Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`[DESCRIPTION]
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`[Invention Title]
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`METHODFOR PREPARING GLY-TB4
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`[Technical Field]
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`The present invention relates to a method of preparing Gly-thymosin B4 (Gly-
`
`TB4), and more particularly, to a method of preparing Gly-TB4, which includes an
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`expression process for expressing GST fusion recombinant human thymosin B4
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`(hereinafter, “GST-TB4”), which is a precursor of Gly-TB4, and a Gly-TB4 purification
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`process.
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`[Background Art]
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`Large-scale protein purification is an issue that has become more and more
`
`important in the biotechnological industry. Generally, proteins are produced bycell
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`culture using a mammalian or bacterial cell line engineered to produce a target protein
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`by inserting a recombinant plasmid containing the gene of the target protein.
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`Since
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`cell
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`lines used herein are living organisms, composite culture media containing
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`saccharides, amino acids and growth factors, which are generally provided from
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`animal serum preparations, will be provided to the cells.
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`It is very difficult to
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`separate a desired protein from the mixture of the compounds providedto the cells and
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`by-products produced from the cells in sufficient purity for human treatment.
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`However, in the case of biologics such as proteins used as drugs, the removal
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`of impurities as well as a product yield is very important. There may be a difference
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`between process-dependent
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`impurities and product-dependent
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`impurities.
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`The
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`process-dependent impurities contain host cell components such as a protein and a
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`nucleic acid, and are derived from a cell culture (e.g., medium ingredient), or a post-
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`treatment process (e.g., a salt or separated chromatography ligand). The product-
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`dependent impurities are molecular variants of products with different properties.
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`For example,
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`such molecular variants
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`include modified forms produced by
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`deamination,
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`incorrect glycosylation or mismatched disulfide bridges as well as
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`shortened forms such as a precursor and hydrolytic degradation products.
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`The
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`product-dependent variants also include a polymer and an aggregate.
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`The term
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`“contaminant” is used to present any material having a chemical, biochemical or
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`microbiological characteristic, which is not directly involved in a preparation process.
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`The contaminant includes viruses that may undesirably appearin a cell culture.
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`Impurities and contaminants cause safety issues in the case of biologics.
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`It
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`happens very often in the case of biologics, and for example, it could be seriousif a
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`therapeutic protein is administered directly into the bloodstream by injection or
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`infusion. Accordingly, host cell ingredients may cause an allergic response or an
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`immunopathological effect.
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`In addition,
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`impurities may lead to undesirable
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`immunogenicity of the administered protein, which triggers an undesirable immune
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`response ofa patient to the therapeutic, for example, to the point of lethal anaphylactic
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`shock. Therefore, there is a need for a suitable purification method that can remove
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`20
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`all undesirable materials to an insignificantlevel.
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`Meanwhile, in the case of biologics, economical aspects cannot be ignored.
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`Accordingly, the production and purification methods used should not threaten the
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`economic feasibility of the biologics produced.
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`Jn addition, the measure of time over
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`which a novel purification method is able to be established plays an importantrole.
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`Substitute Specification — Marked Up
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`That is, in addition to a cost effect, process development needs to be coordinated with
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`preclinical and clinical development of the drug.
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`Therefore, for instance, some
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`preclinical trials and all clinical trials can only be initiated when biologics of sufficient
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`purity are available at sufficient amounts.
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`A method of purifying a protein from cell debris initially depends on the
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`protein’s expression site. One protein may be directly secreted from cells to a
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`surroundingproliferation medium, and another protein may be made in cells.
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`In the
`
`case of the latter protein, the first step of the purification method includesa cell lysis
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`step that can be performed by various methods including mechanical shearing, osmotic
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`shock or enzymatic treatment. Due to such cell lysis, the entire cell contents are
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`released into a cell suspension, and intracellular fragments that are difficult to remove
`
`due to their small size are produced. These intracellular fragments are generally
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`removed by fractional centrifugation or filtration.
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`To a lesser extent than in the
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`above-described case, this problem also appearsin proteins directly secreted due to the
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`release of host cell proteins in cells during natural cell death and protein production.
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`Whena purified solution containing a target protein is obtained, separation of
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`the target protein from another protein produced by cells is generally performed by a
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`combination of different
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`chromatography techniques.
