Indian Patents. 234148:"AN IMPROVED PROCESS FOR REGENERATING A CHROMATOGRAPHIC STATIONARY PHASE"

Title of Invention	"AN IMPROVED PROCESS FOR REGENERATING A CHROMATOGRAPHIC STATIONARY PHASE"
Abstract	Process for regenerating a chromatographic stationary phase.

Full Text	REGENERATION OF CHROMATOGRAPHIC STATIONARY PHASES. FIELD OF THE INVENTION The present invention relates to the field of chromatographic purification. More specifically it pertains to a process for regenerating chromatographic stationary phases. BACKGROUND OF THE INVENTION Polypeptides are increasingly being used as medicaments for the treatment of diseases within all major therapy areas. Treatment of diabetes by chronic insulin administration has been practised for more than 80 years, and therapeutic applications of polypeptides within growth disorders and cancer also have been practised for many years. Economical processes for the large scale production of polypeptides with a purity sufficiently high for therapeutic applications are crucial for further polypeptide-based therapies to reach the mass market and for the existing therapies to become more widely used. Purification of a polypeptide from a mixture is a steps which is normally used several times during the overall manufacturing process for a therapeutic polypeptide. Reverse phase high pressure liquid chromatography (RP-HPLC) is the preferred method for industrial high resolution separation of polypeptides, and the method has proven versatile for the large scale purification of many polypeptides. Since polypeptides for therapeutic use are to be highly purified in order not to cause adverse events upon administration to the patient, it is quite common to use several chromatographic purification steps in the manufacturing process. The stationary phase of chromatographic columns in manufacturing plants are expensive and they are thus used for several chromatographic cycles. However, over time the performance of the chromatographic stationary phase declines, i.e. pressure drop over the column increases prohibitively and the separation factor is impaired. This has been attributed to the gradual build-up of deposits. The problem has been suggested to be overcome by a regeneration process comprising alkaline buffers (J. Chrom. 461, 1989, 45-61), e.g. pH 7.4 and high concentration of organic modifier. Brange et al. (J. Pharm. Sci. 86 (1997) 517-525) discloses dissolving insulin fibrils in acid and in base. For many years the problem has been alleviated by regenerating the chromatographic stationary phase with alkaline solution, e.g. 0.1 molar sodium hydroxide {vide Liliedahl, "Twelve years of silica-based HPLC purification with focus on peptides", at Tides 2000, 10 May 2000 in Las Vegas, USA). This regeneration process may increase the lifetime of silica used for purifying insulin to between 100 to 600 cycles. However, silica materials are not stable when exposed to harsh alkaline conditions, and especially substituted silica materials may not be amenable to regeneration by alkaline solutions. Economically viable processes for purifying pharmaceuticals such as therapeutic polypeptides must include a regeneration process which does not degrade the chromatographic stationary phase. A general and complex process for regenerating particulate materials (clay, sand, silica etc.) from a wide variety of sources has been disclosed in WO 00/61493. It is a 5 step process comprising contacting the material with a) an extractant of organic material, b) an oxidizing agent, followed by c) an acid solution, d) heating the material and e) recovering the material. The process is cumbersome and not amenable for implementation in a chromatographic purification plant. There is a need in the art for more efficient ways of regenerating chromatographic stationary phases so as to increase the lifetime of these expensive raw materials and prevent the pressure drop over chromatographic columns to rise. Especially, regeneration processes which are suited for in-situ regeneration of chromatographic stationary phases in manufacturing plants are needed. SUMMARY OF THE INVENTION The present invention provides a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 75%w/w water. In another aspect the present invention provides a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 1%w/w water. In one embodiment of the invention the organic acid is formic acid. In another embodiment of the invention the organic acid is acetic acid. 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 process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid, an organic solvent and less than about 1%w/w water. In one embodiment of the invention the organic solvent is ethanol. In another embodiment of the invention the organic solvent is 2-propanol. In another embodiment of the invention the organic solvent is acetonitrile. In another embodiment of the 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 has been regenerated by the processes of the invention. In another aspect the present invention relates to a polypeptide product obtained by the processes of the invention. In yet another aspect the present invention relates to a polypeptide product manufactured by a process comprising the regeneration of the chromatographic stationary phase by the processes. In yet another aspect the invention relates to an automated chromatographic equipment comprising piping and control system for implementing the regeneration process. In yet another aspect the present invention relates to a pharmaceutical composition prepared by purifying a polypeptide using a chromatographic stationary phase which has been regenerated by the process. DEFINITIONS The following is a detailed definition of the terms used in the specification. The term "chromatographic stationary phase" as used herein means the solid phase over which the soluble phase passes, i.e. the chromatographic matrix. The chromatographic stationary phase is normally placed within a chromatographic column. Examples of chroma- tographic stationary phases are substituted silica, such as C-4 silica and C-18 silica, as well as polymeric materials. The term "chromatographic eluent" as used herein means the solution which is used for the elution step where the polypeptide being purified is normally released from the chromatographic stationary phase into the eluent. In the normal mode of chromatography a complete cyclus comprises a) equilibration with an equilibration buffer to bring the column in a state where it is ready for a cyclus, b) application of the product holding sample, c) an optional washing step where the chromatographic stationary phase with the bound product is washed, d) elution where the affinity of the product towards the chromatography stationary phase decreases and the product leaves the column in the chromatographic column eluate, and e) an optional regeneration where it is attempted to strip the chromatographic stationary phase from remaining impurities using a regeneration solution. The term "equilibrium buffer" as used herein means the solution which is used for the equilibration step wherein the chromatographic column is prepared for a chromatographic cycle. The term "regeneration solution" as used herein means a solution which is used to regenerate a chromatographic stationary phase. The purpose of the regeneration is keep a satisfactory perfonnance of the chromatographic separation over several chromatographic cycles. Typically critical performance related parameters are the pressure drop over the chromatographic column and the separation factor. A regeneration step may comprise contacting of the chromatographic stationary phase with either a single regeneration solution or with more than one regeneration solution. In the latter case, each of the regeneration solutions as well as the resulting mixtures of these are encompassed by the term "regeneration solution". The term "mixture" as used herein means a composition of matter comprising at least two ingredients. A chromatographic column eluate is a mixture which comprises the chemicals in the eluent together with the product which has been stripped from the column. Another example of a mixture is a solution of a chemical in a solvent, e.g. saline. Yet another example of a mixture is water and a water-miscible organic solvent. Yet 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 "isolating a