RU2350945C2 - Regeneration of chromatographic stationary phase - Google Patents

Abstract

FIELD: physics, measurement.

SUBSTANCE: present invention is related to the field of chromatographic purification. Method is suggested for regeneration of chromatographic stationary phase, in which mentioned chromatographic stationary phase contacts with regeneration solution that contains at least one organic acid and less than 75 wt % of water, where specified organic acid is formic acid. The following are also suggested: Method for production of therapeutical polypeptide, in which at least one chromatographic stage is performed, in which chromatographic stationary phase is regenerated by means of above described regeneration method; Application of method for regeneration of chromatographic stationary phase according to invention in production of therapeutical polypeptide and Chromatographic stationary phase, which is regenerated by means of suggested regeneration method.

EFFECT: provision of efficient method of chromatographic stationary phases regeneration to increase life time of expensive initial materials and to prevent growth of pressure difference at chromatographic fibres, regeneration method, which is suitable for in-situ regeneration of chromatographic stationary phases in production installations.

29 cl, 12 tbl, 4 dwg

Description

FIELD OF THE INVENTION

The present invention relates to the field of chromatographic purification. More specifically, it relates to a method for regenerating a chromatographic stationary phase.

State of the art

Polypeptides are increasingly used as medicines for the treatment of diseases in all major areas of therapy. Diabetes has been treated for over 80 years with continuous insulin administration, and the therapeutic use of polypeptides for growth disorders and cancer has also been ongoing for many years. Cost-effective methods for large-scale production of polypeptides with a purity high enough for therapeutic applications are critical for the further development of polypeptide-based therapies in order to enter the mass market and to make existing therapy more widely used.

Purification of the polypeptide (isolation from the mixture) is a series of steps that are typically used several times during the general process for the production of therapeutic polypeptides. Reverse phase high performance liquid chromatography (RP-HPLC) is the preferred method for the industrial isolation of high resolution polypeptides, and the method, as shown, is suitable for large-scale purification of many polypeptides.

Since polypeptides for therapeutic use must be purified with a high degree of purity so as not to cause harmful effects when administered to a patient, it is completely normal to use several chromatographic purification steps in a manufacturing method. The stationary phases of chromatographic columns in production plants are expensive, and they are thus used for several chromatographic cycles. However, over time, the efficiency of the chromatographic stationary phase decreases, that is, the pressure drop across the column gradually increases and the separation coefficient decreases. This is attributed to the gradual deposition of sediment. The problem is proposed to be overcome by using a regeneration method that includes alkaline buffers (J. Chrom. 461, 1989, 45-61), for example at pH 7.4, and a high concentration of organic modifier.

Brange et al. (J. Pharm. Sci. 86 (1997) 517-525) describes the dissolution of insulin fibrils in acid and base.

For many years, the problem was alleviated by regenerating the chromatographic stationary phase with an alkaline solution, for example, 0.1 molar sodium hydroxide (see Liliedahl, "Twelve years of silica-based HPLC purification with focus on peptides", at Tides 2000, 10 May 2000 in Las Vegas, USA). This regeneration method can increase the life time of the silica used to purify insulin to between 100 and 600 cycles. However, silica materials are not stable when subjected to harsh alkaline conditions, and in particular, materials based on substituted silica may not be suitable for regeneration using alkaline solutions. Economically viable methods for purifying pharmaceutical preparations, such as therapeutic polypeptides, should include a regeneration method that does not degrade the chromatographic stationary phase.

A general and complex method for the recovery of materials containing particles (clay, sand, silica and the like) from a variety of sources is described in International Patent Application WO 00/61493. It is a 5-step process, which involves contacting the material with a) an extractant of organic material, b) an oxidizing agent, and then c) with an acid solution, d) heating the material, and e) recovering the material. The method is time-consuming and not suitable for implementation in the installation for chromatographic purification.

There is a need in the art for more efficient methods for regenerating stationary chromatographic phases in order to increase the lifetime of these expensive starting materials and to prevent the increase in pressure drop across the chromatographic columns. Especially required are regeneration methods that are suitable for the regeneration of in situ chromatographic stationary phases in production plants.

SUMMARY OF THE INVENTION

The present invention provides a method for regenerating a chromatographic stationary phase, wherein said stationary chromatographic phase is contacted with a regeneration solution containing at least one organic acid and less than about 75% w / w. water.

In another aspect, the present invention provides a method for regenerating a chromatographic stationary phase, wherein said stationary chromatographic phase is contacted with a regeneration solution containing at least one organic acid and less than about 1% w / w. water.

In one embodiment of the present invention, the organic acid is formic acid. In another embodiment of the present invention, the organic acid is acetic acid. In yet another embodiment, the regeneration solution contains less than 0.5% water, preferably less than 0.1% water, more preferably less than 0.02% water and most preferably less than 0.001% water.

In another aspect, the present invention relates to a method for regenerating a chromatographic stationary phase, wherein said stationary chromatographic phase is contacted with a regeneration solution containing at least one organic acid, an organic solvent and less than about 1% w / w. water.

In one embodiment of the present invention, the organic solvent is ethanol. In another embodiment of the present invention, the organic solvent is 2-propanol. In another embodiment of the present invention, the organic solvent is acetonitrile. In another embodiment of the present invention, the organic solvent is selected from the group consisting of methanol, 1-propanol and hexylene glycol.

In another embodiment, the regeneration solution contains less than 0.5% water, preferably less than 0.1% water, more preferably less than 0.02% water, and most preferably less than 0.001% water.

In another aspect, the present invention relates to a chromatographic stationary phase, which is regenerated using the methods of the present invention.

In another aspect, the present invention relates to a polypeptide product obtained using the methods of the present invention.

In another aspect, the present invention relates to a polypeptide product produced by a method comprising the regeneration of a stationary chromatographic phase using these methods.

In yet another aspect, the present invention relates to automated chromatographic equipment comprising pipelines and a monitoring system for implementing a regeneration method.

In another aspect, the present invention relates to a pharmaceutical composition obtained by purification of a polypeptide using a stationary chromatographic phase that is regenerated by this method.

Brief Description of the Drawings

Further, the present invention is illustrated by the accompanying drawings, in which:

Figure 1 shows the principle of a confocal device.

Figure 2 depicts a two-dimensional picture of Source 30Q with insulin fibrils stained with Thioflavin T.

Figure 3 shows the principle of measuring the area of fibrils on the Source 30Q. The lighter portions show green light emanating from insulin fibrils on Source 30Q particles.

Figures 4A-B show preparative chromatograms of chromatographic purification III (Fig. 4A, the upper figure, before regeneration with formic acid, and Fig. 4B (lower figure) after regeneration with formic acid).

Definitions

The following is a detailed definition of the terms used in the description.

The term "chromatographic stationary phase", as used here, means the solid phase over which the soluble phase, that is, the chromatographic matrix. A stationary chromatographic phase is usually placed inside a chromatographic column. Examples of stationary chromatographic phases are substituted silica such as C-4 silica, C-12 silica and C-18 silica, as well as polymeric materials such as polystyrene, Source 30Q and Sepharose. Further examples of stationary chromatographic phases are membranes, monolithic materials and filters.

The term “chromatographic eluent,” as used herein, means a solution that is used for the elution step, where the purified polypeptide is typically released from the chromatographic stationary phase to the eluent. In normal chromatography mode, the full cycle includes:

a) balancing using a balancing buffer to bring the column to the state where it is ready for the loop,

b) applying a sample containing the product,

c) an optional washing step, wherein the chromatographic stationary phase with the associated product is washed,

d) elution, where the affinity of the product relative to the chromatographic stationary phase decreases and the product leaves the column in the eluate of the chromatographic column,

e) optional regeneration, where they try to remove the remaining impurities from the chromatographic stationary phase using a regeneration solution.

The term "balancing buffer" as used here, means a solution that is used for the stage of equilibration, in which the chromatographic column is prepared for the chromatographic cycle.

The term “regeneration solution,” as used herein, means a solution that is used to regenerate a chromatographic stationary phase. The purpose of regeneration is to maintain satisfactory chromatographic separation performance over several chromatographic cycles. Typically, critical parameters related to efficiency are the pressure drop across the chromatographic column and the separation coefficient. The regeneration step may include contacting the chromatographic stationary phase with either a single regeneration solution or more than one regeneration solution. In the latter case, each of the regeneration solutions, as well as the mixtures obtained from them, are covered by the term “regeneration solution”.

The term "mixture", as used here, means a composition of a material containing at least two ingredients. The chromatographic column eluate is a mixture that contains chemicals in the eluent, along with the product that is removed from the column. Another example of a mixture is a solution of a chemical reagent in a solvent, for example, saline. Another example of a mixture is water and a water-miscible organic solvent. Another example of a mixture is a solution or suspension of a polypeptide in a solvent, such as water or an organic solvent.

The term “isolation of a polypeptide”, as used herein, means bringing the polypeptide to a state where it has a higher concentration or higher purity than it had before isolation, that is, in the starting material. Thus, an example of the isolation of a polypeptide is the precipitation or crystallization of a polypeptide from a solution and the separation of the precipitate or crystals from the mother liquor.