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`The
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`key
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`to
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`this
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`chromatography technique is that, as proteins may move down a long column at
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`20
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`different speeds,
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`the proteins may be more and more physically separated, or
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`selectively adhered to separation media, thereby being differentially eluted by different
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`solvents.
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`In some cases, when impurities are specifically attached to a column, and
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`the target protein is not attached thereto, that is, present in flow-through, the target
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`protein is separated from the impurities, or when the target protein is specifically
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`Attorney Docket No. 206132-0113-00US
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`attached to the column,
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`the attached target protein may be separated from the
`
`impurities through elution. Numerous kinds of the column chromatography method
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`and materials that can be used in this step are known. However, as the number of
`
`alternative methods increases, a much larger numberofpreliminary trials need to be
`
`carried out to determine optimal material and method in terms of purification effect,
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`yield, biological activity, time and cost.
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`In addition, in the establishment and optimization of a purification method,it
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`is clear that the method has to be adjusted individually according to the biochemical
`
`and biophysical properties of a specific molecule to be purified (target molecule or
`
`target protein) and a biological starting material. The biological starting material
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`from which the target material will be separated is generally formed of a mixture of
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`very complicated materials. To separate and concentrate the target molecule, specific
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`characteristics such as a shape, a size,
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`solubility, a surface charge,
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`surface
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`hydrophobicity and bio-specific affinity to a binding partner are used. For a new
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`target molecule, that is, even for the same target molecule changed only in oneof the
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`previous steps (e.g., a change in composition of a fermentation culture medium), the
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`purification process has to be newly adjusted because the best possible result may not
`
`be achieved from the above-mentioned aspects under the new conditions.
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`At the same time, the numberof alternative processes that can be theoretically
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`20
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`estimated depends on the number of parameters
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`listed above.
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`In column
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`chromatography, for example, in the entire process, a series of chromatography steps,
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`a column material, a pH, a salt content and the properties of various elution buffers
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`used, a protein concentration when chargedin the column, and may other aspects need
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`to be optimized.
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`This makes it
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`impossible to develop an optimal column
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`chromatography process within appropriate cost and time ranges on an industrial scale.
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`Meanwhile, economic feasibility and device-related limitations (e.g., the use of as few
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`kinds of buffers as possible, the necessity of minimum amounts or volumes of the
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`buffers and chromatography materials,
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`the necessity of maintaining a product-
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`containing fraction volumeas small as possible, and the necessity of minimum process
`
`time and wastewater volume) also need to be optimized in each step of the process.
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`Therefore, the column chromatography purification method of a biomolecule
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`may beestablished and optimized rapidly and at low cost, and there is still an urgent
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`need for a process of obtaining satisfactory results under large-scale production and
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`purification conditions.
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`However, Gly-thymosin 34 (Gly-TB4) is a peptide represented by a sequence
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`of 44 amino acids, and has glycine (Gly) added to the N-terminus of conventional
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`thymosin B4 (TB4). Gly-TB4 was found through the known quaternary structure
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`analysis of TB4 and the genetic engineering modification of the N-terminus of the
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`protein, and has different activity from original TB4. The structure and function of
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`TB4 are relatively defined, and TB4 is one of the known major thymusfactors.
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`It is
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`known that TB4 is the first separated polypeptide from thymosin fraction 5 (TF5)
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`extracted from the bovine mammary gland, and has 43 amino acids and a molecular
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`20
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`weight of 4963KD without bisulfide bonds and glycosylation (The Journal of
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`Biological Chemistry, Vol 257, No2, pp1000-1006, 1982, Chemical Characterization
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`of Thymosin beta).
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`Gly-TB4 is obtained by expression and purification of Gly-TB4 through
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`fermentation in bacterial cells manipulated to express conventional Gly-TP4.
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`However, it has been pointed out that a considerable amount of proteins is lost in
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`purification, and Gly-TB4 obtained through the above-mentioned process ultimately
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`has low productivity.
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`In addition, when GST-TB4 is fermented according to a
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`conventional 30 L culture batch record (hereinafter, “NR culture process’)
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`manufactured by Northland Biotech, Beijing, China, disadvantages such as the
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`formation of a medium precipitate and acetate accumulation as well as
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`low
`
`productivity have been pointed out.
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`In addition, when purification is carried out
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`according to the purification process of Northland Biotech, due to a low proliferation
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`yield, a high unit cost, and the low purity of the final product, there were many
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`problems for use as a medicine.