polypeptide" as used herein means to bring the polypeptide in a state where it is of higher concentration or higher purity than it was before isolating it, i.e. in the starting material. Thus, an example of isolating a polypeptide is to precipitate or crystallize the polypeptide from a solution and separate the precipitate or crystals from the mother liquor. The term "organic solvent" as used herein means a solvent which comprises at least one carbon-atom and which is in the fluid state throughout the temperature range from 0°C to 50°C. Non-limiting examples of organic solvents are lower alcohols such as methanol and ethanol, polyhydric alcohols, acetonitrile, hexane and acetone. The term "water miscible organic solvent" as used herein means an organic solvent which 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 hexyleneglycol. The term "organic acid" as used herein means an organic compound which has at least one functional group with a dissociation constant, pKa, of less than 5.0. Examples of organic acids are formic acid, acetic acid, citric acid etc. The term "lower alcohol" as used herein means a C1-6-alcohol which is characterized by having between 1 and 6 carbon atoms and one hydroxyl moiety. The carbon skeleton in the lower alcohol may be straight or branched. Non-limiting examples of lower alcohols are ethanol, n-propanol, iso-propanol, and t-butanol. The term "polyhydric alcohol" as used herein means an alcohol having at least two hydroxyl moieties. Non-limiting examples of polyhydric alcohols 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 which are added to phamia-ceutical compositions in order to stabilize and preserve the composition. Typical excipients are buffers, preservatives and tonicity modifiers. The term "pharmaceutical composition" as used herein means a product comprising an active compound or a salt thereof together with pharmaceutical excipients such as buffer, preservative and tonicity modifier, said pharmaceutical composition being useful for treating a disease or disorder. Thus a pharmaceutical composition is also known in the art as a pharmaceutical formulation. The term "buffer" as used herein refers to a chemical compound which is used in a solution to reduce the tendency of pH of the solution to change over time as would othenwise occur due to chemical reactions. Buffers include chemicals 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 pharmaceuti- cal composition so that the osmotic pressure becomes closer to that of human plasma. Tonicity modifiers include NaCI, glycerol, D-mannitol etc. The term "pharmaceutically acceptable" as used herein means suited for normal pharmaceutical applications, i.e. does not cause adverse events in patients etc. The term "human insulin" as used herein means the human hormone whose structure and properties are well known. Human insulin has two polypeptide chains that are connected by disulphide bridges between cysteine residues, namely the A-chain and the B-chain. The A-chain is a 21 amino acid peptide and the B-chain is a 30 amino acid peptide, the two chains being connected by three disulphide bridges : one between the cysteines in position 6 and 11 of the A-chain, the second between the cysteine in position 7 of the A-chain and the cysteine in position 7 of the B-chain, and the third between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain. The term "polypeptide" as used herein means a compound composed of at least ten constituent amino acids connected by peptide bonds. The constituent amino acids may be from the group of the amino acids encoded by the genetic code and they may natural amino acids which are not encoded by the genetic code, as well as synthetic amino acids. Natural amino acids which are not encoded by the genetic code are e.g. hydroxyproline, y-carboxyglutamate, ornithine, phosphoserine, D-alanine and D-glutamine. Synthetic amino acids comprise amino acids manufactured by chemical synthesis, i.e. D-isomers of the amino acids encoded by the genetic code such as D-alanine and D-leucine, Aib (a-aminoisobutyric acid), Abu (a-aminobutyric acid). Tie (tert-butylglycine), and (3-alanine. The term "therapeutic polypeptide" as used herein means a polypeptide for which there is a recognized potential utility as a therapeutic agent. Therapeutic polypeptides are typically highly purified and they are subjected to clinical studies as part of the regulatory approval process. Examples of therapeutic polypeptides are human insulin, thrombopoetin, erythropoietin and human growth hormone. The term "polypeptide product" as used herein means a composition comprising the polypeptide. Examples of polypeptide products are crystallized polypeptide, precipitated polypeptide, and a solution of the polypeptide. The term "analogue" as used herein in relation to a parent polypeptide means a modified polypeptide wherein one or more amino acid residues of the parent polypeptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the parent polypeptide and/or wherein one or more amino acid residues have been deleted from the parent polypeptide and/or wherein one or more amino acid residues have been added to the parent polypeptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the polypeptide or at the C-terminal of the polypeptide or within the polypeptide. An example of an analogue is Arg34-GLP-1 (7-37) which is a GLP-1 (7-37) polypeptide wherein the Lys at position 34 has been replaced with an Arg. Other examples are porcine or bovine insulin which are both analogues of human insulin. The term "precursor" as used herein in relation to a polypeptide means a modified version of the polypeptide which is being produced. Precursors of a polypeptide are typically amino acid extended versions of the polypeptide, or truncated versions of the polypeptide. These precursor may serve to enhance cellular expression, comprise affinity tags for purification, protect certain reactive groups of the polypeptide being produced, etc. The term "derivative" as used herein in relation to a parent polypeptide means a chemically modified parent polypeptide or an analogue thereof, wherein at least one sub-stituent is not present in the parent polypeptide or an analogue thereof. I.e. a parent polypeptide which has been covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, PEGylations and the like. Examples of derivatives of human insulin are threonine methyl esterB30 human insulin and NεB29Metradecanoyl des(B30) human insulin. The term "lipophilic substituent" as used herein means a substituent comprising 4-40 carbon atoms and having a solubility in water at 20°C in the range from about 0.1 mg/100 ml water to about 250 mg/100 ml water, such as in the range from about 0.3 mg/100 ml water to about 75 mg/100 ml water. For instance, octanoic acid (C8) has a solubility in water at 20°C of 68 mg/100 ml, decanoic acid (CI10) 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 of 0.3 mg/100 ml. The term "piping and control system" as used herein means the physical means (pipes and control valves) and the software controlling the pipes and valves of a process equipment. DESCRIPTION OF THE INVENTION The present invention is concerned with a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 75%w/w water. The present invention is also concerned with a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 1%w/w water. The present invention is also concerned with a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution having a concentration of organic acid which is at least 25%w/w. A number of organic acids may be used in the regeneration solution of the process. A preferred organic acid is formic acid. Another organic acid for the regeneration solution is acetic acid. Another regeneration solution comprises two organic acids, e.g. formic acid and acetic acid. The regeneration solution may furthermore comprise an organic solvent. Preferably the organic solvent is also used in the equilibrium buffer or chromatographic eluent. In one embodiment of the 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 process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising 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 chromatographic stationary phase is preferably contacted with the regeneration solution inside the chromatographic column. In this way a minimum of production capacity is lost due to down-time in connection with the regeneration