Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1997-10-17
2003-09-30
Brumback, Brenda (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S412000, C530S415000, C530S416000
Reexamination Certificate
active
06627737
ABSTRACT:
FIELD OF INVENTION
The present invention relates generally to novel protein recovery and purification methods and more specifically to novel methods for the recovery and purification of factor IX.
BACKGROUND OF THE INVENTION
The advent of recombinant technology now allows for the production of high levels of proteins within suitably transformed host cells. For secreted proteins, purification of the protein of interest involves isolation and purification from the host cell culture medium. Typically, the culture medium contains selected nutrients (e.g. vitamins, amino acids, cofactors, minerals,) and additional growth factors/supplements including insulin and possibly additional exogenous proteins. Conditioned medium contains not only the secreted product of interest, but also significant quantities of additional secreted host cell proteins and other substances (e.g. nucleic acids, membrane vesicles). Although expressed at high levels, the product of interest may represent a minority of all proteins present in conditioned medium. Not unexpectedly, proteins secreted by transformed host cells may possess characteristics quite different from those of the product of interest (e.g. charge, molecular size, amino acid composition). Similarly, selected secreted host cell proteins may exhibit properties very similar to those of the product of interest, thereby placing significant burden on the process used for purification. While developing a process for purification of a recombinant protein from conditioned medium, it is important that conditions used be limited with respect to denaturation of the product of interest (conditions which could be used to exploit minor differences between secreted proteins for major benefit to separation), thereby making it difficult to separate the product of interest from all other host cell proteins present.
In addition to secreted host cell proteins described above, conditioned medium may also contain products derived from the heterologously-expressed gene coding for the product of interest. These are not desirable for the final drug substance and include, for example, product forms lacking certain post-translational modifications such as glycosylation, sulfation, gamma carboxylation, or other modification potentially necessary for biological activity. In addition, proteolytically-degraded forms of the product of interest may be present in conditioned medium which also need to be removed during purification, but which very closely resemble the product of interest. Unfortunately, most approaches, such as ion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography may not provide the extent of resolution of the product of interest necessary for use in human therapeutic situations from these undesired forms. To take full advantage of minor differences between the desired product and contaminants (e.g. small charge differences, small differences in molecular size) the use of strong denaturants is often required. Such denaturants, however, can lead to loss of biological activity, expression of neoantigenic sites, and potentially enhance chemical decomposition of selected post-translational modifications.
In addition to separating the product of interest from molecules with similar properties (e.g. modified forms of the expressed gene), it is also important to recognize the need to separate the desired product from components present in conditioned medium with which it specifically interacts. Where the protein of interest is positively charged, it will tend to bind to any negatively charged molecules present thereby making purification of the protein by traditional methods very difficult.
Of general background interest to the present invention are the following. Yan, U.S. Pat. No. 4,981,952 (Jan. 1, 1991) and Yan, etal. Bio/Technology 8:655 (July 1990) which disclosed the use of pseudo-affinity anion exchange chromatography for the purification of vitamin K-dependent proteins. Josic, et al. J. Chrorn. 632:1 (1993) disclosed the use of heparin affinity chromatography to resolve factor IX from other vitamin K-dependent proteins. Suomela. Thromb. Res. 7:101 (1975); Suomela, Eur. J. Bio. Chem. 71:145 (1976); and Suomela, Thrombos. Haemostis. 35:211 (1976) described the use of hydroxyapatite in the separation of various clotting factors and factor IX plasma variants (based on charge differences due to variation in content of carbohydrate moieties, for example, sialic acid and galactose). However, Reekers, et al. Haemostasis 1:2 (1972) demonstrated the inability of hydroxyapatite to separate factors II, VII and IX from each other and from other plasma proteins. Schwinn, et al. U.S. Pat. No. 4,411,794 disclosed the partial purification of blood clotting factors using hydroxyapatite in the presence of calcium at a concentration of 50-200 mM. Feldman, et al. Biotech. Blood Proteins 227:63 (1993) and Roberts, et al. Vox Sang 67(suppl. 1): 69 (1994) disclosed the reduction of viral infectivity using acidification and copper-charged chelating Sepharose which resulted in low factor IX yields from human plasma.
Typically, researchers have used combinations of traditional chromatographic techniques to purify desired products. Often times, such techniques are not sufficient for purification of a product to the level of purity and consistency desired for a human therapeutic product. Researchers have attempted to overcome this difficulty by use of affinity chromatography wherein a protein of interest is bound to an immobilized ligand with which it interacts specifically. Following appropriate washing, the desired product can be eluted by disruption of the ligand-protein interaction, often resulting in a significantly more pure eluate. However, in the instance of separation of a desired product from modified forms present in conditioned medium, single step affinity chromatographic techniques may not be sufficient, and must be used in conjunction with other affinity resins and/or traditional separation techniques. Even high resolution affinity chromatography steps (e.g., immunoaffinity purification using an immobilized monoclonal antibody) may not afford sufficient resolution of the desired product from other components due to common sites of interaction (e.g., where an epitope which is present in the product of interest, is present as well in a proteolytically-degraded form of the product).
Accordingly, there continues to exist a need in the art for protein purification methods that effectively overcome such difficulties.
BRIEF SUMMARY OF THE INVENTION
Provided by the present invention are methods for the purification of factor IX in a solution comprising the steps of applying the solution containing factor IX to an anion exchange resin, washing said anion exchange resin with a solution having a conductivity that is less than required to elute factor IX from the resin, eluting said anion exchange resin with a first eluant to form a first eluate, applying said eluate to a heparin or heparin-like (e.g., negatively charged matrix) resin, eluting said heparin or heparin-like resin with a second eluant to form a second eluate, applying said second eluate to an hydroxyapatite resin, and then eluting said hydroxyapatite resin with a third eluant to form a third eluate containing the purified factor IX. Optionally, the first eluate can be applied to an hydroxyapatite resin. As yet another option, the method comprises the further steps of applying the third eluate to an immobilized metal affinity resin, and then eluting the immobilized metal affinity resin with a fourth eluant to form a fourth eluate containing the purified factor IX. According to the methods of the invention, the factor IX can be either plasma-derived, expressed by cells in culture, or recombinantly produced as is known to one skilled in the art. Preferably, the first wash comprises a solution having a conductivity that is less than required to elute factor IX from the column and is generally greater than or equal to the conductivity of the load solution and of the first elua
Bonam Duane
Costigan Robert J.
Foster W. Barry
Switzer Mary B.
Walsh Rochelle
Brumback Brenda
Elrifi Ivor R.
Genetics Institute LLC
Gupta Anish
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
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