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`[Disclosure]
`
`[Technical Problem]
`
`The present invention is directed to providing a method of stably obtaining a
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`high-purity Gly-TB4 on a large scale. More specifically,
`
`the present invention
`
`provides a method of preparing glycine-thymosin B4 (Gly-TB4), which includes the
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`following steps: (S1) loading a sample containing GST fusion thymosin B4 (GST-TB4)
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`in a primary anion exchange chromatography column equilibrated with an
`
`equilibration buffer and eluting a fraction attached to the column with the elution
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`20
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`buffer; (S2) performing affinity chromatography on an eluate; ($3) enzymatically
`
`cleaving the eluate by thrombin treatment; (S4) loading the cleaved product in a
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`secondary anion exchange chromatography column equilibrated with an equilibration
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`buffer, and eluting a fraction attached to the column with an elution buffer at a dynamic
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`binding capacity (DBC) of 1.8 mg/mLor less; (S5) loading the eluate in a cation
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`exchange chromatography column equilibrated with an equilibration buffer, washing
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`the column with a wash buffer, and eluting a fraction attached to the column with an
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`elution buffer at a DBC of 5 mg/mLorless; and (S6) filtrating the eluate.
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`However, technical problems to be solved in the present invention are not
`
`limited to the above-described problems, and other problems which are not described
`
`herein will be fully understood by those of ordinary skill in the art from the following
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`descriptions.
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`[Technical Solution]
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`To solve the above-described problem, the present invention provides a method
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`of preparing glycine-thymosin B4 (Gly-TB4).
`
`Morespecifically, the method of preparing Gly-TB4 according to the present
`
`invention includes the following steps:
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`(S1) loading a sample in a primary anion exchange chromatography column
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`equilibrated with an equilibration buffer, washing the column with a wash buffer, and
`
`eluting a fraction attached to the column with the elution buffer;
`
`(S2) performing affinity chromatography on an eluate;
`
`(S3) enzymatically cleaving the eluate by thrombin treatment;
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`20
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`(S4)
`
`loading the
`
`cleaved product
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`in a
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`secondary anion exchange
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`chromatography column equilibrated with an equilibration buffer, washing the column
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`with a washing buffer, and eluting a fraction attached to the column with an elution
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`buffer at a dynamic binding capacity (DBC) of 3 mg/mLorless;
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`(S5)
`
`loading the eluate in a cation exchange chromatography column
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`equilibrated with an equilibration buffer, washing the column with a wash buffer, and
`
`eluting a fraction attached to the column with an elution buffer at a DBC of 3 to 8
`
`mg/mL; and
`
`(S6) filtrating the eluate.
`
`In Step (S4), the cleaved product may be loaded in the column at pH 8 to 9 and
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`a conductivity of 4 mS/cm orless.
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`Thefraction attached to the column in the anion exchange chromatography in
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`Step (S4) is eluted with a buffer of pH 7 to 9, which contains 10 to 30 mM Tris-HCl
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`and 30 to 130 mM NaCl,at a flow rate of 100 to 300 cm/hr.
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`In the cation exchange chromatography in Step (S5), the cleaved productis
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`loaded in the column at pH 4 to 7 and a conductivity of 4 mS/cm orless.
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`In the cation exchange chromatography in Step (S5), the fraction attached to
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`the column is eluted with a buffer of pH 4 to 5, which contains 10 to 30 mM acetate
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`and 150 to 210 mM NaCl, at a flow rate of 100 to 300 cm/hr.
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`In Step (S3), the ratio of the target protein to thrombin may be 5 to 0.5:1
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`(unit:mg).
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`The reaction temperature in Step (S3) may be 13 to 27 °C.
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`The purity of the final purified extract according to the method may be 97% or
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`20
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`more.
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`In Step (S1), the sample may be obtained by the followingsteps:
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`culturing host cells expressing the target protein; lysing the host cells; and
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`washing andfiltering the lysate.
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`Substitute Specification — Marked Up
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`The main culture of the host cells may be fed-batch culture at OD6vo of 40 to
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`65 and an induction temperature of 25 to 35 °C in theinitiation of induction.
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`Cell productivity according to the method may be 180 g pellet/L or more, and
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`the productivity of the target protein may be 54 mg/g pellet or more.
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`Hereinafter, the present invention will be describedin detail.