step. Thus, the process of regenerating the chromatographic stationary phase can be performed without repacking the column. In one embodiment, the chromatographic stationary phase is fluidized during said regeneration. In another embodiment the chromatographic eluent or equilibrium buffer is displaced by a water miscible organic solvent before said chromatographic stationary phase is contacted with said regeneration solution. Preferably said water miscible organic solvent is also present in the chromatographic eluent or equilibrium buffer. Preferably said water miscible organic solvent is also present in the regeneration solution. In another embodiment the chromatographic stationary phase is contacted with said regeneration solution outside the chromatographic column. This procedure is more cumbersome than performing the regeneration process inside the column, but it may nevertheless be useful if precipitated material is trapped between the chromatographic stationary phase particle. In the latter case, the precipitated material can be removed from the chromatographic stationary phase without dissolving said material. In one embodiment the chromatographic stationary phase is a RP-HPLC matrix. The chromatographic stationary phase for RP-HPLC are mechanically very rigid materials which may be silica or substituted silica such as C4, C6, C8, C16, C18, C30 or phenyl silica, or it may be a pressure stable polymeric material which is substituted or unsubstituted. The chromatographic stationary phase, be it a silica based matrix or a polymeric material, may also be present in the columns as monolithic rods with macropores and mesopores. Suitable silica material for use as chromatographic stationary phase is spherical particles with a narrow pore size distribution and particle sizes in the range from 8µm to 30µm, such as 10pm, 13pm, 16pm, and 18pm. Typically pore sizes in the range of 60A to 300A, such as 100Ǻ, 120Ǻ, 150Ǻ or 200Ǻ, are used. For pressure stable polymeric materials the pore size may be even higher, e.g. 400A, 600A or 1000Ǻ. The chromatographic column is packed with the stationary phase and after appropriate testing of the quality of the packing, the column is equilibrated with the buffer used in the binding mode. Production scale chromatographic columns typicallly have diameters of 15 to 100 cm, and such systems have dynamic axial compression. For production of small volume polypeptides the production columns may have a diameter of e.g. 15cm, 20cm or 25cm. For production of large volume polypeptides the production columns may have a diameter of e.g. 40cm, 60cm, 80cm or larger. In one embodiment of the process 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, such as 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 the process for regenerating a chromatographic stationary phase, said chromatographic stationary phase is contacted with said regeneration solution until the pressure drop over 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 the process for regenerating a chromatographic stationary phase, contacting of said chromatographic stationary phase with the regeneration solution is performed at a temperature in the range from about 5°C to 50°C, preferably from 10°C to 40°C, more preferably from 15°C to 30°C, such as from 18°C to 25°C. In another embodiment of the process for regenerating a chromatographic stationary phase, the life time 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 the process for regenerating a chromatographic stationary phase, said process is applied to said chromatographic stationary phase for every 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 process for regenerating a chromatographic stationary phase, said process is applied to said chromatographic stationary phase whenever the pressure drop over the length of the chromatographic column exceeds a threshold value. Another aspect of the present invention is a process for the production of a therapeutic polypeptide or a precursor thereof, said process comprising at least one chromatographic step wherein the chromatographic stationary phase is regenerated by a regeneration process as described above. In one embodiment of the process for the production of a therapeutic polypeptide or a precursor thereof, said therapeutic polypeptide is a derivative comprising a lipophilic substituent. In another embodiment of the process for the production of a therapeutic polypeptide or a precursor thereof, said therapeutic polypeptide is a derivative comprising a lipophilic substituent attached to the e-amino group of a lysine residue. In another embodiment of the process for the production of a therapeutic polypeptide or a precursor thereof, 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, analogues thereof and derivatives thereof. In another embodiment said polypeptide is selected from the group consisting of Lys26(Nε-(γ-Glu(Nα-hexadecanoyl)))-GLP-1(7-37), Arg34-GLP-1(7-37), and exendin-4. 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 fusion polypeptide between GLP-1(7-37) or an analogue thereof and a human serum albumin fragment or an analogue thereof. In another embodiment said polypeptide is a fusion polypeptide between exendin-4(1-39) or an analogue thereof and a human serum albumin fragment or an analogue thereof. In another embodiment said polypeptide is a fusion polypeptide comprising the Fc portion of an immunoglobulin or a fragment thereof. In another embodiment said polypeptide is a fusion polypeptide between GLP-1 (7-37) or an analogue thereof and a fragment of the Fc portion of an immunoglobulin or an analogue thereof. In another embodiment said polypeptide is a fusion polypeptide between exendin-4(1-39) or an analogue thereof and a fragment of the Fc portion of an immuneoglobulin or an analogue thereof. In another embodiment said polypeptide is selected from the group consisting of human insulin, a human insulin precursor, a human insulin analog, a human insulin analog precursor, a GLP-1 (7-37) analogue, an exendin-4(1-39) analogue, and derivatives thereof. In another embodiment said polypeptide is selected from a human insulin derivative comprising at least one methoxy or ethoxy moiety. In another embodiment said polypeptide is selected from the group consisting of threonine methyl esterB30 human insulin, threonine ethyl esterB30 human insulin, AspB28 human insulin, threonine methyl esterB30 AspB28 human insulin, threonine ethyl esterB30 AspB28 human insulin, LysB28 ProB29 human insulin, MetB-1ArgB0LysB28 ProB29 human proinsulin, LysB3 GluB29 human insulin, GlyA21 ArgB31 ArgB32 human insulin, des(B30) human insulin, NεB29-tetradecanoyl des(B30) human insulin, NεB29-litocholoyl-y-glutamyl des(B30) human insulin, NεB29-octanoyl des(B30) human insulin, and. NεB29-octanoyl human insulin. In yet another embodiment said polypeptide is selected from human serum albumin, erythropoietin, TNF-a, an interleukin, IGF-1, IGF-2, human growth hormone, somatostatin, human amylin and analogues thereof. The polypeptides being purified on chromatographic stationary phases regenerated by the processes of the present invention may be produced by a variety of techniques known in the art of polypeptide production. Polypeptides larger than 3000 Dalton are usually produced by fermentation or cell culture, whereas smaller polypeptide may be produced by chemical peptide synthesis. Other important factors determining the optimal production method are also the amount of polypeptide to be produced and the structure of the polypeptide, e.g. disul- phide bonds and other modifications. Fermentation or cell culture derived polypeptides are commonly produced by cultivation of recombinant host cells, e.g. bacteria, fungi mammalian cells, insect cells or plant cells in appropriate cultivation media. The cultivation medium may be a more or less chemically defined medium containing the necessary nutrients for growth and product formation of the host cells, e.g. sugar, nitrogen source, salts, vitamins and other growth factors. Once the microorganisms or the cells have been cultivated in the medium and they have optionally been disrupted, the cultivation medium contains the desired product in a mixture with remnant medium components, host cell derived impurities and product related impurities. Host cell derived impurities are mainly polypeptides, nucleic acids and cellular debris. The product is