Methods for purifying a target protein from one or more impurities in a sample

Medicilon's protein scientists have been working on protein expression and purification for many years. We can start your project even you have nothing in hand but the name of your protein. In Medicilon's laboratories, protein purification is performed in scales from micrograms and milligrams. All Protein Purification Services start with the analysis of physico-chemical and biological properties of a target protein resulting in the development of tailored procedures for its extraction, purification and characterization.Email:marketing@medicilon.com.cn Web:www.medicilon.com

The present invention relates, at least in part, to improved methods of protein purification. In particular, the present invention relates, at least in part, to methods for purifying an Fc region containing protein from a composition comprising the Fc region containing protein and one or more impurities, where the methods eliminate the need for a holding tank and/or a buffer exchange step.

Field of the Invention

The present invention relates, at least in part, to methods of protein purification. In particular, the present invention relates, at least in part, to methods for purifying a target protein (e.g., Fc region containing proteins such as, e.g., antibodies and fragments thereof and antibody-like molecules such as, e.g., immunoadhesins) from a composition comprising the target protein and one or more impurities.

Background:Efficient and economic large scale purification of proteins, e.g., therapeutic proteins including antibodies, is an increasingly important consideration for the biotechnology and pharmaceutical industries. Generally, the processes for protein purification are quite elaborate and expensive and include many different steps. For example, typically, proteins are produced using cell culture methods, e.g., using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. In general, separation of the desired protein from the media components which are fed to the cells and from the by-products of the cells themselves as well as any other impurities that may exist poses a formidable challenge. Such separation is especially important when the therapeutic proteins are meant for use in humans and have to be approved by the Food and Drug Administration.

In general, the purification processes that are currently being used, include at least the following steps: cell lysis to recover an intracellular protein or recovery of a protein from the media in case of a secreted protein; removal of cellular debris using differential centrifugation or filtration to obtain a clarified sample containing the protein of interest; use of a variety of chromatography media in a multi-step process to separate a protein of interest from the various impurities in the sample. [0005] Commonly used chromatography methods include one or more of affinity chromatography media, ion exchange chromatography media, hydrophobic interaction, hydrophilic interaction, size exclusion and mixed mode (i.e., combination of various chromatography methods). For example, for purification of monoclonal antibodies, several processes have been described, most of which include an initial Protein A affinity capture step followed by one or more ion exchange polishing steps. Further, other chromatography technologies, such as: bind and elute hydrophobic interaction chromatography (HIC); flow- through hydrophobic interaction chromatography (FTHIC); mixed mode chromatography techniques, e.g., bind and elute weak cation and anion exchange (Abx), bind and elute hydrophobic and ion exchange interaction and flow-through hydrophobic and ion exchange mixed mode interaction (FTMM), both of which can utilize resins such as Capto Adhere, Capto MMC, HEA Hypercel, PPA Hypercel, may be used. Additionally, hydrophobic charge induction (HCI) chromatography along with others and combinations of various techniques can be used for polishing. Generally, it is important for the polishing steps to contain an anion exchange step to provide an additional, orthogonal virus removal step.

Although, protein purification methods involving a combination of various chromatography steps have been described, as discussed above, these methods require the use of a holding tank and/or a buffer exchange step between each chromatography step. Typically, the target protein is eluted into a holding tank where the buffer/solution conditions are adjusted such that they are suitable for the next chromatography step (referred to as buffer exchange). The conditions of the buffer in the holding tank, e.g., pH., salt etc., are typically adjusted as necessary, prior to loading onto to the next chromatography media or a filtration membrane.

Summary of the Invention

The present invention provides, at least in part, improved methods of purifying or separating a protein of interest from one or more impurities, where the methods eliminate the need for a holding tank and/or a buffer exchange step between various chromatography steps.

In various embodiments, the present invention provides for the purification of

Fc region containing proteins (e.g., monoclonal antibodies and similar proteins) with a robust template, however, avoids intermediate holding tank or buffer exchange steps. Accordingly, the purification process may be referred to as a "connected process." The process encompasses the use of appropriate conditions and chromatography media to achieve a continuous, holding tank free process.