The term "organic solvent", as used here, means a solvent that contains at least one carbon atom and which is in a liquid state in the temperature range from 0 ° C to 50 ° C. Non-limiting examples of organic solvents are lower alcohols such as methanol and ethanol, polyols, acetonitrile, hexane and acetone.

The term “water miscible organic solvent”, as used herein, means an organic solvent that has a solubility in water at 20 ° C. of at least 1 g / L. Non-limiting examples of water-miscible organic solvents are ethanol, 1-propanol, 2-propanol, acetonitrile and hexylene glycol.

The term "organic acid", as used here, means an organic compound that has at least one functional group with a dissociation constant, pK _a less than 5.0. Examples of organic acids are formic acid, acetic acid, citric acid and the like.

The term "lower alcohol", as used here, means a C _1-6 alcohol, which is characterized in that it has 1-6 carbon atoms and one hydroxyl residue. The carbon skeleton in lower alcohol may be straight or branched. Non-limiting examples of lower alcohols are ethanol, n-propanol, isopropanol and tert-butanol.

The term "polyhydric alcohol" as used here means an alcohol having at least two hydroxyl residues. Non-limiting examples of polyols are hexylene glycol (4-methyl-2,4-pentanediol) and neopentyl alcohol (2,2-dimethyl-1,3-propanediol).

The term “excipient,” as used herein, means compounds that are added to pharmaceutical compositions to stabilize and preserve the composition. Typical excipients are buffers, preservatives, and tonicity modifiers.

The term "pharmaceutical composition", as used here, means a product containing the active compound or its salt, together with pharmaceutical excipients, such as a buffer, preservative and tonicity modifier, where the specified pharmaceutical composition is suitable for the treatment of a disease or disorder. Thus, a pharmaceutical composition is also known in the art as a pharmaceutical preparation.

The term “buffer,” as used herein, refers to a chemical compound that is used in a solution to lower the tendency of the pH of the solution to change over time, as would otherwise be the case with chemical reactions. Buffers include reagents such as sodium phosphate, TRIS, glycine and sodium citrate.

The term “tonicity modifier”, as used herein, refers to a chemical compound in a pharmaceutical composition that serves to modify the osmotic pressure of the pharmaceutical composition so that the osmotic pressure becomes closer to the pressure of human plasma. Tonicity modifiers include NaCl, glycerin, D-mannitol and the like.

The term "pharmaceutically acceptable", as used here, means suitable for conventional pharmaceutical applications, that is, not adversely affecting patients, and the like.

The term "human insulin" as used here, means a human hormone, the structure and properties of which are well known. Human insulin has two polypeptide chains that are connected by disulfide bridges between cysteine residues, namely, the A chain and the B chain. The A chain is a peptide of 21 amino acids, and the B chain is a peptide of 30 amino acids, two chains are connected via three disulfide bridges: one between cysteines at position 6 and 11 of the A chain, the second between cysteine at position 7 of the A chain and cysteine at position 7 of the B chain, and a third between cysteine at position 20 of the A chain and cysteine at position 19 of the B chain.

The term "polypeptide", as used here, means a compound consisting of at least ten constituent amino acids linked via peptide bonds. The constituent amino acids can be a group of amino acids encoded by the genetic code, and they can be natural amino acids that are not encoded by the genetic code, as well as synthetic amino acids. Natural amino acids that are not encoded by the genetic code are, for example, hydroxyproline, γ-carboxyglutamate, ornithine, phosphoserine, D-alanine and D-glutamine. Synthetic amino acids include amino acids obtained by chemical synthesis, i.e. D-isomers of amino acids encoded by the genetic code, such as D-alanine and D-leucine, Aib (α-aminoisobutyric acid), Abu (α-aminobutyric acid), Tle ( tert-butyl glycine) and β-alanine.

The term “therapeutic polypeptide,” as used herein, means a polypeptide that has potential utility as a therapeutic agent. Therapeutic polypeptides are generally of a high degree of purity and are undergoing clinical trials as part of the legislative approval process. Examples of therapeutic polypeptides include human insulin, thrombopoietin, erythropoietin, and human growth hormone.

The term “product polypeptide,” as used herein, means a composition comprising a polypeptide. Examples of polypeptide products are crystallized polypeptide, precipitated polypeptide, and polypeptide solution.

The term “analogue,” as used herein, with respect to a parent polypeptide, means a modified polypeptide, where one or more amino acid residues of the parent polypeptide are replaced by other amino acid residues and / or where one or more amino acid residues are removed from the parent polypeptide, and / or where one or several amino acid residues are removed from the parent polypeptide, and / or where one or more amino acid residues are added to the parent polypeptide. Such attachment or removal of amino acid residues may occur at the N-terminus of the polypeptide, or at the C-terminus of the polypeptide, or within the polypeptide. An example of an analogue is Arg ³⁴ -GLP-1 (7-37), which is the GLP-1 polypeptide (7-37), where Lys at position 34 is replaced by Arg. Other examples are pork or bovine insulin, both of which are analogues of human insulin.

The term "precursor", as used here, in relation to a polypeptide means a modified version of the polypeptide that is produced. Polypeptide precursors are typically extended amino acid versions of a polypeptide or shortened versions of a polypeptide.

These precursors can serve to enhance cell expression, contain affinity tags for purification, protect certain reactive groups of the produced polypeptide, and the like.

The term “derivative,” as used herein, with respect to a parent polypeptide, means a chemically modified parent polypeptide or an analog thereof, where at least one substituent is not present in the parent polypeptide or its analog, that is, the parent polypeptide that is covalently modified . Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, PEGylation products and the like. Examples of human insulin derivatives are human insulin threonine methyl ester ^B30 and human N ^εB29- tetradecanoyl-des (B30) human insulin.

The term “lipophilic substituent,” as used herein, means a substituent containing 4-40 carbon atoms and having a solubility in water at 20 ° C. ranging from about 0.1 mg / 100 ml of water to about 250 mg / 100 ml of water, for example, in the range of about 0.3 mg / 100 ml of water to about 75 mg / 100 ml of water. For example, octanoic acid (C8) has a solubility in water at 20 ° C of 68 mg / 100 ml, decanoic acid (C10) has a solubility in water at 20 ° C of 15 mg / 100 ml, and octadecanoic acid (C18) has a solubility in water at 20 ° C 0.3 mg / 100 ml.

The term "piping and control system", as used here, means physical means (pipes and control valves) and software that controls the pipes and valves of the equipment used in the method.

Description of the invention

The present invention relates to a method for regenerating a chromatographic stationary phase, wherein said stationary chromatographic phase is contacted with a regeneration solution containing at least one organic acid and less than about 75% w / w. water. The present invention also relates to a method for regenerating a chromatographic stationary phase, wherein said stationary chromatographic phase is contacted with a regeneration solution containing at least one organic acid and less than about 1% w / w. water. The present invention also relates to a method for regenerating a chromatographic stationary phase, wherein said stationary chromatographic phase is contacted with a regeneration solution having an organic acid concentration of at least 25% w / w, a number of organic compounds can be used in the regeneration solution of the method acids. A preferred organic acid is formic acid. Another organic acid for the regeneration solution is acetic acid. Another regeneration solution contains two organic acids, for example formic acid and acetic acid.

The regeneration solution may also contain an organic solvent. Preferably, the organic solvent is also used in equilibration buffer or chromatographic eluent. In one embodiment of the present invention, the organic solvent is ethanol. In another embodiment, the organic solvent is 2-propanol. In another embodiment, the organic solvent is acetonitrile. In another embodiment, the organic solvent is selected from the group consisting of methanol, 1-propanol and hexylene glycol.

In another embodiment, the organic acid is formic acid, and the organic solvent is ethanol. In another embodiment, the organic acid is formic acid, and the organic solvent is acetonitrile. In yet another embodiment, the organic acid is formic acid, and the organic solvent is 2-propanol. In yet another embodiment, the organic acid is formic acid, and the organic solvent is hexylene glycol. In another embodiment, the organic acid is acetic acid, and the organic solvent is ethanol. In another embodiment, the organic acid is acetic acid and the organic solvent is acetonitrile. In yet another embodiment, the organic acid is acetic acid, and the organic solvent is 2-propanol. In yet another embodiment, the organic acid is acetic acid, and the organic solvent is hexylene glycol.

In another embodiment, the present invention relates to a method for regenerating a chromatographic stationary phase, wherein said stationary chromatographic phase is contacted with a regeneration solution containing at least one organic acid and less than 0.5% water, preferably less than 0.1% water , more preferably less than 0.02% water and most preferably less than 0.001% water.

The stationary chromatographic phase is preferably contacted with a regeneration solution in a chromatographic column. Thus, the minimum production capacity is lost due to downtime in connection with the regeneration stage. Thus, a method for regenerating a chromatographic stationary phase can be carried out without re-packing the column. In one embodiment, the stationary chromatographic phase is liquefied during said regeneration. In another embodiment, the chromatographic eluent or equilibration buffer is displaced with a water-miscible organic solvent before said stationary chromatographic phase is brought into contact with said regeneration solution. Preferably, said water-miscible organic solvent is also present in the chromatographic eluent or in equilibration buffer. Preferably, said water miscible organic solvent is also present in the regeneration solution.