separated from these non-related impurities in the recovery or early purification steps. In the final purification steps (polishing) where impurities closely related to the product polypeptide are separated from the product polypeptide, chromatographic steps are extensively used. Synthesis of polypeptides may also be performed via solid phase synthesis by Merrifield-type chemistry, by solution phase methods, or by semisynthetic methods known in the art. One or more chemical conversion steps may be performed in-between the recovery and the final purification steps. Such chemical modifications may by the hydrolysis of a precursor polypeptide wherein the amino acid extension on the polypeptide is cleaved of the polypeptide. Such amino acid extensions may be used for increasing the host cell expression in the case of culture derived polypeptides, or it may be used to specifically purify the polypeptide, such as by affinity chromatography e.g. IMAC purification of histidine tagged polypeptides. The chemical conversion can also be the chemical modification to produce a polypeptide which is a derivative, e.g. by acylation, PEGylation or esterification. Such chemical modifications are well known in the art (see e.g. WO 98/08871, WO 99/43706, US 5,424286, 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 processes for regenerating a chromatographic stationary phase for decreasing the pressure drop over the length of the chromatographic column. Another aspect of the present invention is the use of the above processes for regenerating a chromatographic stationary phase for the manufacture of a therapeutic polypeptide. Another aspect of the present invention is a chromatographic stationary phase which has been regenerated by contacting said chromatographic stationary phase with a regeneration solution, said regeneration solution comprising at least one organic acid and less than about 75%w/w water. Another aspect of the present invention is a chromatographic stationary phase which has been regenerated by contacting said chromatographic stationary phase with a regeneration solution, said regeneration solution comprising at least one organic acid and less than about 1%w/w water. In one embodiment the chromatographic stationary phase has been regenerated by a process as described above. In another embodiment the chromatographic stationary phase has been regenerated by a process, wherein 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 a further embodiment the regenerated chromatographic stationary phase is a silica, or a substituted silica material. In another aspect the present invention relates to a polypeptide product manufactured by a process comprising the steps of a) purifying a polypeptide or a precursor thereof using the chromatographic stationary phase produced by the regeneration process of the present invention, and b) isolating said polypeptide or a precursor thereof to give the resulting polypeptide product. In another aspect the present invention relates to a polypeptide product manufactured by a process wherein is used a chromatographic stationary phase regenerated according to the process of the present invention. In another aspect the present invention relates to an automated chromatographic equipment comprising piping and control system for implementing the regeneration process according to the present invention. In another aspect the present invention relates to a pharmaceutical composition prepared by a process comprising the steps of a) first purifying a polypeptide or a precursor thereof using a chromatographic sta tionary phase regenerated by the process according to the present invention, b) then drying said polypeptide, and c) finally admixing with a pharmaceutically acceptable excipient. In another aspect the present invention relates to a pharmaceutical composition prepared by a process comprising the steps of a) first purifying a polypeptide or a precursor thereof using a chromatographic stationary phase regenerated by the process wherein said chromatographic stationary phase is contacted with a regeneration solution comprising 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 said polypeptide, and c) finally admixing with a pharmaceutically acceptable excipient. We Claim: 1. A process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 75% w/w water, wherein said organic acid is formic acid. 2. A process for regenerating a chromatographic stationary phase as claimed in claim 1 wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 1 % w/w water. 3. The process as claimed in any one of claims 1-2, wherein the concentration of said organic acid is at least about 25% w/w. 4. The process as claimed in any one of claims 1-3, wherein said regeneration solution comprises an organic solvent. 5. The process as claimed in claim 4, wherein said organic solvent is ethanol. 6. The process as claimed in claim 4, wherein said organic solvent is 2-propanol. 7. The process as claimed in claim 4, wherein said organic solvent is acetonitrile. 8. The process as claimed in claim 4, wherein said organic solvent is selected from the group consisting of methanol, 1-propanol, and hexylene glycol. 9. The process as claimed in any one of claims 1 -8, wherein 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. 10. The process as claimed in any one of claims 1-9, wherein said chromatographic stationary phase is contacted with said regeneration solution inside the chromatographic column. 11. The process as claimed in claim 10, wherein said chromatographic stationary phase is regenerated without repacking the column. 12. The process as claimed in claim 10, wherein said chromatographic stationary phase is fluidized during said regeneration. 13. The process as claimed in any one of claims 10-12, wherein the chromatographic eluent or equilibrium buffer is displaced by a water miscible organic solvent before said chromatographic stationary phase is contacted with said regeneration solution. 14. The process according to claim 13, wherein said organic solvent is also present in the chromatographic eluent or equilibrium buffer. 15. The process as claimed in any one of claims 13-14, wherein said water miscible organic solvent is also present in the regeneration solution. 16. The process as claimed in any one of claims 1-9, wherein said chromatographic stationary phase is contacted with said regeneration solution outside the chromatographic column. 17. The process as claimed in any one of claims 1-16, wherein said chromatographic stationary phase is a RP-HPLC matrix. 18. The process as claimed in any one of claims 1-15, wherein said chromatographic stationary phase is a silica or substituted silica material. 19. The process as claimed in claim 18, wherein said chromatographic stationary phase is CI6 or CI8 substituted silica. 20. The process as claimed in claim 18, wherein said chromatographic stationary phase is C4, C8 or phenyl-substituted silica. 21. The process as claimed in claim 18, wherein said chromatographic stationary phase is a polymeric material. 22. The process as claimed in any of the preceding claims, wherein 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, most preferably from 1 minute to 24 hours, from 1 minute to 5 hours, from 1 minute to 2 hours, from 10 minutes to 60 minutes. 23. The process as claimed in any one of claims 1-11, wherein said chromatographic stationary phase is contacted with said regeneration solution until the pressure drop over the length of the chromatographic column at normal flow rate decreases by at least 10%, preferably at least 25%, most preferably at least 50%. 24. The process as claimed in any of the preceding claims, wherein contacting of said chromatographic stationary phase with said regeneration solution is performed at a temperature in the range from about 5°C to 50°C, preferably from 10°C to 40°C, more preferably from 15°C to 30°C and most preferably from 18°C to 25°C. 25. The process as claimed in any one of claims 1-24, wherein the life time 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. 26. The process as claimed in any one of claims 1-25, wherein said process is applied to said chromatographic stationary phase for every 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. 27. Use of the process as claimed in claims 1- 26 for regenerating a chromatographic stationary phase used for the production of a therapeutic polypeptide.