`
`The present invention relates to a method of preparing glycine-thymosin B4
`
`(Gly-TB4), which includes the followingsteps:
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`(S1) loading a sample in a primary anion exchange chromatography column
`
`equilibrated with an equilibration buffer, washing the column with a wash buffer, and
`
`eluting a fraction attached to the column with an elution buffer;
`
`(S2) performing affinity chromatography on an eluate;
`
`(S3) enzymatically cleaving the eluate by thrombin treatment;
`
`(S4)
`
`loading the
`
`cleaved product
`
`in a
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`secondary anion exchange
`
`chromatography column equilibrated with an equilibration buffer, washing the column
`
`with a wash buffer, and eluting the fraction attached to the column with an elution
`
`buffer at a dynamic binding capacity (DBC) of 1.8 mg/mLorless;
`
`(S5) loading the eluate in a cation exchange chromatography equilibrated with
`
`an equilibration buffer, washing the column with a wash buffer, and eluting a fraction
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`20
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`attached to the column with an elution buffer at a dynamic binding capacity (DBC) of
`
`5 mg/mLorless; and
`
`(S6) filtering the eluate.
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`The term “glycine-thymosin B4
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`(Gly-TB4)” used herein is_ preferably
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`mammalian, and most preferably human Gly-TB4 protein or a polypeptide thereof.
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`The aminoacid sequence of the human Gly-TB4 protein is disclosed in GenBank No.
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`NP_066932.1, and the genetic sequence of the human Gly-TP4 protein is disclosed in
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`GenBank No. NM_021109.
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`The term “sample” used herein refers to a mixture of a target protein and one
`
`or more types of contaminants, and the “target protein” includes Gly-TB4, a
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`polypeptide thereof or a precursor thereof, and preferably, the precursor of the Gly-
`
`TB4 refers to GST fusion thymosin 34 (GST-TB4). According to one embodiment of
`
`the present invention, the sample contains GST fusion thymosin 84 (GST-TB4).
`
`In the present invention, at least 4 cycles of chromatography are performed.
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`Preferably, 4 cycles of chromatography are performed. Most preferably, 2 cycles of
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`anion chromatography, one cycle of affinity chromatography and one cycle of cation
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`chromatography are performed.
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`In addition, before or after Step $1), before or after
`
`Step $2), before or after Step $3), before or after Step S4), before or after Step S5), or
`
`before or after Step S6), any one or more chromatography techniques selected from
`
`the group consisting of affinity chromatography,
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`ion exchange chromatography,
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`hydrophobic interaction chromatography and mixed mode chromatography may be
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`20
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`further performed.
`
`In the present invention,all types of chromatography are preferably performed
`
`according to a binding/elution (B/E) mode. The term “B/E mode”refers to a working
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`method of chromatography in which a target protein binds to a chromatography
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`material by applying a solution containingthe target protein to be purified (e.g., sample)
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`to the chromatography material. Accordingly, the target protein is maintained on the
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`chromatography material, whereas a material that does not correspond to the target
`
`protein is removed along with a flow-through or supernatant.
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`Thetarget protein is
`
`then recovered from the chromatography material by an elution buffer.
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`In the present
`
`invention, according to the B/E mode, it is possible to obtain a high-purity target
`
`protein with high efficiency.
`
`The term “application”refers to a partial step of a purification method in which
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`a solution containing a target protein is in contact with a chromatography material.
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`This means that (a) a solution is added to a chromatography apparatus containing a
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`chromatography material, or (b) a chromatography material is added to asolution.
`
`In
`
`(a), the solution enables an interaction between the chromatography material and a
`
`material contained in the solution by passing through the apparatus.
`
`For example,
`
`according to conditions such as pH, conductivity, a salt concentration, a temperature
`
`and/or a flow rate, some materials in the solution may bind to the chromatography
`
`material, and other materials may be recovered from the chromatography material.
`
`Materials remaining in the solution or recovered from the chromatography material
`
`may be found in the flow-through. The term “flow-through” refers to a solution
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`obtained after passing through the apparatus, which may be a buffer (or solution)
`
`applied to wash a column or cause the elution of a material binding to the
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`20
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`chromatography material.
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`The apparatus is a column or cassette.
