BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a method for removing water from a compound solution and performing conjugate acid conversion, in particular using a nanometer film for reverse osmosis removal of water and performing conjugate acid substitution of the compound, and the compound is particularly a peptide compound.

2. Description of Related Art

Peptides and proteins are compounds formed by the condensation reaction of different amino acids. There are many types of peptides and proteins and they play a very important role in the organism. In addition to fermentation, the methods for preparing peptides and proteins include solid phase chemical synthesis and liquid phase chemical synthesis. In solid phase chemical synthesis and liquid phase chemical synthesis, acidic substances are usually used to remove protective groups or solid supports. The most commonly used acidic substance is trifluoroacetic acid, so trifluoroacetic acid is the most common residual substance.

In order to modify and optimize the properties of a compound, the compound is usually reacted with a conjugate acid (i.e., a counter ion) to form a compound salt so as to change its solubility, dissolution rate, stability, and crystal habits. Trifluoroacetic acid is not a common conjugate acid that can be used to form compound salts. Therefore, it is necessary to reduce the trifluoroacetic acid content of synthesized peptides or proteins and replace trifluoroacetic acid with a conjugate acid with dosage stability or high bioavailability. At present, there are two methods to remove trifluoroacetic acid from peptides and proteins. First, column chromatography allows peptides or proteins to stay on the stationary phase material in the column, and an acidic solution other than trifluoroacetic acid (i.e., a compound conjugate acid) is used as the eluent for replacement. Second, to let the peptide or protein solution flow through an ion exchange resin, and the cations contained on the surface of the resin can retain trifluoroacetic acid. In operation, the ion exchange resin is washed with strong base and then washed with the required conjugate acid, and then the peptide or protein is allowed to flow into the resin, and the peptide or protein solution containing less trifluoroacetic acid is collected after elution. However, unlike chromatographic separation, ion exchange resin cannot provide an effective chromatographic purification process and can only be used for replacement by the conjugate acid on the resin.

BRIEF SUMMARY OF THE INVENTION

If the water in the synthesized peptide or protein aqueous solution can be effectively reduced and the organic solvent and trifluoroacetic acid can be removed, the downstream purification process can be more flexible and improve efficiency. The reason is that chromatographic purification is carried out in the downstream purification process, which usually produces more aqueous solutions, and peptides or proteins are easily soluble in high water phase. It usually takes a long time to remove water and organic solvents with a vacuum concentrator. If lyophilization is used, the organic solvent in the aqueous solution will affect the freezing efficiency and even damage the pump. Therefore, the chromatographic purification process of peptides or proteins has the problems of multiple steps, cumbersome process, long operation time and high production cost.

Therefore, the present invention intends to provide a peptide or protein solution that facilitates the downstream purification process more efficiently. The objective of the invention is to provide a method for removing water from a compound solution and performing conjugate acid conversion in the same environment, which can effectively remove water and trifluoroacetic acid from the peptide or protein aqueous solution.

That is, the method for removing water from a compound solution and performing conjugate acid conversion of the present invention includes the steps of: (a) removing water: providing a compound solution containing an acidic substance A, pressurizing the compound solution to penetrate at least one nanometer film for reverse osmosis, and then discharging the aqueous solution obtained by reverse osmosis; (b) providing conjugate acid: providing an acidic substance B aqueous solution different from the acidic substance A to the nanometer film; (c) acid conversion and water removal: pressurizing for reverse osmosis, and discharging the aqueous solution obtained by reverse osmosis, acidic substance A and excess acidic substance B; (d) cleaning: adding water, pressurizing for reverse osmosis, and then discharging the aqueous solution obtained by reverse osmosis and excess acidic substance B; and (e) collection: adding water to clean the compound concentrate.

In a preferred embodiment, the acidic substance A is trifluoroacetic acid.

In a preferred embodiment, the acidic substance B is hydrochloric acid, hydrofluoric acid, sulfuric acid, acetic acid, benzoic acid, benzenesulfonic acid, bromic acid, hypobromous acid, camphorsulfonic acid, chloride, citric acid, methanesulfonic acid, fumaric acid, glucoheptonic acid, sodium glucoheptonate, hippuric acid, iodide, isethionic acid, lactic acid, lactobionic acid, sodium laureth sulfate, malic acid, maleic acid, mesylate, methanesulfonic acid, naphthoic acid, naphthalenesulfonic acid, nitric acid, stearic acid, oleic acid, sodium oleate, oxalic acid, palmitic acid, phosphoric acid, polygalacturonic acid, succinic acid, sulfuric acid, sulfosalicylic acid, tartaric acid, or p-toluenesulfonyl.

In a preferred embodiment, any one or more of the steps (a) to (d) are repeated at least twice.

In a preferred embodiment, the compound is a protein or peptide.

In a preferred embodiment, the peptide comprises etetcalcetide, teriparatide, or abaloparatide.

In a preferred embodiment, the nanometer film includes a nanofiltration membrane or a reverse osmosis membrane.

In a preferred embodiment, the material of the nanometer film includes cellulose acetate fiber, polysulfone, polyvinylidene fluoride, polyethersulfone resin, or a composite film of a combination thereof.

In a preferred embodiment, the step (e) is further followed by a step (f) lyophilization: freeze drying the compound concentrate containing the acidic substance B to obtain a salt of the compound.

In a preferred embodiment, a cleaning step is further performed between the step (a) and the step (b): washing the nanometer film with water and then discharging the washed aqueous solution.

Compared with the conventional technology, the present invention provides a method for removing water from a compound solution and performing conjugate acid conversion in the same environment, which can effectively remove water and acidic substances (especially trifluoroacetic acid) from the peptide or protein aqueous solution. The method can facilitate the purification of downstream peptides or proteins and improve the efficiency of the downstream purification process.