Full Text

REGENERATION OF CHROMATOGRAPHIC STATIONARY PHASES.
FIELD OF THE INVENTION
The present invention relates to the field of chromatographic purification. More specifically it pertains to a process for regenerating chromatographic stationary phases.
BACKGROUND OF THE INVENTION
Polypeptides are increasingly being used as medicaments for the treatment of diseases within all major therapy areas. Treatment of diabetes by chronic insulin administration has been practised for more than 80 years, and therapeutic applications of polypeptides within growth disorders and cancer also have been practised for many years. Economical processes for the large scale production of polypeptides with a purity sufficiently high for therapeutic applications are crucial for further polypeptide-based therapies to reach the mass market and for the existing therapies to become more widely used.
Purification of a polypeptide from a mixture is a steps which is normally used several times during the overall manufacturing process for a therapeutic polypeptide. Reverse phase high pressure liquid chromatography (RP-HPLC) is the preferred method for industrial high resolution separation of polypeptides, and the method has proven versatile for the large scale purification of many polypeptides.
Since polypeptides for therapeutic use are to be highly purified in order not to cause adverse events upon administration to the patient, it is quite common to use several chromatographic purification steps in the manufacturing process. The stationary phase of chromatographic columns in manufacturing plants are expensive and they are thus used for several chromatographic cycles. However, over time the performance of the chromatographic stationary phase declines, i.e. pressure drop over the column increases prohibitively and the separation factor is impaired. This has been attributed to the gradual build-up of deposits. The problem has been suggested to be overcome by a regeneration process comprising alkaline buffers (J. Chrom. 461, 1989, 45-61), e.g. pH 7.4 and high concentration of organic modifier.
Brange et al. (J. Pharm. Sci. 86 (1997) 517-525) discloses dissolving insulin fibrils in acid and in base.

For many years the problem has been alleviated by regenerating the chromatographic stationary phase with alkaline solution, e.g. 0.1 molar sodium hydroxide {vide Liliedahl, "Twelve years of silica-based HPLC purification with focus on peptides", at Tides 2000, 10 May 2000 in Las Vegas, USA). This regeneration process may increase the lifetime of silica used for purifying insulin to between 100 to 600 cycles. However, silica materials are not stable when exposed to harsh alkaline conditions, and especially substituted silica materials may not be amenable to regeneration by alkaline solutions. Economically viable processes for purifying pharmaceuticals such as therapeutic polypeptides must include a regeneration process which does not degrade the chromatographic stationary phase.
A general and complex process for regenerating particulate materials (clay, sand, silica etc.) from a wide variety of sources has been disclosed in WO 00/61493. It is a 5 step process comprising contacting the material with a) an extractant of organic material, b) an oxidizing agent, followed by c) an acid solution, d) heating the material and e) recovering the material. The process is cumbersome and not amenable for implementation in a chromatographic purification plant.
There is a need in the art for more efficient ways of regenerating chromatographic stationary phases so as to increase the lifetime of these expensive raw materials and prevent the pressure drop over chromatographic columns to rise. Especially, regeneration processes which are suited for in-situ regeneration of chromatographic stationary phases in manufacturing plants are needed.
SUMMARY OF THE INVENTION
The present invention provides a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 75%w/w water. In another aspect the present invention provides a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 1%w/w water.

In one embodiment of the invention the organic acid is formic acid. In another embodiment of the invention the organic acid is acetic acid. 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 process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid, an organic solvent and less than about 1%w/w water.
In one embodiment of the invention the organic solvent is ethanol. In another embodiment of the invention the organic solvent is 2-propanol. In another embodiment of the invention the organic solvent is acetonitrile. In another embodiment of the 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 has been regenerated by the processes of the invention.
In another aspect the present invention relates to a polypeptide product obtained by the processes of the invention.
In yet another aspect the present invention relates to a polypeptide product manufactured by a process comprising the regeneration of the chromatographic stationary phase by the processes.
In yet another aspect the invention relates to an automated chromatographic equipment comprising piping and control system for implementing the regeneration process. In yet another aspect the present invention relates to a pharmaceutical composition prepared by purifying a polypeptide using a chromatographic stationary phase which has been regenerated by the process.
DEFINITIONS
The following is a detailed definition of the terms used in the specification.
The term "chromatographic stationary phase" as used herein means the solid phase over which the soluble phase passes, i.e. the chromatographic matrix. The chromatographic stationary phase is normally placed within a chromatographic column. Examples of chroma-