`
`In (b),
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`the
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`chromatography material is, for example, a solid, and allows the interaction between
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`the chromatography material and a material in the solution by being addedto a solution
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`containing a material to be purified. After the interaction,
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`the chromatography
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`material may be removedbyfiltration, and a material binding to the chromatography
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`Attorney Docket No. 206132-0113-00US
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`material is also removed from the solution, whereas a material that does not bind to
`
`the chromatography material remains in the solution.
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`In the chromatography step of the present invention, equilibration, loading,
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`washingandelution processesare included, and in each process, a buffer may be used.
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`The term “buffer” used herein is a solution resistant to a pH change by the
`
`action of an acid-base complex ingredient.
`
`For example, various buffers that can be
`
`used according to desired pH of the buffer are disclosed in the document[Buffers. A
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`Guide for the Preparation and Use of Buffers in Biological System, Gueffroy, D., Ed.
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`Calbiochem Corporation (1975)]. Generally, a pharmaceutically acceptable bufferis
`
`used.
`
`In one aspect, the buffer is selected from a phosphate buffer consisting of
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`phosphoric acid and/or a salt thereof, an acetate buffer consisting of acetic acid and a
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`salt thereof, a citrate buffer consisting of citric acid and/orasalt thereof, a morpholine
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`buffer, a 2-(N-morpholino)ethanesulfonic acid buffer, a histidine buffer, a glycine
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`buffer and a tris(hydroxymethyl)aminomethane(tris) buffer.
`
`In another aspect, the
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`buffer is selected from a Tris buffer, a Tris-hydrochloric acid buffer, a citrate buffer,
`
`and a histidine buffer. The buffer may contain, for example, an inorganic salt such
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`as sodium chloride, sodium sulfate, calcium chloride, calcium sulfate, ammonium
`
`chloride or ammonium sulfate.
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`20
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`In the present invention, the term “equilibration buffer” is used before being
`
`loaded in a column,and the equilibration buffer may be, for example, any one selected
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`from the group consisting of sodium citrate, sodium acetate, sodium chloride, sodium
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`phosphate, sodium sulfate, calcium chloride, potassium sulfate, potassium phosphate,
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`Tris, Tris-hydrochloric acid (HCI), 2-(N-morpholino)ethanesulfonic acid (MES), 4-(2-
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`hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 3-[(3-cholamidopropy])-
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`dimethylammonio]-1-propanesulfonate (CHAPS), but the present invention is not
`
`limited thereto.
`
`The term “wash buffer” used herein refers to a buffer used to wash or re-
`
`equilibrate an ion exchangeresin before elution of a target protein. The “washing”
`
`of an ion exchange resin meanspenetrating or passing a suitable (wash) buffer into or
`
`by an ion exchange resin. For example, the wash buffer may be any oneselected
`
`from the group consisting of sodium citrate, sodium acetate, sodium chloride, sodium
`
`phosphate,
`
`sodium sulfate, potassium chloride, potassium sulfate, potassium
`
`phosphate, Tris, Tris-hydrochloric acid (HCl), 2-(N-morpholino)ethanesulfonic acid
`
`(MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 3-[(3-
`
`cholamidopropy1)-dimethylammonio]-1-propanesulfonate (CHAPS), but the present
`
`invention is not limited thereto.
`
`The term “elution” used herein is for elution of a molecule (e.g., the target
`
`protein or contaminant) from an ion exchangeresin (or column), and the removal of a
`
`molecule from an ion exchangeresin (or column) by changing the ion concentration
`
`of a buffer surrounding the ion exchangeresin (or column)so that the buffer competes
`
`with the molecules for a charged part of the ion exchange resin.
`
`The elution is
`
`performed with a sufficient amount of buffer to cause efficient elution of the target
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`20
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`protein so that the elution buffer containing the target protein is recovered.
`
`The
`
`“efficient elution” refers to elution of 75% or more, or 80% or more, for example, 85%
`
`or more of the solution containing the target protein loaded in the resin from theresin.
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`In the present invention, the term “elution buffer” is used to elute the target protein
`
`from the ion exchange resin (or column). The elution buffer may be, for example,
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`any one or moreselected from the group consisting of sodium citrate, sodium acetate,
`
`sodium chloride, sodium phosphate, sodium sulfate, calcium chloride, potassium
`
`sulfate,
`
`potassium phosphate, Tris, Tris-hydrochloric
`
`acid
`
`(HCl),
`
`2-(N-
`
`morpholino)ethanesulfonic
`
`acid
`
`(MES),
`
`4-(2-hydroxyethyl)-1-
`
`piperazineethanesulfonic
`
`acid
`
`(HEPES)
`
`and
`
`3-[(3-cholamidopropy])-
`
`dimethylammonio]-1-propanesulfonate (CHAPS), but the present invention is not
`
`limited thereto.