In another embodiment, the stationary chromatographic phase is contacted with said regeneration solution outside the chromatographic column. This procedure is more time-consuming than the implementation of the regeneration method inside the column, but, nevertheless, it can be useful if the deposited material is captured between the particles of the chromatographic stationary phase. In the latter case, the precipitated material can be removed from the chromatographic stationary phase without dissolving the specified material.

In one embodiment, the stationary chromatographic phase is an RP-HPLC matrix. Chromatographic stationary phase for RP-HPLC is mechanically very hard materials, which can be silica gel or substituted silicon dioxide, such as C4, C6, C8, C10, C12, C16, C18, C30 or phenyl substituted silicon dioxide, or it can be high pressure stable polymer material that is substituted or unsubstituted. The stationary chromatographic phase, whether it is a matrix based on silicon dioxide or a polymeric material, can also be present in columns in the form of monolithic rods with macropores and mesopores. A silica material suitable for use as a stationary chromatographic phase is spherical particles with a narrow pore size distribution and particle sizes ranging from 3 μm to 100 μm, for example from 5 μm to 100 μm, for example from 8 μm to 30 μm, for example 10 μm, 13 μm, 15 μm, 16 μm, 18 μm and 20 μm. Typically, pore sizes ranging from 60 Å to 300 Å are used, for example, 100 Å, 120 Å, 150 Å, 175 Å, 200 Å or 300 Å. For polymer materials that are stable under high pressure, the pore size can be from 10 Å or even higher, for example, 50 Å, 100 Å, 400 Å, 600 Å, 1000 Å or 3000 Å. In one embodiment, the high-pressure stable polymer material is Source 30Q or XAD 1180. The chromatographic column is packed with a stationary phase, and, after an appropriate test of the quality of the packing, the column is balanced using a buffer used in binding mode. Industrial chromatographic columns typically have diameters of 15 to 100 cm, and such systems can have dynamic axial compression. For the production of a small volume of polypeptides, production columns may have a diameter of, for example, 15 cm, 20 cm or 25 cm. For the production of a large volume of polypeptides, production columns may have a diameter of, for example, 40 cm, 60 cm, 80 cm or more.

In another embodiment of the present invention, the chromatographic stationary phase that is regenerated is a membrane, monolithic materials, filters, or the like.

In one embodiment of a method for regenerating a chromatographic stationary phase, said chromatographic stationary phase is contacted with said regeneration solution for at least 1 second, preferably for at least 1 minute, more preferably for at least 5 minutes, for example, from 1 minute to 24 hours, from 1 minute to 5 hours, from 1 minute to 2 hours, from 10 minutes to 60 minutes.

In another embodiment of a method for regenerating a chromatographic stationary phase, said chromatographic stationary phase is contacted with said regeneration solution until the pressure drop along the length of the chromatographic column at normal flow rate decreases by at least 10%, preferably at least 25%, even more preferably at least 50%.

In another embodiment of a method for regenerating a chromatographic stationary phase, contacting said chromatographic stationary phase with a regeneration solution is carried out at a temperature ranging from about 0 ° C to 70 ° C, from 5 ° C to 50 ° C, for example, from 10 ° C to 40 ° C, for example, from 15 ° C to 30 ° C or from 18 ° C to 25 ° C. In another embodiment of a method for regenerating a chromatographic stationary phase, the lifetime of said chromatographic stationary phase is at least 500 chromatographic cycles, preferably at least 700 chromatographic cycles, more preferably at least 1000 chromatographic cycles, most preferably at least 2000 chromatographic cycles.

In another embodiment of a method for regenerating a chromatographic stationary phase, the method is applied to the specified chromatographic stationary phase during each chromatographic cycle, at least once every 2 chromatographic cycles, at least once every 5 chromatographic cycles, at least once every 20 chromatographic cycles, at least once every 50 chromatographic cycles, or at least once every 100 chromatographic cycles.

In another embodiment of the present invention, the number of regeneration processes carried out on a chromatographic stationary phase is at least 25, at least 50, at least 100, at least 200, at least 400, or, at at least 1000.

In another embodiment of a method for regenerating a chromatographic stationary phase, the method is applied to the specified chromatographic stationary phase every time when the pressure drop along the length of the chromatographic column exceeds a threshold value.

Another aspect of the present invention is a method for manufacturing a therapeutic polypeptide or a precursor thereof, said method comprising at least one chromatographic step in which the chromatographic stationary phase is regenerated by a regeneration method as described above. In one embodiment of a method of manufacturing a therapeutic polypeptide or a precursor thereof, said therapeutic polypeptide is a derivative containing a lipophilic substituent. In another embodiment of a method of manufacturing a therapeutic polypeptide or a precursor thereof, said therapeutic polypeptide is a derivative containing a lipophilic substituent attached to the amino group of the lysine residue. In another embodiment of a method of manufacturing a therapeutic polypeptide or its precursor, said therapeutic polypeptide is selected from the group consisting of glucagon, glucagon-like peptide 1, glucagon-like peptide 2, exendin-4, TFF peptides, human insulin, its analogues and its derivatives. In another embodiment, said polypeptide is selected from the group consisting of Lys ²⁶ (N ^ε - (γ-Glu (N ^α- hexadecanoyl))) - GLP-1 (7-37), Arg ³⁴ -GLP-1 (7-37 ), exendin-4, Lys ¹⁷ Arg ^30- GLP-2 (1-33), Arg ³⁰ Lys ¹⁷ N ^ε (β-Ala (N ^α- hexadecanoyl)) GLP-2 (1-33) and Gly ^2- GLP -2 (1-33). In another embodiment, said polypeptide is exendin-4. In another embodiment, said polypeptide is a fusion polypeptide comprising human serum albumin or a fragment thereof. In another embodiment, said polypeptide is a GLP-1 fusion polypeptide (7-37) or an analogue thereof and a human serum albumin fragment or analogue thereof. In another embodiment, said polypeptide is an exendin-4 (1-39) fusion polypeptide or an analogue thereof and a human serum albumin fragment or analogue thereof. In another embodiment, said polypeptide is a fusion polypeptide comprising an immunoglobulin Fc portion or fragment thereof. In another embodiment, said polypeptide is a GLP-1 fusion polypeptide (7-37) or an analogue thereof and an Fc fragment of an immunoglobulin or an analogue thereof. In another embodiment, said polypeptide is an exendin-4 (1-39) fusion polypeptide or an analog thereof and an Fc fragment of an immunoglobulin or an analog thereof.

In another embodiment, said polypeptide is selected from the group consisting of human insulin, human insulin precursor, human insulin analogue, human insulin analogue precursor, GLP-1 analogue (7-37), exendin-4 analogue (1-39) and their derivatives . In another embodiment, said polypeptide is selected from a human insulin derivative containing at least one methoxy or ethoxy group. In another embodiment, said polypeptide is selected from the group consisting of

human insulin threonine methyl ester ^B30 ,

human insulin threonine ethyl ester ^B30 ,

Asp ^B28 human insulin,

human insulin threonine methyl ester ^B30 Asp ^B28 ,

human insulin threonine ethyl ester ^B30 Asp ^B28 ,

Lys ^B28 Pro ^B29 Human Insulin,

Met ^B-1 Arg ^B0 Lys ^B28 Pro ^B29 human proinsulin,

Lys ^B3 Glu ^B29 Human Insulin,

Gly ^A21 Arg ^B31 Arg ^B32 human insulin,

des (B30) human insulin,

N ^εB29- tetradecanoyl des (B30) -insulin human

N ^εB29 -lithocholoyl-γ-glutamyl-des (B30) -insulin of a person,

N ^εB29 -Octanoyl-des- (B30) -Human insulin and

N ^εB29 -Octanoyl-human insulin.

In yet another embodiment, said polypeptide is selected from human serum albumin, erythropoietin, TNF-α, interleukin, IGF-1 (IGF-insulin-like growth factor), IGF-2, human growth hormone, somatostatin, human amylin and its analogues.

Polypeptides purified on stationary chromatographic phases, regenerated by the method of the present invention, can be obtained using various technologies known in the field of polypeptide production. Polypeptides larger than 3000 Daltons are usually produced by fermentation or cell culture, while smaller polypeptides can be produced by chemical synthesis of peptides. Other important factors determining the optimal production method are also the amount of polypeptide to be produced and the structure of the polypeptide, for example disulfide bonds and other modifications. Polypeptides obtained by fermentation or cell culture are usually produced by culturing recombinant host cells, for example bacteria, fungi, mammalian cells, insect cells or plant cells, in appropriate culture media. The culture medium may be a more or less chemically defined medium containing the necessary nutrients for the growth and formation of the product of the host cells, for example, sugar, a source of nitrogen, salts, vitamins and other growth factors. After the cultivation of microorganisms or cells in the medium and their optional destruction, the culture medium contains the desired product in a mixture with other components of the medium, impurities obtained from host cells, and impurities associated with the product. The impurities obtained from the host cells are mainly polypeptides, nucleic acids and cell debris. The product is separated from these unnecessary impurities at the stages of extraction or pre-treatment. At the stages of final purification (completion), where impurities that are closely associated with the product polypeptide are separated from the product polypeptide, chromatographic stages are widely used.