tographic stationary phases are substituted silica, such as C-4 silica and C-18 silica, as well as polymeric materials.
The term "chromatographic eluent" as used herein means the solution which is used for the elution step where the polypeptide being purified is normally released from the chromatographic stationary phase into the eluent. In the normal mode of chromatography a complete cyclus comprises
a) equilibration with an equilibration buffer to bring the column in a state where it is ready for a cyclus,
b) application of the product holding sample,
c) an optional washing step where the chromatographic stationary phase with the bound product is washed,
d) elution where the affinity of the product towards the chromatography stationary phase decreases and the product leaves the column in the chromatographic column eluate, and
e) an optional regeneration where it is attempted to strip the chromatographic stationary phase from remaining impurities using a regeneration solution.
The term "equilibrium buffer" as used herein means the solution which is used for the equilibration step wherein the chromatographic column is prepared for a chromatographic cycle.
The term "regeneration solution" as used herein means a solution which is used to regenerate a chromatographic stationary phase. The purpose of the regeneration is keep a satisfactory perfonnance of the chromatographic separation over several chromatographic cycles. Typically critical performance related parameters are the pressure drop over the chromatographic column and the separation factor. A regeneration step may comprise contacting of the chromatographic stationary phase with either a single regeneration solution or with more than one regeneration solution. In the latter case, each of the regeneration solutions as well as the resulting mixtures of these are encompassed by the term "regeneration solution".
The term "mixture" as used herein means a composition of matter comprising at least two ingredients. A chromatographic column eluate is a mixture which comprises the chemicals in the eluent together with the product which has been stripped from the column. Another example of a mixture is a solution of a chemical in a solvent, e.g. saline. Yet another example of a mixture is water and a water-miscible organic solvent. Yet 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 "isolating a polypeptide" as used herein means to bring the polypeptide in a state where it is of higher concentration or higher purity than it was before isolating it, i.e. in the starting material. Thus, an example of isolating a polypeptide is to precipitate or crystallize the polypeptide from a solution and separate the precipitate or crystals from the mother liquor.
The term "organic solvent" as used herein means a solvent which comprises at least one carbon-atom and which is in the fluid state throughout the temperature range from 0°C to 50°C. Non-limiting examples of organic solvents are lower alcohols such as methanol and ethanol, polyhydric alcohols, acetonitrile, hexane and acetone.
The term "water miscible organic solvent" as used herein means an organic solvent which 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 hexyleneglycol.
The term "organic acid" as used herein means an organic compound which has at least one functional group with a dissociation constant, pKa, of less than 5.0. Examples of organic acids are formic acid, acetic acid, citric acid etc.
The term "lower alcohol" as used herein means a C1-6-alcohol which is characterized by having between 1 and 6 carbon atoms and one hydroxyl moiety. The carbon skeleton in the lower alcohol may be straight or branched. Non-limiting examples of lower alcohols are ethanol, n-propanol, iso-propanol, and t-butanol.
The term "polyhydric alcohol" as used herein means an alcohol having at least two hydroxyl moieties. Non-limiting examples of polyhydric alcohols 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 which are added to phamia-ceutical compositions in order to stabilize and preserve the composition. Typical excipients are buffers, preservatives and tonicity modifiers.
The term "pharmaceutical composition" as used herein means a product comprising an active compound or a salt thereof together with pharmaceutical excipients such as buffer, preservative and tonicity modifier, said pharmaceutical composition being useful for treating a disease or disorder. Thus a pharmaceutical composition is also known in the art as a pharmaceutical formulation.
The term "buffer" as used herein refers to a chemical compound which is used in a solution to reduce the tendency of pH of the solution to change over time as would othenwise occur due to chemical reactions. Buffers include chemicals 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 pharmaceuti-

cal composition so that the osmotic pressure becomes closer to that of human plasma. Tonicity modifiers include NaCI, glycerol, D-mannitol etc.
The term "pharmaceutically acceptable" as used herein means suited for normal pharmaceutical applications, i.e. does not cause adverse events in patients etc.
The term "human insulin" as used herein means the human hormone whose structure and properties are well known. Human insulin has two polypeptide chains that are connected by disulphide bridges between cysteine residues, namely the A-chain and the B-chain. The A-chain is a 21 amino acid peptide and the B-chain is a 30 amino acid peptide, the two chains being connected by three disulphide bridges : one between the cysteines in position 6 and 11 of the A-chain, the second between the cysteine in position 7 of the A-chain and the cysteine in position 7 of the B-chain, and the third between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain.
The term "polypeptide" as used herein means a compound composed of at least ten constituent amino acids connected by peptide bonds. The constituent amino acids may be from the group of the amino acids encoded by the genetic code and they may natural amino acids which are not encoded by the genetic code, as well as synthetic amino acids. Natural amino acids which are not encoded by the genetic code are e.g. hydroxyproline, y-carboxyglutamate, ornithine, phosphoserine, D-alanine and D-glutamine. Synthetic amino acids comprise amino acids manufactured by chemical synthesis, i.e. D-isomers of the amino acids encoded by the genetic code such as D-alanine and D-leucine, Aib (a-aminoisobutyric acid), Abu (a-aminobutyric acid). Tie (tert-butylglycine), and (3-alanine.
The term "therapeutic polypeptide" as used herein means a polypeptide for which there is a recognized potential utility as a therapeutic agent. Therapeutic polypeptides are typically highly purified and they are subjected to clinical studies as part of the regulatory approval process. Examples of therapeutic polypeptides are human insulin, thrombopoetin, erythropoietin and human growth hormone.
The term "polypeptide product" as used herein means a composition comprising the polypeptide. Examples of polypeptide products are crystallized polypeptide, precipitated polypeptide, and a solution of the polypeptide.
The term "analogue" as used herein in relation to a parent polypeptide means a modified polypeptide wherein one or more amino acid residues of the parent polypeptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the parent polypeptide and/or wherein one or more amino acid residues have been deleted from the parent polypeptide and/or wherein one or more amino acid residues have been added to the parent polypeptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the polypeptide or at the C-terminal

of the polypeptide or within the polypeptide. An example of an analogue is Arg34-GLP-1 (7-37) which is a GLP-1 (7-37) polypeptide wherein the Lys at position 34 has been replaced with an Arg. Other examples are porcine or bovine insulin which are both analogues of human insulin.
The term "precursor" as used herein in relation to a polypeptide means a modified version of the polypeptide which is being produced. Precursors of a polypeptide are typically amino acid extended versions of the polypeptide, or truncated versions of the polypeptide. These precursor may serve to enhance cellular expression, comprise affinity tags for purification, protect certain reactive groups of the polypeptide being produced, etc.
The term "derivative" as used herein in relation to a parent polypeptide means a chemically modified parent polypeptide or an analogue thereof, wherein at least one sub-stituent is not present in the parent polypeptide or an analogue thereof. I.e. a parent polypeptide which has been covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, PEGylations and the like. Examples of derivatives of human insulin are threonine methyl esterB30 human insulin and NεB29Metradecanoyl des(B30) human insulin.
The term "lipophilic substituent" as used herein means a substituent comprising 4-40 carbon atoms and having a solubility in water at 20°C in the range from about 0.1 mg/100 ml water to about 250 mg/100 ml water, such as in the range from about 0.3 mg/100 ml water to about 75 mg/100 ml water. For instance, octanoic acid (C8) has a solubility in water at 20°C of 68 mg/100 ml, decanoic acid (CI10) 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 of 0.3 mg/100 ml.
The term "piping and control system" as used herein means the physical means (pipes and control valves) and the software controlling the pipes and valves of a process equipment.
DESCRIPTION OF THE INVENTION
The present invention is concerned with a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 75%w/w water. The present invention is also concerned with a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 1%w/w water.

