With the advent of recombinant protein technology, a protein of interest can be produced using cultured eukaryotic or prokaryotic host cell lines engineered to express the protein. The use of the desired recombinant protein for pharmaceutical applications is generally contingent on being able to reliably recover adequate levels of the protein from impurities such as host cell proteins, protein variants, and compounds from the culture medium.
Conventional protein purification methods are designed to separate the protein of interest from impurities based on differences in size, charge, solubility, and degree of hydrophobicity. Such methods include chromatographic methods such as affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, and hydroxyapatite chromatography. These methods often employ a separation medium that can be designed to selectively adhere either the protein of interest or the impurities. In the bind-elute mode, the desired protein selectively binds to the separation medium and is differentially eluted from the medium by different solvents. In the flow-through mode, the impurities specifically bind to the separation medium while the protein of interest does not, thus allowing the recovery of the desired protein in the “flow-through.”
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 chromatography for protein purification. In some instances, leakage of the Protein A capture agent results in contamination of the eluted protein product, while in other instances, affinity capture does not separate protein variants, such as 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 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.