remove aggregates by stepwise elution two protocols are tested. The first protocol consisted of the sample loading, the wash with the equilibration buffer and the low pH elution. The wash stage of the second protocol B included the wash with 1.0 M arginine. (Yada et al. ,2016 Choosing the right protein A affinity chromatography media can remove aggregates efficiently, doi: 10.1002/biot.201600427)

In this literature it is stated that there is no capacity for aggregate removal for protein A stage. At this stage, some additional substances such as arginine are added to the buffer during elution in order to prevent aggregation, and the elution process with low pH is prevented from aggregating the product.

In another publication, Zhang et al. disclosed that they developed a method that could significantly improve Protein A's ability to remove aggregates. This method involves adding calcium chloride/polyethylene glycol (PEG) or sodium chloride/PEG combination to wash and elution buffers. (Zhang Y et al. A method for improving protein A affinity chromatography's aggregate removal capability, doi: 10.1016/j.pep.2019.02.017)

Considering the state of the art, a special purifying method that does not contain arginine or other non-economical chemical substances is still needed to remove process-related and product-related impurities especially the protein aggregates that may be specifically or non- specifically attached to the column.

Objects and Brief Description of the Invention

The main object of the present invention is to provide an improved method for purifying an antibody from a harvested cell culture fluid comprising the antibody and removing one or more impurities using Protein A affinity chromatography which eliminates all aforesaid problems and brings additional advantages to the relevant prior art.

Another object of the present invention is to provide an improved method that removes product related impurities formed during the upstream and downstream process.

Another object of the present invention is to provide an improved method that provides a product with a higher quality profile to be loaded onto the column in the next chromatographic steps.

Another object of the present invention is to provide an improved method to increase the dynamic binding capacity (because of the decreased impurity profile) of the resins to be used in the next stages.

Another object of the present invention is to provide an improved method for removing process-related and product-related impurities without the use of an additional substance such as arginine or an arginine derivative. Another object of the present invention is to provide an optimized, economical and industry viability purification method.

Another object of the present invention is to obtain more than > 90-95% monomer purity of the desired monoclonal antibody.

Another object of the present invention is to decrease the purification stages to two instead of three with this process.

Detailed description of the Invention

In accordance with the objects outlined above, detailed features of the present invention are given herein.

The present invention relates to a method for purifying an antibody comprising the steps of;

- providing a harvested cell culture fluid comprising said antibody and one or more impurities,

- loading said fluid onto a protein A affinity chromatography column for binding,

- washing said protein A affinity chromatography column, wherein column wash comprising three steps; a) first wash with buffer at suitable pH and salt concentration, b) second wash with a buffer of the same pH with different salt concentration than the first buffer, wherein the second wash buffer component selected from the group comprising sodium chloride, sodium phosphate, potassium chloride, glycine, L-histidine, tris buffers, HEPES, phosphate buffers or mixtures thereof, at a concentration of about 0.01 to about 2.0 M, c) third wash with a buffer of the different pH than the first and second wash steps, wherein the pH of the wash buffer is between 4,5 and 6,8,

- contacting the washed column with an elution buffer under thereby forming an eluate comprising the antibody,

- collecting the eluate comprising the antibody.

The term "purifying" an antibody from a composition comprising the antibody and one or more impurities is meant increasing the degree of purity of the antibody in the composition by removing (completely or partially) at least one impurity from the composition. The term "polypeptide" as used herein refers to a sequential chain of amino acids linked together via peptide bonds.

The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies and multispecific antibodies so long as they exhibit the desired biological activity.

According to an embodiment of the present invention, wherein the antibody is specific for anti-HER antibody, anti-VEGF antibody, anti-TNF antibody, anti-CD20 antibody, anti-CD52 antibody, anti-lgE antibody.

According to another embodiment of the present invention, wherein said antibody is selected from trastuzumab, bevacizumab, pertuzumab, adalimumab, infliximab, certolizumab, rituximab, alemtuzumab, omalizumab, palivizumab, natalizumab, eculizumab, denosumab, panitumumab, pembrolizumab, ipilimumab, nivolumab, canakinumab, golimumab, ustekinumab, basiliximab, cetuximab, nimotuzumab, etanercept, raxibacumab, belimumab, ofatumumab, siltuximab, , tocilizumab, durvalumab, avelumab, brodalumab, dupilumab, emicizumab, ocrelizumab, sarilumab, guselkumab, reslizumab, ixekizumab, evolocumab, obiltoxaximab, olaratumab, bezlotoxumab, atezolizumab, daratumumab, elotuzumab, secukinumab, mepolizumab, alirocumab, idarucizumab, ramucirumab, obinutuzumab, efalizumab, vedolizumab, ibritumomab, basiliximab , dinutuximab, necitumumab.