`
`The conductivity and/or pH of the elution buffer are/is the
`
`conductivity and/or pH at which the target protein is eluted from aresin (column).
`
`In
`
`the present invention, the conductivity and/or pH is a degreeof elution of virtually all
`
`contaminants and target proteins using a resin.
`
`The term “conductivity” used herein refers to an ability of an aqueous solution
`
`allowing current flow between two electrodes.
`
`In the solution, current flows by ion
`
`transport. Accordingly, when an ion amount present in the aqueous solution is
`
`increased, the solution may have a higher conductivity. The measurement unit of
`
`conductivity is mmhos (mS/cm), and the conductivity may be measured using a
`
`commercially available conductivity meter, for example, Orion. The conductivity of
`
`the solution may be changed by changing the ion concentration of the solution.
`
`For
`
`example, to obtain a desired conductivity, the concentration of a buffer material in the
`
`20
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`solution and/or a salt (e.g., sodium citrate, sodium acetate, sodium chloride, sodium
`
`phosphate, sodium sulfate, calcium chloride, potassium sulfate, potassium phosphate,
`
`Tris, Tris-hydrochloric acid (HCI), 2-(N-morpholino)ethanesulfonic acid (MES), 4-(2-
`
`hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 3-[(3-cholamidopropy])-
`
`dimethylammonio|-1-propanesulfonate (CHAPS)) may be changed.
`
`Preferably, a
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`14
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`desired conductivity may be obtained by changing salt concentrations of various types
`
`of buffers.
`
`The term “dynamic binding capacity (DBC)”used herein refers to the capacity
`
`(Q) of a target molecule binding to a certain amount of resin under conditions for
`
`chromatography.
`
`Since the binding ability between materials causes changes in
`
`equilibrium and dissociation constants (K) according to a temperature, pH or a flow
`
`rate, the binding ability of a target molecule in a resin should be known to calculate a
`
`required amount of resin or a load amount of a specimen per column. DBC may be
`
`used as reference data to determine how much material to be separated or purified.
`
`DBC measuresthe point at which elutionstarts after all target molecules are bound to
`
`a column, and generally, the time point at which the concentration of 10% of the loaded
`
`target molecules is reachedis set as a starting point (Qs, 10%: Binding Capacity at 10%
`
`Breakthrough point). According to the characteristics of the target molecule, a
`
`method for an identity test and measurement may be, for example, an analysis method
`
`such as ELISA, SEC or UV absorbance, and after setting the 10% breakthrough point,
`
`the total volume from the moment of loading to the corresponding starting point is
`
`calculated to calculate the total amount and load amount (Vio% * Co) of the target
`
`material, and then divided by a resin volume (Vc): Qs, 10% = Vi0% * Co/ Ve.
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`20
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`The sample of the target protein obtained according to the method of the
`
`present invention may be subjected to an additional purification step if necessary.
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`The “purification” used herein refers to an increase in purity of the target
`
`protein by completely or partially removing one or more types of contaminants from
`
`the samplein whichthetarget protein is mixed with one or more types of contaminants.
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`Accordingto the present invention, the purity of the final purified extract is 97%
`
`or more.
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`Hereinafter, the present invention will be described by step. However, in
`
`order to avoid excessive repetition, overlapping description in each step is provided
`
`only oncein the first mentioned part and omitted.
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`The method includes (S1) loading a sample containing GST fusion thymosin
`
`B4 (GST-TB4) in a primary anion exchange chromatography column equilibrated with
`
`an equilibration buffer, washing the column with a wash buffer, and eluting a fraction
`
`attached to the column with the elution buffer.
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`In the present invention, an anion exchange resin in the anion exchange
`
`chromatography step may be one or morepositively-charged ligands attached to a solid
`
`phase, which are substituted with, for example, diethylamino ethyl (DEAE) or
`
`quaternary ammonium groups, but
`
`the present
`
`invention is not
`
`limited thereto.
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`Preferably, any one of anion exchangeresins having a group havingastrongly basic
`
`20
`
`quaternary ammonium group and a weakly basic DEAE group may be selected and
`
`used.