The synthesis of polypeptides can also be carried out using solid-phase synthesis by a chemical mechanism such as Merrifield, using methods using the phase of the solution, or using semisynthetic methods known in this field. One or more stages of chemical conversion can be carried out between the stages of extraction and final purification. Such chemical modifications may be the hydrolysis of a polypeptide precursor in which the extension segment of amino acids on the polypeptide is cleaved from the polypeptide. Such lengthening segments of amino acids can be used to increase expression of host cells in the case of polypeptides derived from culture, or they can be used to specifically purify the polypeptide, for example, by affinity chromatography, for example, IMAC purification of histidine-labeled polypeptides. Chemical conversion can also be a chemical modification to obtain a polypeptide, which is a derivative, for example, by acylation, PEGylation or esterification. Such chemical modifications are well known in the art (see, for example, International Patent Applications WO 98/08871, WO 99/43706, US Patent No. 5,424,286, International Patent Applications WO 00/09666, WO 00/66629, WO 01 / 04156 and WO 02/90388).

Another aspect of the present invention is the use of the above methods for the regeneration of a chromatographic stationary phase in order to reduce the pressure drop along the length of the chromatographic column.

Another aspect of the present invention is the use of the above methods of regenerating a chromatographic stationary phase to produce a therapeutic polypeptide.

Another aspect of the present invention is a chromatographic stationary phase, which is regenerated by contacting said chromatographic stationary phase with a regeneration solution, wherein said regeneration solution contains at least one organic acid and less than about 75% w / w. water.

Another aspect of the present invention is a chromatographic stationary phase that is regenerated by contacting said chromatographic stationary phase with a regeneration solution, said regeneration solution containing at least one organic acid and less than about 1% w / w. water.

In one embodiment, the implementation of the chromatographic stationary phase is regenerated using the method as described above. In another embodiment, the stationary chromatographic phase is regenerated using a method in which said regeneration solution contains less than 0.5% water, preferably less than 0.1% water, more preferably less than 0.02% water and most preferably less than 0.001 % water. In another embodiment, the regenerated chromatographic stationary phase is silica or substituted silica material.

In another aspect, the present invention relates to a polypeptide product produced using a method comprising the steps of

a) purification of the polypeptide or its precursor using a chromatographic stationary phase obtained by the regeneration method of the present invention, and

b) isolating said polypeptide or precursor thereof to obtain a polypeptide product.

In another aspect, the present invention relates to a polypeptide product produced using a method that uses a chromatographic stationary phase regenerated in accordance with the method of the present invention.

In another aspect, the present invention relates to automatic chromatographic equipment comprising pipelines and a monitoring system for implementing the regeneration method in accordance with the present invention.

In another aspect, the present invention relates to a pharmaceutical composition obtained using a method comprising the steps of

a) first purification of the polypeptide or its precursor using a chromatographic stationary phase, regenerated by the method in accordance with the present invention,

b) then drying the specified polypeptide and

c) finally, mixing with a pharmaceutically acceptable excipient.

In another aspect, the present invention relates to a pharmaceutical composition obtained using a method comprising the steps of

a) first purifying the polypeptide or its precursor using a chromatographic stationary phase regenerated by a method in which said chromatographic stationary phase is contacted with a regeneration solution containing at least one organic acid and less than 0.5% water, preferably less than 0 1% water, more preferably less than 0.02% water, and most preferably less than 0.001% water, and

b) then drying the specified polypeptide and,

c) finally, mixing with a pharmaceutically acceptable excipient.

EXAMPLES

The following abbreviations are used for commercially available chemicals and materials.

HCOOH: Formic Acid

EtOH: ethanol

AcOH: acetic acid

ODDMS: Octadecyldimethyl-substituted Silica Particles (Silicon Dioxide ODDMS)

Formic acid used for examples 1-68, 70-71, 73-74 and 76 has a specified purity of 98-100% (according to the manufacturer), and formic acid used for example 72 has a purity of 99.9%.

The abbreviation CV means column volumes, as is known in the field of chromatography.

Examples 1-68

The general set of experiments of examples 1-68 is as follows:

1. Determination of the back pressure and the height of the depression (the minimum point between the peaks on the curve) for a column where there are no problems with pressure (a new unused column).

2. Artificially creating problems with pressure and efficiency.

3. Determination of back pressure and the height of the depression between the peaks for the column where there are problems with pressure.

4. Column regeneration.

5. Determination of the back pressure and the height of the depression between the peaks for the column after regeneration.

Determination of back pressure and trough height between peaks.

The same type of silica gel is used for all of the experiments discussed in this section. Silica gel is 15 μm silica gel ODDMS 200Å. For the most part, the same gel load is used (load no. 205144) (all columns starting from 874-).

Silica gel is filled into steel columns 10 mm × 250 mm and examined in a functional test using des-B30-insulin as the test substance and eluting with water-ethanol mixtures containing calcium chloride and potassium chloride salts (see table 1). The back pressure during elution and the height of the cavity between the peaks of des-B30-insulin and the nearest impurity (before the peak of insulin) are used as parameters in the study of how the column regained its effectiveness.

The elution time of des-B30-insulin may undergo some experimental scatter due to small changes in temperature and other experimental parameters. When the elution time of des-B30-insulin is the same in different experiments, the height of the depression between the peaks is an excellent parameter for comparing the separation efficiency for the columns. When the elution time of des-B30-insulin changes from one experiment to another, the height of the depression between the peaks is a less good measure of column separation efficiency. All other things being equal, the longer the retention time, the smaller the height of the depression between the peaks.

Table 1
Solutions used in the study of functionality entrance Buffer Type of Content (w / w) A11 one Balancing buffer 20% ethanol A12 2 Elution Buffer A 25% ethanol, 1.5% KCl, 0.4% CaCl ₂ and 0.15% triethanolamine (pH 7.4, with HCl) B1 3 Elution Buffer B 35% ethanol, 1.5% KCl, 0.4% CaCl ₂ and 0.15% triethanolamine (pH 7.4, with HCl) A13 four Regeneration buffer 70% ethanol and 6.9% acetic acid A18 Application Des-B30-Insulin Na ₂ EDTA solution, pH 7.5, ethanol

The study of functionality is carried out at 23 ± 2 ° C on Äktaexplorer 100A with Unicorn 4.0 as the control software and the following column cycle is carried out (see table 2):

table 2
Functionality Column Cycle (CV - Column Volumes) Act CV (count) Volume (ml) Balancing 3 58.9 Loading 0.8 fifteen Flushing 1 one 19.6 Elution 10 196.3 Flushing 2 0.5 9.8 Rinse 2 39.3

A column packed with silica gel (loading 205144) is examined and a back pressure of 3.4 ± 0.1 MPa is measured. Similarly, backpressure is determined for other columns before artificially creating pressure problems. Experiments show that if these values are restored after processing, the column restores its effectiveness.

The height of the depression between the peaks is also determined for individual columns before and after the creation of problems with pressure and efficiency and after regeneration of the column.

Artificially creating problems with pressure and efficiency:

Problems with pressure and efficiency are created in 1 of 3 ways:

1. The column is used in studies of functionality, but is removed from the system after loading and incubated at 70 ° C for 1-16 hours. After that, the back pressure and the height of the depression between the peaks are measured, performing the remainder of the study of functionality.

2. Silica gel ODDMS (25 g) was mixed in a beaker with des-B30-insulin (0.14 g), 0.1M Tris buffer (22 ml) and ethanol (15 ml) at 50 ° C for 1 hour. After that, the gel is decanted and filled into a steel column (10 mm × 250 mm). The back pressure and the height of the trough between the peaks are measured by performing functional studies.

This method can also be carried out at lower temperatures, but it requires a longer reaction time.

3. The combination of 1 and 2. First, the gel is processed as in 2, but after filling the column is treated as in 1.

The methods used for individual columns are listed below in table 4.

Column regeneration (i.e. eliminating pressure and efficiency issues):

Column regeneration is also performed on the Äktaexplorer 100A. The column cycle for this method can be seen in Table 3 below. After regeneration, the column is again tested in a functional study and the back pressure is determined.

Table 3
Regeneration column cycle Act Solvent CV (count) Volume (ml) Flushing 1 EtOH 3 58.9 Regeneration See table 4 and 5 2 39.3 Leave standing - 0 30 minutes Regeneration See table 4 and 5 2 39.3 Flushing 2 EtOH 3 58.9

Various solvents were tested at 22 ° C and 40 ° C (the entire HPLC system was placed in a refrigerator at a predetermined temperature of ± 1 ° C). The specific conditions for individual columns are presented in table 4, and the results of the study of functionality are presented in table 5.

