Study for the purification of fc-containing proteins

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The present invention is in the field of protein purification. More specifically, it relates to the purification of Fc -containing proteins. The method comprises at least a step of purification via cation exchange chromatography.

Proteins have become commercially important as drugs that are generally called "biologicals". One of the greatest challenges is the development of cost effective and efficient processes for purification of proteins on a commercial scale. While many methods are now available for large-scale preparation of proteins, crude products, such as cell culture supematants, contain not only the desired product but also impurities, which are difficult to separate from the desired product. Although cell culture supematants of cells expressing recombinant protein products may contain less impurities if the cells are grown in serum-free medium, the host cell proteins (HCPs) still remain to be eliminated during the purification process. Additionally, the health authorities request high standards of purity for proteins intended for human administration.

A number of chromatographic systems are known that are widely used for protein purification.

Ion exchange chromatography systems are used for separation of proteins primarily on the basis of differences in charge.

Anion exchangers can be classified as either weak or strong. The charge group on a weak anion exchanger is a weak base, which becomes de-protonated and, therefore, loses its charge at high pH. DEAE-sepharose is an example of a weak anion exchanger, where the amino group can be positively charged below pH ~ 9 and gradually loses its charge at higher pH values. Diethylaminoethyl (DEAE) or diethyl-aminoethyl (QAE) have chloride as counter ion, for instance. A strong anion exchanger, on the other hand, contains a strong base, which remains positively charged throughout the pH range normally used for ion exchange chromatography (pH 1-14). Q-sepharose is an example for a strong anion exchanger.

Cation exchangers can also be classified as either weak or strong. A strong cation exchanger contains a strong acid that remains charged from pH 1 - 14; whereas a weak cation exchanger contains a weak acid , which gradually loses its charge as the pH decreases below 4 or 5. Carboxymethyl (CM) and sulphopropyl (SP) have sodium as counter ion, for example.

Hydrophobic interaction chromatography (HIC) is used to separate proteins on the basis of hydrophobic interactions between the hydrophobic moieties of the protein and insoluble, immobilized hydrophobic groups on the matrix. Generally, the protein preparation in a high salt buffer is loaded on the HIC column. The salt in the buffer interacts with water molecules to reduce the salvation of the proteins in solution, thereby exposing hydrophobic regions in the protein which are then adsorbed by hydrophobic groups on the matrix. The more hydrophobic the molecule, the less salt is needed to promote binding. Usually, a decreasing salt gradient is used to elute proteins from a column. As the ionic strength decreases, the exposure of the hydrophilic regions of the protein increases and proteins elute from the column in order of increasing hydrophobicity.

Hydrophobic charge induction chromatography (HCIC) is another mode of chromatography based on the pH dependent behavior of heterocyclic ligands that bnize at low pHs. While adsorption on this mode of chromatography occurs via hydrophobic interactions, desorption is facilitated by lowering the pH to produce charge repulsion between the ionizable ligand and the bound protein.

Yet a further way of purifying proteins is based on the affinity of a protein of interest to another protein that is immobilized to a chromatography resin. Examples for such immobilized ligands are the bacterial cell wall proteins Protein A and Protein G, having specificity to the Fc portion of certain immunoglobulins. Although both Protein A and Protein G have a strong affinity for IgG antibodies, they have varying affinities to other immunoglobulin classes and isotypes as well.

Affinity chromatography on protein A allows the clearance of more than 99.5 % of the impurities such as host cell proteins (HCPs), DNA, viruses, incomplete forms of the antibodies in only one step. However, the major disadvantage of this purification technique is the cost of the resin. It is approximately 30 times more expensive than ion exchange resins and can represent nearly 35 % of the total cost of the raw material used for large scale purification. Protein A resin also presents some stability problems as Protein A residues, which are potentially immunogenic, are found in the eluate and need therefore to be cleared. Protein A resin is also difficult to sanitize as the ligand is easily denatured by common sanitization solutions like sodium hydroxide and this represents a major problem in production in the event of contamination as reuse of the resin may be detrimentally affected. Combinatorial chemistry has enabled the synthesis of a wide variety of ligands which can mimic the action of protein A e.g. the triazine derivatives that mimic the Phe-132, Tyr-133 dipeptide binding site in the hydrophobic core structure of Protein A (marketed as MAbsorbent A1 P, A2P, and A3P by Prometic). A further way of purifying antibodies uses affinity ligands developed by making use of Camelidae heavy chain antibody fragments.

In the field of antibody protein purification, Follman and Fahrner (2004) have determined that the same host cell protein removal obtained with a process incorporating Protein A chromatography can be achieved using a process with no affinity chromatography steps. They identified three non-affinity purification processes including hydrophobic interaction chromatography, anion- exchange chromatography and cation -exchange chromatography that remove CHOPs to levels comparable to the traditional Protein A process. They also disclose a method for protein purification that invdves the combination of non-affinity chromatography and high performance tangential flow filtration (HPTFF). After a first purification (capture) step on cation exchange chromatography the host cell protein content was about 14,000 ppm.