Method for purifying bone morphogenetic protein
Inventors
Shim, Young-Bock • Kim, Yeong-Schick • Choi, Yon-Rak • Jang, Ju-Woong
Assignees
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Publication Number
US-8648178-B2
Publication Date
2014-02-11
Expiration Date
Abstract
The present invention relates to a method for purifying a protein belonging to the TGF-β, superfamily, preferably BMP, and more preferably BMP-2. According to the invention, the number of purification steps is reduced and the purification process is simplified, compared to the conventional BMP-2 purification method. Thus, the time required for purification can be shortened and the cost can be reduced. In addition, the invention solves the problem that as the time for purification increases and the number of purification steps increases, BMP-2 is degraded by protease or lost during purification steps, resulting in a decrease in the final yield of BMP-2. Thus, the invention increases the final yield of BMP-2. In addition, according to the invention, although the number of purification steps is reduced, BMP-2 having high purity is obtained in high yield by optimizing and using filtrations and chromatographies, and columns, types and concentrations of buffers, and a cut-off size of membrane used in diafiltration, which are different from those of the conventional BMP-2 purification method.
Core Innovation
Disclosed is an improved method for purifying bone morphogenetic protein 2 (BMP-2) from a BMP-2-containing solution. The method reduces purification steps and purification time while preventing yield loss associated with proteolysis and/or process losses.
The purification method concentrates the BMP-2-containing solution using a 30 kDa cut-off membrane filter, then subjects the concentrated solution to hydrophobic interaction chromatography using butyl sepharose 4 fast flow. After hydrophobic interaction chromatography, the process includes diafiltering the solution using a 30 kDa cut-off membrane filter with a Tris/NaCl/L-arginine/glycerol buffer.
The resulting solution is then subjected to size exclusion chromatography to obtain purified BMP-2, using Sephacryl S-100 with Tris/NaCl buffers. The document reports improved purity and identity as demonstrated by SDS-PAGE and Western blot analyses, and comparative performance versus a commercial BMP-2 standard, including final yield.
Claims Coverage
The independent claim covers a multi-step BMP-2 purification workflow with four sequential inventive operations: 30 kDa cut-off membrane concentration, hydrophobic interaction chromatography, diafiltration, and size exclusion chromatography. Overall, the independent claim specifies a defined order of processing to obtain purified BMP-2.
Concentrating BMP-2 using a 30 kDa cut-off membrane filter
Pre-treating by concentrating a solution containing BMP 2 using a 30 kDa cut-off membrane filter.
Hydrophobic interaction chromatography of the concentrated BMP-2 solution
Subjecting the solution obtained in step a) to hydrophobic interaction chromatography.
Diafiltering after hydrophobic interaction chromatography
Diafiltering the solution obtained in step b).
Size exclusion chromatography to obtain purified BMP-2
Subjecting the solution obtained in step c) to size exclusion chromatography to obtain purified BMP-2.
The claim set covers purifying BMP-2 by combining 30 kDa cut-off membrane concentration with hydrophobic interaction chromatography, followed by diafiltration and completion with size exclusion chromatography, with dependent claims further refining specific resins and buffer condition constraints.
Stated Advantages
Reduces purification steps and purification time.
Prevents yield loss from proteolysis and/or process losses.
Provides high final yield and high purity of purified BMP-2, with identity confirmation using SDS-PAGE and Western blot.
Documented Applications
Purification of bone morphogenetic protein 2 (BMP-2) from a BMP-2-containing solution, including material associated with Chinese hamster ovary (CHO) cell culture fluid.
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