Specific experimental conditions for individual columns

Table 4
Experimental conditions for individual columns Example No. Column No. Artificially creating problems using Method No. Regeneration solvent Temperature (° C) one 204377/1 one HCOOH 22 2 874-32 / 1 2 HCOOH 22 3 874-32 / 2 3 HCOOH 22 four 874-32 / 7 2 HCOOH 22 5 874-33 / 9 2 HCOOH 22 6 874-33 / 6 3 HCOOH 22 7 874-24 / 22 2 HCOOH 40 8 874-24 / 10 3 AcOH 22 9 874-24 / 15 2 AcOH 22 10 874-24 / 23 2 AcOH 40 eleven 874-31 / 1 2 AcOH 40 12 203635/5 one HCOOH / AcOH 99: 1 22 13 874-24 / 6 3 HCOOH / AcOH 99: 1 22 fourteen 205019/1 one HCOOH / AcOH 99: 1 40 fifteen 204770/1 one HCOOH / AcOH 3: 1 22 16 874-24 / 26 2 HCOOH / AcOH 3: 1 40 17 204888/1 one HCOOH / AcOH 1: 1 22 eighteen 874-24 / 27 2 HCOOH / AcOH 1: 1 40 19 204923/1 one HCOOH / AcOH 1: 3 22 twenty 874-24 / 28 2 HCOOH / AcOH 1: 3 40 21 203251/3 one HCOOH / EtOH 99: 1 22 22 874-24 / 4 2 HCOOH / EtOH 99: 1 22 23 204815/1 one HCOOH / EtOH 99: 1 40 24 204815/1 one HCOOH / EtOH 3: 1 22 25 204888/1 one HCOOH / EtOH 3: 1 40 26 874-24 / 5 3 HCOOH / EtOH 1: 1 22 27 874-24 / 16 2 HCOOH / EtOH 1: 1 22 28 204923/1 one HCOOH / EtOH 1: 1 40 29th 204550/1 one HCOOH / EtOH 1: 3 22 thirty 874-24 / 17 2 HCOOH / EtOH 1: 3 22 31 204770/1 one HCOOH / EtOH 1: 3 40 32 203635/5 one HCOOH / Water 99: 1 22 33 874-24 / 24 2 HCOOH / Water 99: 1 40 34 204377/1 one HCOOH / Water 95: 5 22 35 874-24 / 29 2 HCOOH / Water 95: 5 40 36 202985/2 one HCOOH / water 4: 1 22 37 874-24 / 30 2 HCOOH / water 4: 1 40 38 204550/1 one HCOOH / water 1: 1 22 39 874-24 / 31 2 HCOOH / water 1: 1 40 40 874-33 / 5 3 HCOOH / water 1: 1 40 41 874-24 / 7 3 HCOOH / water 1: 3 22 42 874-24 / 18 2 HCOOH / water 1: 3 22 43 204770/1 one HCOOH / water 1: 3 40 44 874-24 / 8 3 AcOH / Water 99: 1 22 45 874-24 / 19 2 AcOH / Water 99: 1 40 46 874-24 / 11 3 AcOH / water 3: 1 22 47 874-24 / 20 2 AcOH / water 3: 1 40 48 874-31 / 2 2 AcOH / water 3: 1 40 49 874-33 / 4 3 AcOH / water 3: 1 40 fifty 874-24 / 12 3 AcOH / water 1: 1 22 51 874-24 / 25 2 AcOH / water 1: 1 40 52 874-31 / 3 2 AcOH / water 1: 1 40 53 874-24 / 13 3 HCOOH / phenol / water 1: 1: 1 22 54 874-24 / 21 2 HCOOH / phenol / water 1: 1: 1 22 55 874-32 / 3 2 1,500 ppm Na-hypochlorite 22 56 874-33 / 11 3 1,500 ppm Na-hypochlorite 22 57 874-33 / 14 2 1,500 ppm Na-hypochlorite 22 58 874-32 / 4 2 Formic acid 22 59 874-33 / 13 3 Formic acid 22 60 874-33 / 18 2 Formic acid 22 61 874-32 / 5 2 1,5M formaldehyde 22 62 874-33 / 12 3 1,5M formaldehyde 22 63 874-33 / 15 2 1,5M formaldehyde 22 64 874-32 / 6 2 6M guanidine hydrochloride 22 65 874-33 / 10 3 6M guanidine hydrochloride 22 66 874-33 / 17 2 6M guanidine hydrochloride 22 67 874-33 / 19 2 EtOH (60% w / w) /
0.1M NaOH 22 68 874-33 / 20 3 EtOH (60% w / w) /
0.1M NaOH 22

Functionality study results for individual columns

Table 5
The back pressure and the height of the hollow between the peaks obtained during studies of the functionality, performed before and after the artificial creation of problems, and after regeneration of the columns in accordance with the conditions listed in table 4.
RT is the retention time for des-B30-insulin, and the depression height between the peaks (HV) is given for des-B30-insulin and the nearest impurity. Example No. Back Pressure (MPA) Efficiency To problems After problems After regeneration To problems After problems After regeneration RT ml Hv
mAU RT ml Hv
mAU RT ml Hv
mAU one 3.8 6.2 3.8 140.2 38,4 144.3 36.1 2 3.4 5.8 3.6 141.2 39.6 146.6 62.0 138.6 44.5 3 3.4 > 10 3,7 141.2 39.6 - - 137.9 45.9 four 3.4 4.4 3.4 141.2 39.6 146.6 62.0 139.0 43,2 5 3.4 4.7 3.4 141.7 41.9 134.6 63.0 139.2 42.9 6 3.4 > 10 3.6 141.7 41.9 - - 140.1 45.8 7 3.4 4.7 3.4 134.5 54.3 136.5 47.8 137.2 48.6 8 3.4 > 10 > 10 134.5 54.3 - - - - 9 3.4 4.7 4.4 134.5 54.3 136.5 47.8 135.9 48,4 10 3.4 4.7 3.9 134.5 54.3 136.5 47.8 135.0 50.1 eleven 3.4 4.2 4.2 134.5 54.3 149.5 44.7 12 3.0 3,7 2,8 136.1 42.7 130.3 51.9 13 3.4 > 10 3,5 134.5 54.3 - - 145.2 44,4 fourteen 3,5 > 10 3,5 139.9 44.1 - - 135.9 44.8 fifteen 3,5 > 10 3.3 138.7 40.6 - - 127.3 54.7 16 3.4 4.7 3.4 134.5 54.3 136.5 47.8 139.3 45.0 17 3.0 > 10 2.9 139.2 30,4 - - 130.2 36,2 eighteen 3.4 4.7 3.4 134.5 54.3 136.5 47.8 137.1 47.0 19 3,1 > 10 3.0 130.2 36,2 - - 133.3 57.2 twenty 3.4 4.7 3,5 134.5 54.3 136.5 47.8 136.6 47.5 21 2.2 3.4 2,3 131.6 32.9 133.8 35,2 22 3.4 5.2 3.4 134.5 54.3 136.5 47.8 135.1 50.1 23 3.4 5.8 3.3 148.5 29.6 142.0 65.0 24 3.3 6.2 3,1 135.2 35,4 135.3 34.0 25 3,1 9 3,1 157.0 24.7 - - 151.3 31.9 26 3.4 > 10 > 10 134.5 54.3 - - - - 27 3.4 4.7 3,5 134.5 54.3 136.5 47.8 136.2 46.9 28 3.2 > 10 3,5 139.7 44.7 - - 148.0 35.7 29th 2,8 > 10 > 10 142.4 23,2 - - - - thirty 3.4 4.7 4.1 134.5 54.3 136.5 47.8 137.6 44,2 31 3.6 > 10 > 10 147.4 38.7 - - - - 32 3,1 6.9 3,1 143.8 34.7 136.3 41,4 33 3.4 4.7 3.3 134.5 54.3 136.5 47.8 132.1 55.8 34 3,7 > 10 3.9 140.6 41.6 - - 145.8 34.5 35 3.4 4.7 3.6 134.5 54.3 136.5 47.8 139.8 42.0 36 2,4 5.1 2,3 145.8 22.0 134.1 32,0 37 3.4 4.7 3,5 134.5 54.3 136.5 47.8 140.3 45.4 38 2.6 4.6 2.7 126.6 27,2 135.9 34.5 39 3.4 4.7 3.4 134.5 54.3 136.5 47.8 140,4 42,2 40 3.4 > 10 4.1 141.7 41.9 - - 141.8 64.0 41 3.4 > 10 > 10 134.5 54.3 - - - - 42 3.4 4.7 3.3 134.5 54.3 136.5 47.8 137.0 45.6 43 3.4 5.8 3.8 131.9 47.0 145.0 40.8 44 3.4 > 10 > 10 134.5 54.3 - - - - 45 3.4 4.7 4.3 134.5 54.3 136.5 47.8 135.2 42.9 46 3.4 > 10 5.2 134.5 54.3 - - 134.2 119.3 47 3.4 4.7 3.4 134.5 54.3 136.5 47.8 134.4 64.0 48 3.4 4.2 3,5 134.5 54.3 137.3 47.7 49 3.4 > 10 5,6 141.7 41.9 - - 141.9 73,4 fifty 3.4 > 10 9 134.5 54.3 136.5 47.8 - - 51 3.4 4.7 3.4 134.5 54.3 136.5 47.8 134.9 50.7 52 3.4 4.2 3,5 134.5 54.3 126.9 62.7 53 3.4 > 10 4.1 134.5 54.3 - - 137.7 46.1 54 3.4 4.7 3.4 134.5 54.3 136.5 47.8 138.6 45.0 55 3.4 4.4 3.8 141.2 39.6 146.6 62.0 138.0 46.2 56 3.4 > 10 > 10 141.7 41.9 - - - - 57 3.4 4.7 4.1 141.7 41.9 134.6 63.0 149.8 39.3 58 3.4 4.4 3.6 141.2 39.6 146.6 62.0 138.2 46.1 59 3.4 > 10 3,5 141.7 41.9 - - 135.5 52.3 60 3.4 4.7 3.3 141.7 41.9 134.6 63.0 135.1 54.1 61 3.4 4.4 3,7 141.2 39.6 146.6 62.0 137.7 49.4 62 3.4 > 10 > 10 141.7 41.9 - - - - 63 3.4 4.7 3.9 141.7 41.9 134.6 63.0 147.6 40.7 64 3.4 4.4 3,5 141.2 39.6 146.6 62.0 139.4 45.0 65 3.4 > 10 > 10 141.7 41.9 - - - - 66 3.4 4.7 3.8 141.7 41.9 134.6 63.0 137.4 53,4 67 3.4 4.7 3,5 141.7 41.9 134.6 63.0 138.4 39.1 68 3.4 > 10 3.9 141.7 41.9 134.6 - 138.8 49.3