In a more preferred embodiment, the present invention relates to a method for purifying an antibody that binds specifically to human epidermal growth factor receptor-2 (HER-2) from a composition comprising the antibody and one or more impurities.

According to one embodiment of the present invention, wherein said antibody is trastuzumab and bevacizumab.

The "composition" to be purified herein comprises the antibody of interest and one or more impurities. The composition may be "partially purified" by subjecting to one or more purification steps or may be obtained directly from a host cell or organism producing the antibody (e.g. the composition may comprise harvested cell culture fluid).

The term "impurity" refers to any foreign or undesirable molecule that is present in a solution such as a load fluid. An impurity can be a biological macromolecule such as host cell proteins, DNA, RNA, endotoxin and leached Protein A, product-related impurities such as dimer/aggregate, endogenous retrovirus and adventitious viruses such as parvovirus, pseudorabies virus. The term "aggregate" is a material that is different from the desired polypeptide product which may be formed when subjected to different environmental conditions such as buffer type, temperature, pH and salt concentration during upstream, downstream, and storage processes.

A “buffer” is a solution that resists changes in pH by the action of its acid-base conjugate components.

The term “wash buffer” is used herein to refer to the buffer that is passed over the protein A column following loading and prior to elution of the protein of interest.

The "elution buffer" is used to elute protein from the immobilized Protein A. Preferably the elution buffer has a low pH and thereby disrupts interactions between Protein A and the protein of interest.

According to an embodiment of the present invention, a Protein A column is washed with three steps with wash buffers which have different concentrations, components, pH.

According to an embodiment, the Protein A column is washed by using a first wash buffer.

According to an embodiment, said first wash buffer components are selected from the group comprising 0.1-2000 mM sodium chloride, 0.01-500 mM sodium phosphate, 0.01-500 mM phosphate buffers, 0.01-500 mM potassium chloride, 0.01-2000 mM glycine, 0.01-500 mM L- histidine, 0.01-500 mM tris buffers, 0.01-1000 mM HEPES or mixtures thereof.

According to the preferred embodiment of the present invention, said first wash buffer components comprise 150 mM sodium chloride and 50 mM sodium phosphate buffer.

According to the preferred embodiment of the present invention, said first wash buffer is performed at a pH ranging from about pH 6 and pH 7,8, preferably 6 to 7,5, most preferably 7,4.

According to an embodiment of the present invention, the Protein A column is washed by using a second wash buffer.

According to an embodiment of the present invention, said second wash buffer components are selected from the group comprising 0.1-2000 mM sodium chloride, 0.01-500 mM sodium phosphate, 0.01-500 mM phosphate buffers, 0.01-500 mM potassium chloride, 0.01-2000 mM glycine, 0.01-500 mM L-histidine, 0.01-500 mM tris buffers, 0.01-1000 mM HEPES or mixtures thereof. According to the preferred embodiment of the present invention, said second wash buffer components comprise 1 M sodium chloride and 50 mM sodium phosphate buffer.

According to the preferred embodiment of the present invention, wherein the second wash buffer is performed at a pH ranging from about pH 5,5 and pH 7,8, preferably 6 to 7,5, most preferably 7,4.

According to an embodiment of the present invention, the Protein A column is washed by using a third wash buffer.

According to an embodiment of the present invention, said third wash buffer component is selected from the group comprising 0.001-2 M sodium chloride, 10-2000 mM sodium acetate, 10-2000 mM acetate, 10-2000 mM tris-HCI, glycine, 10-500 mM maleic acid, 10-2000 mM citric acid, 10-2000 mM sodium citrate, 10-500 mM lactic acid, 10-500 mM L-histidine, IQ- 500 mM lactic acid, 10-500 mM formic acid, 10-500 mM succinic acid, 10-2000 mM acetic acid, 10-500 mM malonic acid, 10-500 mM MES, 10-1000 mM sodium phosphate, 10-2000 mM tris buffers mixtures thereof.

According to the preferred embodiment of the present invention, said third wash buffer component comprises 50 mM sodium acetate.

According to the preferred embodiment of the present invention, wherein the third wash buffer is performed at a pH from about ranging pH 4,5 to pH 6,8, preferably 5 to 6, most preferably 5,6.

It has been surprisingly seen that using a high salt wash buffer and another low pH (higher than pH of the elution buffer) wash buffer in the process and eluting the protein of interest from the column at low buffer pH condition removes specific impurities such as aggregates, dimers and monomers.

According to an embodiment of the present invention, wherein said wash buffers do not contain arginine or derivative of arginine or other additional substances.

It also has been observed that process-related and product-related impurities are removed without the use of an additional substance such as arginine or an arginine derivative or other additional substances.