The present invention is also concerned with a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution having a concentration of organic acid which is at least 25%w/w. A number of organic acids may be used in the regeneration solution of the process. A preferred organic acid is formic acid. Another organic acid for the regeneration solution is acetic acid. Another regeneration solution comprises two organic acids, e.g. formic acid and acetic acid.
The regeneration solution may furthermore comprise an organic solvent. Preferably the organic solvent is also used in the equilibrium buffer or chromatographic eluent. In one embodiment of the 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 process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising 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 chromatographic stationary phase is preferably contacted with the regeneration solution inside the chromatographic column. In this way a minimum of production capacity is lost due to down-time in connection with the regeneration step. Thus, the process of regenerating the chromatographic stationary phase can be performed without repacking the column. In one embodiment, the chromatographic stationary phase is fluidized during said regeneration. In another embodiment the chromatographic eluent or equilibrium buffer is displaced by a water miscible organic solvent before said chromatographic stationary phase is contacted with said regeneration solution. Preferably said water miscible organic solvent is also present in the

chromatographic eluent or equilibrium buffer. Preferably said water miscible organic solvent is also present in the regeneration solution.
In another embodiment the chromatographic stationary phase is contacted with said regeneration solution outside the chromatographic column. This procedure is more cumbersome than performing the regeneration process inside the column, but it may nevertheless be useful if precipitated material is trapped between the chromatographic stationary phase particle. In the latter case, the precipitated material can be removed from the chromatographic stationary phase without dissolving said material.
In one embodiment the chromatographic stationary phase is a RP-HPLC matrix. The chromatographic stationary phase for RP-HPLC are mechanically very rigid materials which may be silica or substituted silica such as C4, C6, C8, C16, C18, C30 or phenyl silica, or it may be a pressure stable polymeric material which is substituted or unsubstituted. The chromatographic stationary phase, be it a silica based matrix or a polymeric material, may also be present in the columns as monolithic rods with macropores and mesopores. Suitable silica material for use as chromatographic stationary phase is spherical particles with a narrow pore size distribution and particle sizes in the range from 8µm to 30µm, such as 10pm, 13pm, 16pm, and 18pm. Typically pore sizes in the range of 60A to 300A, such as 100Ǻ, 120Ǻ, 150Ǻ or 200Ǻ, are used. For pressure stable polymeric materials the pore size may be even higher, e.g. 400A, 600A or 1000Ǻ. The chromatographic column is packed with the stationary phase and after appropriate testing of the quality of the packing, the column is equilibrated with the buffer used in the binding mode. Production scale chromatographic columns typicallly have diameters of 15 to 100 cm, and such systems have dynamic axial compression. For production of small volume polypeptides the production columns may have a diameter of e.g. 15cm, 20cm or 25cm. For production of large volume polypeptides the production columns may have a diameter of e.g. 40cm, 60cm, 80cm or larger.
In one embodiment of the process 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, such as 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 the process for regenerating a chromatographic stationary phase, said chromatographic stationary phase is contacted with said regeneration solution until the pressure drop over 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 the process for regenerating a chromatographic stationary phase, contacting of said chromatographic stationary phase with the regeneration solution is performed at a temperature in the range from about 5°C to 50°C, preferably from 10°C to 40°C, more preferably from 15°C to 30°C, such as from 18°C to 25°C.
In another embodiment of the process for regenerating a chromatographic stationary phase, the life time 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 the process for regenerating a chromatographic stationary phase, said process is applied to said chromatographic stationary phase for every 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 process for regenerating a chromatographic stationary phase, said process is applied to said chromatographic stationary phase whenever the pressure drop over the length of the chromatographic column exceeds a threshold value.
Another aspect of the present invention is a process for the production of a therapeutic polypeptide or a precursor thereof, said process comprising at least one chromatographic step wherein the chromatographic stationary phase is regenerated by a regeneration process as described above. In one embodiment of the process for the production of a therapeutic polypeptide or a precursor thereof, said therapeutic polypeptide is a derivative comprising a lipophilic substituent. In another embodiment of the process for the production of a therapeutic polypeptide or a precursor thereof, said therapeutic polypeptide is a derivative comprising a lipophilic substituent attached to the e-amino group of a lysine residue. In another embodiment of the process for the production of a therapeutic polypeptide or a precursor thereof, 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, analogues thereof and derivatives thereof. In another embodiment said polypeptide is selected from the group consisting of Lys26(Nε-(γ-Glu(Nα-hexadecanoyl)))-GLP-1(7-37), Arg34-GLP-1(7-37), and exendin-4. 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 fusion polypeptide between GLP-1(7-37) or an analogue thereof and a human serum albumin fragment or an analogue thereof. In another embodiment said polypeptide is a fusion polypeptide between exendin-4(1-39) or an analogue thereof and a human serum albumin fragment or an analogue thereof. In another

embodiment said polypeptide is a fusion polypeptide comprising the Fc portion of an immunoglobulin or a fragment thereof. In another embodiment said polypeptide is a fusion polypeptide between GLP-1 (7-37) or an analogue thereof and a fragment of the Fc portion of an immunoglobulin or an analogue thereof. In another embodiment said polypeptide is a fusion polypeptide between exendin-4(1-39) or an analogue thereof and a fragment of the Fc portion of an immuneoglobulin or an analogue thereof.
In another embodiment said polypeptide is selected from the group consisting of human insulin, a human insulin precursor, a human insulin analog, a human insulin analog precursor, a GLP-1 (7-37) analogue, an exendin-4(1-39) analogue, and derivatives thereof. In another embodiment said polypeptide is selected from a human insulin derivative comprising at least one methoxy or ethoxy moiety. In another embodiment said polypeptide is selected from the group consisting of
threonine methyl esterB30 human insulin, threonine ethyl esterB30 human insulin, AspB28 human insulin,
threonine methyl esterB30 AspB28 human insulin, threonine ethyl esterB30 AspB28 human insulin, LysB28 ProB29 human insulin, MetB-1ArgB0LysB28 ProB29 human proinsulin, LysB3 GluB29 human insulin, GlyA21 ArgB31 ArgB32 human insulin, des(B30) human insulin, NεB29-tetradecanoyl des(B30) human insulin, NεB29-litocholoyl-y-glutamyl des(B30) human insulin, NεB29-octanoyl des(B30) human insulin, and. NεB29-octanoyl human insulin.
In yet another embodiment said polypeptide is selected from human serum albumin, erythropoietin, TNF-a, an interleukin, IGF-1, IGF-2, human growth hormone, somatostatin, human amylin and analogues thereof.
The polypeptides being purified on chromatographic stationary phases regenerated by the processes of the present invention may be produced by a variety of techniques known in the art of polypeptide production. Polypeptides larger than 3000 Dalton are usually produced by fermentation or cell culture, whereas smaller polypeptide may be produced by chemical peptide synthesis. Other important factors determining the optimal production method are also the amount of polypeptide to be produced and the structure of the polypeptide, e.g. disul-