Example 69

Column Life - Chromatographic Solvents / Eluents

The lifetime of substituted silica gels can be limited by chemical decomposition of the gel by solutions applied to the chromatographic column, for example, buffers and regeneration solutions. Chemical decomposition of substituted silica gels can be observed by the appearance of silicon at the outlet of the column or by a decrease in the carbon content in the gel, indicating a loss of substitution. The following experiments demonstrate the appearance of silicon in the column effluent and a decrease in the carbon content of the gels during prolonged washing with three chromatographic solutions: 20% ethanol in water, eluent 1 and eluent 2. The composition of eluent 2 is 31% w / w. ethanol, 1.5% wt./wt. KCl, 0.40% w / w CaCl ₂ , 0.15% w / w triethanolamine, and the pH was adjusted to pH 7.4 using HCl. The eluent composition 1 is a salt, a buffer, ethanol and a pH of 3.0.

The ODDMS silica gel charge is packed in five 4.0 × 250 mm steel columns using standard chromatographic column packing procedures. The columns are then equilibrated with 3 CV ethanol before continuous washing with chromatographic eluent begins. Then, five columns are washed with chromatographic eluent (20% EtOH, eluent 1 or eluent 2) for 1, 3, 7, 12 and 16 days, respectively. After washing for an appropriate time, each column was then equilibrated with 3 CV ethanol, and silica gel was removed from the columns. A sample of the used silica gel is analyzed for carbon, and a sample of the used regeneration solution is analyzed for silicon. Results for day No. 0 are the result (carbon content) of silica gel used for packing columns.

Table 6
Estimation of column life during prolonged flushing with typical chromatographic eluents by measuring remaining carbon and silicon released Column number Chromatographic solution Duration
(days) Measured With
(%) Measured Si (mg) Volume (ml) Relative Content C
(%) 100 Å ODDMS
4.0 * 250mm
204614/15
204614/16
204614/17
204614/23
20% EtOH
20% EtOH
20% EtOH
20% EtOH 0
one
3
7
12 19.40
19.14
19.16
19.26
19.28 0.00
0.05
0.13
0.31
0.25 0
136
425
1540
1270 100.00
98.66
98.76
99.28
99.38 204614/24 20% EtOH 16 19.27 0.41 1360 99.33
204614/18
204614/20
204614/21
204614/19
Eluent 1
Eluent 1
Eluent 1
Eluent 1 0
one
3
7
12 19.40
18.67
18.85
18.76
18.64 0.00
0.15
0.36
0.87
1.33 0
147
360
870
1325 100.00
96.24
97.16
96.70
96.08 204614/22 Eluent 1 16 18.67 0.86 860 96.24 Column number 20% EtOH
(% v / v) Duration
(days) Measured With
(%) Measured Si (mg) Volume (ml) Relative Content C
(%) 200 Å ODDMS
4.0 * 250mm
204630/14
204630/13
204630/15
204630/16
20% EtOH
20% EtOH
20% EtOH
20% EtOH 0
one
3
7
12 9.80
9.61
9.66
9.72
9.69 0.00
0,03
0.09
0.09
0.18 0
160
430
425
610 100.00
98.06
98.57
99.18
98.88 204630/18 20% EtOH 16 9.69 0.28 930 98.88
204630/19
204630/17
204630/20
204630/21
Eluent 2
Eluent 2
Eluent 2
Eluent 2 0
one
3
7
12 9.80
9.55
9.55
9.54
9.59 0.00
0.13
0.40
0.85
1.29 0
125
400
850
1290 100.00
97.45
97.45
97.35
97.86 204630/23 Eluent 2 16 9.49 0.70 700 96.84

Example 70

Column Life - Formic Acid Regeneration Solutions

The lifetime of substituted silica gels may be limited by chemical decomposition of the gel with solutions applied to the chromatographic column, for example, buffers and regeneration solutions. Chemical decomposition of substituted silica gels can be observed by the appearance of silicon at the outlet of the column and by a decrease in the carbon content in the gel, indicating a loss of substitution. The following experiments demonstrate the appearance of silicon in the column effluent and a decrease in the carbon content of the gels during prolonged flushing with regeneration solutions containing formic acid. Thus, compared with the situation using chromatographic buffers or solvents, regeneration solutions containing formic acid are no longer negatively affecting the column matrix.

The ODDMS silica gel charge is packed in five 4.0 × 250 mm steel columns using standard chromatographic column packing procedures. The columns are then equilibrated with 3 CV ethanol before continuous washing with formic acid begins. Then, five columns are washed with formic acid (100% or 80%) for 1, 3, 7, 12, and 16 days, respectively. After washing for an appropriate time, each column was then equilibrated with 3 CV ethanol, and silica gel was removed from the column. A sample of the used silica gel is analyzed for carbon, and a sample of the used regeneration solution is analyzed for silicon. The results for day No. 0 are the result (carbon content) of silica gel used for packing the columns.

Table 7
Estimation of column life during continuous regeneration with formic acid-containing solutions by measuring the remaining carbon and silicon released Column Type Column No. Formic acid
(%) Duration
(days) Measured With
(%) Measured Si (mg) Volume (ml) Relative Content C
(%) 100 Å ODDMS
4.0 * 250mm 0
204614/7
204614/5
204614/6
204614/3
204614/2 -
one hundred
one hundred
one hundred
one hundred
one hundred 0
one
3
7
12
12 19.40
19.00
18.90
19.26
18.97
18.94 0.00
0.82
1.46
1.95
5.63
3.92 0
124
510
620
1430
1100 100.00
97.94
97,42
99.28
97.78
97.63 0
204614/8
204614/10
204614/9
204614/11
204614/12 -
80
80
80
80
80 0
one
3
7
12
16 19.40
19.08
19.08
18.93
17.04
18.61 0.00
1,67
2.62
4,55
6.07
6.86 0
177
410
970
1080
2090 100.00
98.35
98.35
97.58
Nd
95.93 200 Å ODDMS
4.0 * 250mm 0
204630/8
204630/5
204630/6
204630/3
204630/2 -
one hundred
one hundred
one hundred
one hundred
one hundred 0
one
3
7
12
12 9.80
9.56
9.50
9.41
9.67
9.63 0.00
0.62
1.77
1,02
1.31
1.22 0
85
510
800
1020
1655 100.00
97.55
96.94
96.02
98.67
98.27 0
204630/7
204630/10
204630/9
204630/11
204630/12 -
80
80
80
80
80 0
one
3
7
12
16 9.80
9.64
9.42
9.43
8.67
9.18 0.00
0.56
0.77
1.10
1.40
1.46 0
141
440
920
1470
2000 100.00
98.37
96.12
96.22
Nd
93.67

Example 71

Using Confocal Laser Scanning Microscopy to Detect Insulin Fibrils on a Source 30Q Ion Exchanger

goal

The purpose of using confocal laser scanning microscopy is to visually determine how effective various regenerated solvents are when removing insulin fibrils from Source 30Q. Sophisticated image processing software makes it possible to measure the area of insulin fibrils in each picture, which gives a more accurate estimate, instead of a visual assessment of each picture.