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`For example, as a strongly basic anion exchangeresin, Q Sepharose Fast Flow,
`
`Q Sepharose High Performance, Resource Q, Source 15Q, Source 30Q, Mono Q, Mini
`
`Q, Capto Q, Capto Q ImpRes, Q HyperCel, Q Cermic HyperD F, Nuvia Q, UNOsphere
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`Q, Macro-Prep High Q, Macro-Prep 25 Q, Fractogel EMD TMAE(S), Fractogel EMD
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`TMAEHicap (M), Fractogel EMD TMAE (M), Eshmono Q, Toyopearl QAE-550C,
`
`Toyopearl SuperQ-650C, Toyopearl GigaCap Q-650M, Toyopearl Q-600C AR,
`
`Toyopearl SuperQ-650M, Toyopearl SuperQ-6508, TSKgel SuperQ-5PW (30),
`
`TSKgel SuperQ-5PW (20), or TSKgel SuperQ-S5PW may be used, but the present
`
`invention is not limited thereto, and an anion exchange resin known in the art may be
`
`used.
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`In the present invention, as a primary anion exchangeresin, Fractogel EMD
`
`TMAE(M)is used, but the present invention is not limited thereto.
`
`A suitable volume of a resin used in the anion exchange chromatography step
`
`is reflected by column dimensions, that is, the diameter of the column andthe height
`
`of the resin, and for example, varies according to an amountofthe target protein in the
`
`applied solution and binding performance of the used resin.
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`Before the anion
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`exchange chromatography, the anion exchangeresin is preferably equilibrated with a
`
`buffer to allow the resin to bind with a counter ion thereof.
`
`According to an embodimentof the present invention, in the anion exchange
`
`chromatography in step ($1), the sample may be loaded in the column at pH 8 to 9 and
`
`a conductivity of 4 mS/cm or less. More preferably, the sample may be loadedin the
`
`column at pH 8 and a conductivity of 2 mS/cm orless.
`
`In the present invention, the anion exchange chromatography in step (S1) is
`
`20
`performed using a buffer having pH8to 9.
`
`According to an embodimentof the present invention, the column for the anion
`
`exchange chromatography in step (S1) of the present invention may be equilibrated
`
`with 10 to 30 mM and preferably, 20 mM Tris-HCl to be pH 7 to 9, and preferably,
`
`pH 8.
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`In addition, according to an embodiment of the present invention, the ion
`
`exchange resin of the anion exchange chromatography in step (S1) of the present
`
`invention is washed with a buffer having pH 7 to 9, and preferably, pH 8, which
`
`contains 10 to 30 mM andpreferably, 20 mM Tris-HCl.
`
`According to an embodiment of the present invention, a fraction attached to
`
`the column in the anion exchange chromatography in step (S1) of the present invention
`
`is eluted with a buffer of pH 7 to 9, and preferably, pH 8, which contains 10 to 30 mM
`
`and preferably, 20 mM Tris-HCl and 200 to 400 mM andpreferably, 300 mM NaCl,
`
`at a flow rate of 100 to 300 cm/hr, and preferably, 200 cm/hr.
`
`According to an embodiment of the present invention, in the anion exchange
`
`chromatography of step (S1), a DBCis 5 to 9, and preferably, 7.5 mg GST-TB4/mL of
`
`resin.
`
`The method includes (S2) performing affinity chromatography on an eluate.
`
`The term “affinity chromatography” used herein refers to a protein separation
`
`technique in which a target protein specifically binds to a ligand specific therefor.
`
`The ligand is generally called a bio-specific ligand, and specifically interacts with the
`
`target protein (e.g., enzyme-substrate, enzyme-inhibitor, or antigen-antibody).
`
`In
`
`some embodiments, bio-specific ligands are attached to a chromatography resin by
`
`20
`
`covalent bonds, and as a solution contacts the chromatographyresin, the ligands have
`
`access to the target protein in the solution. The target protein generally possesses
`
`specific binding affinity to the bio-specific ligand during chromatography, but other
`
`solutes and/or proteins in the mixture do not clearly or specifically bind to the ligand.
`
`Binding ofthe target protein to an immobilized ligand allows a contaminantprotein or
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`Attorney Docket No. 206132-0113-00US
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`Substitute Specification — Marked Up
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`protein impurities to pass through the chromatography resin, but holds the target
`
`protein