Confocal principle

The principle of using CLSM is that the sample is stained with a fluorescent dye. The stained sample is placed under a microscope, and a fluorescent dye is excited using a laser. The emitted light emanating from the fluorescent dye is detected and an image is formed. A confocal laser scanning microscope is the same as a conventional confocal microscope, using a laser as a light source. Light from a laser passes through a microscope objective through a dichromatic beam splitter. A dichromatic beam splitter is a device that allows the light from the laser to pass through the lens down onto the sample, and the emitted light coming from the sample to pass through the dichromatic beam splitter and go up into the tubes of the photomultiplier tubes, where light is detected and an image is formed. A confocal point hole is located between the dichromatic beam splitter and the photomultiplier tubes. The confocal pinhole acts like a filter that stops all light coming from outside the focal regions so that it does not reach the tubes of the photomultiplier tubes. This means that the image formed here comes from a very narrow focal plane. By setting the confocal pinhole, it is possible to move the focal plane of the image up and down the sample and obtain a series of images along the optical (z) axis of the microscope. This series of images can be assembled into a three-dimensional image of the sample.

Source 30Q

Source 30Q is a strong anion exchanger based on monodisperse beads with a particle size of 30 microns. The matrix consists of polystyrene / divinylbenzene. Source 30Q gives weak intrinsic fluorescence at wavelengths above 650 nm. This means that Source 30Q particles can be seen without any staining.

Thioflavin t

The dye used to stain insulin fibrils is Thioflavin T, which is used to stain amyloid proteins. Thioflavin T has a wide emission spectrum with a maximum emission ranging from less than 490 nm to more than 530 nm. This means that one can detect differences between the light emanating from insulin fibrils (green) and intrinsic fluorescence light emanating from particles (red).

Method for staining insulin fibrils on Source 30Q

0.0385 g of dry Source 30Q is placed in a 25-30 ml glass beaker.

500 μl of 96% ethanol added to Source 30Q

4.5 ml of 0.05 M acetic acid was added, adjusted to pH 3 with NaOH.

The mixture is stirred by gently shaking the glass.

Add 20 μl of 116.62 mm Thioflavin T in Milli-Q-water

The mixture is gently shaken for 5 minutes.

The sample is now stained and ready for analysis under the microscope.

A small drop of the mixture, approximately 40 μl, is placed in a well on a glass slide and placed under a microscope.

Get 10 two-dimensional paintings of each sample from different sections of the hole.

Equipment

Microscope conditions

Lens: 40 *; oil; NA?

Laser: argon, 488 nm

Laser Intensity: 100%

Confocal point hole: 20 μm

Emission Filter (No. 2): 515 / 30M by Chroma

Emission Filter (No. 3): Chroma HQ650LP

Microscope: Nikon TE300 equipped with Nikon's PCM2000 confocal scanning head (photomultiplier).

Software: Nikon EZ2000 Viewer 2.5.77

Image Processing Software: AnalySIS 3.00

Image processing

The area of insulin fibrils is measured in 10 two-dimensional pictures from each sample. To ensure that each sample is processed in the same way, image processing is carried out automatically, by forming a macro. The macro is shown below. The area of each individual fibril from 10 paintings of each sample is brought together in an Excel worksheet. The total fibril area from each sample is collected in a bar graph for comparison.

The macro for measuring the area of fibrils:

Op.Display = 1;

docActivate ("picture name");

DbLoadlmage ();

MaximizeContrast ();

ShadingCorrection (N * N average filter size; 3; lower limit; 1900; upper limit; 2000);

BinarizeColorlmage (ColorThresholds: = NULL, Phase: = - 1);

Invert ();

EdgeEnhance (size; 5; percent; 70);

SetFrame (Left: = O, Top: = 0, Right: = 1023, Bottom = 1023);

Detect ();

ParticleResults ();

Op.Display = 6;

Option BurnOverlay = TRUE;

SaveAs (FileName :);

docActivate ("Sheet *");

SaveAs (FileName :);

Close (AskForSave: = TRUE);

Table 8
The measured fibril area on the used Source 30Q gel before and after gel regeneration using various regeneration solutions Regeneration solution Fibril area (arbitrary units) No 485 No 458 NaOAc (0.25% w / w NaOAc, 0.24% w / w tris, 42.5% w / w EtOH, pH 7.5 154 NaOH (1M) 101 HCOOH: water (50:50) 68 HCOOH (100%) 0 HCOOH: EtOH (50:50) 0

Complete removal of insulin fibrils is obtained using pure formic acid or a mixture of formic acid and ethanol. A mixture of formic acid: water, 50:50, does not completely remove fibrils, although this mixture is a more effective regeneration solution than sodium hydroxide (1M).

Example 72

Standard Procedure for Regeneration of RP-HPLC Columns with Formic Acid on an Industrial Scale

The procedure described below applies to the two types of columns used in the four stages of chromatographic purification in industrial-scale purification of human insulin.

Column type 1: Layer height 32.5 cm.

1. The column is washed with approximately 1.5 CV 20% w / w. ethanol in water at a flow rate of 4.6 CV / h to remove buffer salts.

2. The flow direction in the column is reversed.

3. The column is washed with approximately 1.1 CV absolute ethanol at a flow rate of 4.5 CV / hour.

4. 1.1 CV of pure formic acid is pumped into the column at a flow rate of 2.1 CV / hour.

5. The flow is stopped and the column is allowed to stand with formic acid for 30 minutes.

6. The column is washed with approximately 1.1 CV absolute ethanol at a flow rate of 4.5 CV / hour.

7. The column is washed with approximately 1.1 CV 20% w / w. ethanol in water at a flow rate of 4.5 CV / hour.

8. The flow direction in the column is again switched to normal.

9. The column is balanced using at least 4 CV 20% w / w. ethanol in water with a flow rate of 4.6 CV / hour.

The column is now ready for use again.

Column Type 2: Layer Height 37.5 cm

1. The column is washed with approximately 1.5 CV 20% w / w. ethanol in water at a flow rate of 4.6 CV / h to remove buffer salts.

2. The flow direction in the column is reversed.

3. The column is washed with approximately 1.0 CV absolute ethanol at a flow rate of 3.9 CV / hour.

4. 0.92 CV of pure formic acid is pumped into the column at a flow rate of 1.9 CV / hour.

5. The flow is stopped and the column is allowed to stand with formic acid for 30 minutes.

6. The column is washed with approximately 1.0 CV absolute ethanol at a flow rate of 3.9 CV / hour.

7. The column is washed with approximately 1.0 CV 20% w / w. ethanol in water at a flow rate of 3.9 CV / hour.

8. The flow direction in the column is again switched to normal.

9. The column is balanced with a minimum of 4.0 CV 20% w / w. ethanol in water with a flow rate of 4.6 CV / hour.

The column is now ready for use again.

On type 1 columns, regeneration using formic acid is carried out after every 16th process, as a preventive action, to suspend the deposition of components, increase back pressure of the column and increase dead volumes. Due to the application of the regeneration procedure with formic acid in the first stage of chromatographic purification, the life time of the column layer increases to 2000 cycles.

On type 2 columns, regeneration with formic acid is usually carried out after every 60th process or, if there is an increase in back pressures, an increase in dead volumes and precipitation of impurities. Due to the application of the regeneration procedure using formic acid, the column lifetime increases to 600 cycles at the III stage of chromatographic purification and up to 900 cycles at the IV stage of chromatographic purification.

The column matrix used in stages I, III, and IV of the chromatographic purification consists of particles of octadecyldimethyl-substituted silica (ODDMS silica). The described standard procedure applies to all types of substituted silica based matrices used on an industrial scale.

The procedure is also used in formic acid regeneration methods for column arrays based on Source Q material.

Examples of effects on chromatographic purification III and IV are shown below.

Table 9
Chromatographic purification III: Impact on dead volume and back pressure Before regeneration with formic acid (100%) Dead volume
(weight) Back pressure
(maximum pressure at the feed pump) 208 kg 27 bar After regeneration with formic acid (100%) Dead volume Back pressure
(maximum pressure at the feed pump) 165 kg 17 bar

Regeneration reduces dead volume by 21% and back pressure by 37%.

Figures 4A-B show the effect of regeneration on a preparative chromatogram for chromatographic purification III.

When applying regeneration with formic acid, an improvement is achieved in the separation and peak shape of human insulin (the peak of insulin is a large peak starting at time 8.20, in the upper figure, and at time 14.02, in the lower figure).

Example 73

Regeneration DEAE Sepharose

The DEAE Sepharose charge, which has been used in a large number of purification cycles in a method for purifying human growth hormone, is regenerated using pure formic acid (100% HCOOH). Before regeneration, Sepharose is brown, indicating the deposition of material on the gel. Regenerated Sepharose is much lighter in color. Samples of regenerated Sepharose were compared with Sepharose before regeneration using a colorimeter (Minolta CR-300) to quantify color differences, see results in Table 10.

Table 10
Assessment of DEAE Sepharose regeneration when it is regenerated as a suspension using formic acid regeneration solution Analyzed Material Sepharose Color coordinates L (light) A (red) b (yellow) Used gel before regeneration 66.81 2,37 17.22 Used gel after 3 hours of regeneration 84,99 0.66 11.55 Used gel after 24 hours of regeneration 87.94 0.35 7.64 Used gel after 24 hours of regeneration, when a fresh regeneration solution is applied at the 12th hour. 85.85 0.69 8.81 Unused gel, i.e. a gel that has never been used for chromatography 86.44 -0.06 2.01

Example 74

Regeneration of used silica gel containing glucagon aggregates

Glucagon and glucagon-like peptides (GLP-1 and GLP-2) are particularly susceptible to aggregation when they form fibrils, i.e. aggregates of β-folded structures. In the present example, a model experiment is carried out where human glucagon is loaded onto a silica gel column (as described in Examples 1-68). The glucagon solution is allowed to remain in the column for 3 days at 30 ° C to artificially create problems with pressure and efficiency, repeating the problems encountered during the industrial production of glucagon. The pressure on the column according to measurements is 3.5 MPa, using eluent 2, as described in example 69, with a flow rate of 9 ml / min at 22 ° C.

After a regeneration cycle using formic acid (100%), the pressure on the column decreased to 2.67 MPa, illustrating the effectiveness of formic acid as a regeneration solution.

Example 75

The column lifetime is a regeneration solution containing 0.1 M NaOH and 60% w / w. ethanol in water

The experiment is carried out as described in example 70, except for the regeneration solution, which in this experiment is alkaline ethanol, as is customary in the regeneration of chromatographic stationary phases.

The flow of the regeneration solution is about 0.1 ml / min, and the experiments are stopped after 4 days, because the pressure on the columns was> 10 MPa. The results before the failure of the columns are shown in table 11.

Table 11
Estimation of column life during continuous regeneration using a regeneration solution containing 0.1 M NaOH and 60% w / w. ethanol in water Column Type Column No. Duration
(days) Measured With
(%) Measured Si
(mg) Volume (ml) Relative Content C
(%) 200 Å ODDMS
4.0 * 250mm 0
204630/26
204630/25
204630/24 0
one
2
four 9.80
9.80
9.77
9.59 0,0
16,2
27.0
31.9 0
116
180
228 one hundred
one hundred
99.7
97.9

Example 76

Regeneration XAD 1180

The life time of the XAD 1180 reverse phase polymer resin used to concentrate the insulin precursor from the clarified fermentation broth is limited due to the strong accumulation of hydrophobic contaminants such as colored compounds, peptides and anti-foaming additives from fermentation. The usual regeneration cycle is based on washing with a regeneration solution containing 80% ethanol in 0.1 M citric acid, pH 3.0, followed by heating together with a 5% NaOH solution at 80 ° C. This regeneration method is not effective in removing accumulated contaminants.

The removal of the associated anti-foaming additive (Pluronic PE 6100 and P2000) from the used resin was evaluated using experiments with different concentrations and contact times of ethanol and isopropanol in a static mode and compared with formic acid regeneration (table 12). Formic acid treatment is excellent at removing adsorbed contaminants compared to standard industrial regeneration solvents.

Table 12
The effectiveness of various regeneration solutions in removing adsorbed contaminants of antifoam additives from the used XAD 1180 stationary chromatographic phase Regeneration solvent Conc. Time
(clock) Tempera Tour (° C) The concentration of antifoam additives on the resin (ppm) Ethanol 84% 2 25 20023 Ethanol 92% 2 25 13058 Ethanol 99% 2 25 8042 Ethanol 84% 6 25 25083 Ethanol 84% 2 fifty 14712 Ethanol 99% 6 fifty 9911 Isopropanol 70% 2 25 15899 Isopropanol 99% 2 25 3262 Formic acid 99% 2 25 670

Claims (29)

1. The method of regeneration of a chromatographic stationary phase, in which the specified chromatographic stationary phase is in contact with a regeneration solution containing at least one organic acid and less than about 75% wt./wt. water, where the specified organic acid is formic acid. 2. The method of regenerating the chromatographic stationary phase according to claim 1, wherein said chromatographic stationary phase is contacted with a regeneration solution containing at least one organic acid and less than about 1% w / w. water. 3. The method according to claim 1, wherein the concentration of said organic acid is at least about 25% w / w. 4. The method according to claim 1, wherein said regeneration solution contains an organic solvent. 5. The method according to claim 4, wherein said organic solvent is selected from the group consisting of ethanol, 2-propanol, acetonitrile, methanol, 1-propanol and hexylene glycol. 6. The method according to claim 1, in which the specified chromatographic stationary phase is in contact with the specified regeneration solution inside the chromatographic column. 7. The method according to claim 6, in which the specified chromatographic stationary phase is regenerated without re-packing columns. 8. The method according to claim 6, in which the specified chromatographic stationary phase liquefies during the specified regeneration. 9. The method of claim 6, wherein the chromatographic eluent or equilibration buffer is displaced with a water-miscible organic solvent before said stationary chromatographic phase is contacted with said regeneration solution. 10. The method according to claim 9, wherein said organic solvent is also present in the chromatographic eluent or equilibration buffer. 11. The method of claim 9, wherein said water miscible organic solvent is also present in the regeneration solution. 12. The method according to claim 1, in which the specified chromatographic stationary phase is in contact with the specified regeneration solution outside the chromatographic column. 13. The method according to claim 1, in which the specified chromatographic stationary phase is a matrix for RP-HPLC. 14. The method according to claim 1, in which the specified chromatographic stationary phase is a material such as silicon dioxide or substituted silicon dioxide. 15. The method according to 14, in which the specified chromatographic stationary phase is a C4-, C8-, C16-, C18- or phenyl-substituted silicon dioxide. 16. The method according to 14, in which the specified chromatographic stationary phase is a polymeric material. 17. The method according to claim 1, wherein said stationary chromatographic phase is contacted with said regeneration solution for at least 1 second, preferably for at least 1 minute, more preferably for at least 5 minutes, for example, from 1 minute to 24 hours, from 1 minute to 5 hours, from 1 minute to 2 hours, from 10 minutes to 60 minutes. 18. The method according to claim 1, wherein said stationary chromatographic phase is contacted with said regeneration solution until the pressure drop along the length of the chromatographic column at normal flow rate decreases by at least 10%, preferably at least , 25%, most preferably at least 50%. 19. The method according to claim 1, in which the contacting of the specified chromatographic stationary phase with the specified regeneration solution is carried out at a temperature in the range from about 5 to 50 ° C, preferably from 10 to 40 ° C, more preferably from 15 to 30 ° C, for example from 18 to 25 ° C. 20. The method according to claim 1, in which the lifetime of the specified chromatographic stationary phase is at least 500 chromatographic cycles, preferably at least 700 chromatographic cycles, more preferably at least 1000 chromatographic cycles, most preferably at least 2000 chromatographic cycles. 21. The method according to claim 1, in which the method is applied to the specified chromatographic stationary phase during each chromatographic cycle, at least once every 2 chromatographic cycles, at least once every 5 chromatographic cycles, at least at least once every 20 chromatographic cycles, at least once every 50 chromatographic cycles, or at least once every 100 chromatographic cycles. 22. A method of manufacturing a therapeutic polypeptide or its precursor, comprising at least one chromatographic step, in which the chromatographic stationary phase is regenerated by the regeneration method according to claim 1. 23. The method of claim 22, wherein said therapeutic polypeptide is a derivative containing a lipophilic substituent. 24. The method of claim 22, wherein said therapeutic polypeptide is selected from the group consisting of glucagon, glucagon-like peptide 1, glucagon-like peptide 2, exendin-4, TFF peptides, human insulin, its analogues and its derivatives. 25. The method according to item 22, wherein said polypeptide or its precursor is selected from the group consisting of Lys ²⁶ (N ^ε - (γ-Glu (N ^α -hexadecanoyl))) - GLP-1 (7-37), Arg ³⁴ -GLP-1 (7-37), exendin-4, Lys ¹⁷ Arg ³⁰ -GLP-2 (1-33), Arg ³⁰ Lys ¹⁷ N ^ε (β-Ala (N ^α- hexadecanoyl)) GLP-2 ( 1-33) and Gly ² -GLP-2 (1-33). 26. The method according to item 22, in which the specified polypeptide or its precursor is selected from the group consisting of
human insulin threonine methyl ester ^B30 ,
human insulin threonine ethyl ester ^B30 ,
Asp ^{B28 of} human insulin,
threonine methyl ester ^B30 Asp ^B28 human insulin,
threonine ethyl ester ^B30 Asp ^B28 human insulin,
Lys ^B28 Pro ^B29 Human Insulin,
Met ^B-1 Arg ^B0 Lys ^B28 Pro ^B29 human proinsulin,
Lys ^B3 Glu ^B29 Human Insulin,
Gly ^A21 Arg ^B31 Arg ^B32 human insulin,
des (B30) human insulin,
N ^εB29- tetradecanoyl des (B30) -insulin human
N ^εB29 -lithocholoyl-γ-glutamyl-des (B30) -insulin of a person,
N ^εB29 -Octanoyl-des- (B30) -Human insulin and
N ^εB29 -Octanoyl-human insulin. 27. The application of the method according to claim 1 for the regeneration of a stationary chromatographic phase used in the manufacture of a therapeutic polypeptide. 28. Chromatographic stationary phase, which is regenerated by the method according to claim 1. 29. The chromatographic stationary phase according to claim 28, wherein said stationary chromatographic phase is a material such as silicon dioxide or substituted silicon dioxide.

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