phide bonds and other modifications. Fermentation or cell culture derived polypeptides are commonly produced by cultivation of recombinant host cells, e.g. bacteria, fungi mammalian cells, insect cells or plant cells in appropriate cultivation media. The cultivation medium may be a more or less chemically defined medium containing the necessary nutrients for growth and product formation of the host cells, e.g. sugar, nitrogen source, salts, vitamins and other growth factors. Once the microorganisms or the cells have been cultivated in the medium and they have optionally been disrupted, the cultivation medium contains the desired product in a mixture with remnant medium components, host cell derived impurities and product related impurities. Host cell derived impurities are mainly polypeptides, nucleic acids and cellular debris. The product is separated from these non-related impurities in the recovery or early purification steps. In the final purification steps (polishing) where impurities closely related to the product polypeptide are separated from the product polypeptide, chromatographic steps are extensively used.
Synthesis of polypeptides may also be performed via solid phase synthesis by Merrifield-type chemistry, by solution phase methods, or by semisynthetic methods known in the art. One or more chemical conversion steps may be performed in-between the recovery and the final purification steps. Such chemical modifications may by the hydrolysis of a precursor polypeptide wherein the amino acid extension on the polypeptide is cleaved of the polypeptide. Such amino acid extensions may be used for increasing the host cell expression in the case of culture derived polypeptides, or it may be used to specifically purify the polypeptide, such as by affinity chromatography e.g. IMAC purification of histidine tagged polypeptides. The chemical conversion can also be the chemical modification to produce a polypeptide which is a derivative, e.g. by acylation, PEGylation or esterification. Such chemical modifications are well known in the art (see e.g. WO 98/08871, WO 99/43706, US 5,424286, 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 processes for regenerating a chromatographic stationary phase for decreasing the pressure drop over the length of the chromatographic column.
Another aspect of the present invention is the use of the above processes for regenerating a chromatographic stationary phase for the manufacture of a therapeutic polypeptide.
Another aspect of the present invention is a chromatographic stationary phase which has been regenerated by contacting said chromatographic stationary phase with a regeneration solution, said regeneration solution comprising at least one organic acid and less than about 75%w/w water. Another aspect of the present invention is a chromatographic stationary

phase which has been regenerated by contacting said chromatographic stationary phase with a regeneration solution, said regeneration solution comprising at least one organic acid and less than about 1%w/w water.
In one embodiment the chromatographic stationary phase has been regenerated by a process as described above. In another embodiment the chromatographic stationary phase has been regenerated by a process, wherein 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 a further embodiment the regenerated chromatographic stationary phase is a silica, or a substituted silica material.
In another aspect the present invention relates to a polypeptide product manufactured by a process comprising the steps of
a) purifying a polypeptide or a precursor thereof using the chromatographic stationary phase produced by the regeneration process of the present invention, and
b) isolating said polypeptide or a precursor thereof to give the resulting polypeptide product.
In another aspect the present invention relates to a polypeptide product manufactured by a process wherein is used a chromatographic stationary phase regenerated according to the process of the present invention.
In another aspect the present invention relates to an automated chromatographic equipment comprising piping and control system for implementing the regeneration process according to the present invention.
In another aspect the present invention relates to a pharmaceutical composition prepared by a process comprising the steps of
a) first purifying a polypeptide or a precursor thereof using a chromatographic sta
tionary phase regenerated by the process according to the present invention,
b) then drying said polypeptide, and
c) finally admixing with a pharmaceutically acceptable excipient.
In another aspect the present invention relates to a pharmaceutical composition prepared by
a process comprising the steps of
a) first purifying a polypeptide or a precursor thereof using a chromatographic stationary phase regenerated by the process wherein said chromatographic stationary phase is contacted with a regeneration solution comprising 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 said polypeptide, and
c) finally admixing with a pharmaceutically acceptable excipient.

We Claim:
1. A process for regenerating a chromatographic stationary phase wherein said
chromatographic stationary phase is contacted with a regeneration solution comprising at
least one organic acid and less than about 75% w/w water, wherein said organic acid is
formic acid.
2. A process for regenerating a chromatographic stationary phase as claimed in claim 1 wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one organic acid and less than about 1 % w/w water.
3. The process as claimed in any one of claims 1-2, wherein the concentration of said organic acid is at least about 25% w/w.
4. The process as claimed in any one of claims 1-3, wherein said regeneration solution comprises an organic solvent.
5. The process as claimed in claim 4, wherein said organic solvent is ethanol.
6. The process as claimed in claim 4, wherein said organic solvent is 2-propanol.
7. The process as claimed in claim 4, wherein said organic solvent is acetonitrile.
8. The process as claimed in claim 4, wherein said organic solvent is selected from the group consisting of methanol, 1-propanol, and hexylene glycol.
9. The process as claimed in any one of claims 1 -8, wherein 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.

10. The process as claimed in any one of claims 1-9, wherein said chromatographic stationary phase is contacted with said regeneration solution inside the chromatographic column.
11. The process as claimed in claim 10, wherein said chromatographic stationary phase is regenerated without repacking the column.

12. The process as claimed in claim 10, wherein said chromatographic stationary phase is fluidized during said regeneration.
13. The process as claimed in any one of claims 10-12, wherein the chromatographic eluent or equilibrium buffer is displaced by a water miscible organic solvent before said chromatographic stationary phase is contacted with said regeneration solution.
14. The process according to claim 13, wherein said organic solvent is also present in the chromatographic eluent or equilibrium buffer.
15. The process as claimed in any one of claims 13-14, wherein said water miscible organic solvent is also present in the regeneration solution.
16. The process as claimed in any one of claims 1-9, wherein said chromatographic stationary phase is contacted with said regeneration solution outside the chromatographic column.
17. The process as claimed in any one of claims 1-16, wherein said chromatographic stationary phase is a RP-HPLC matrix.
18. The process as claimed in any one of claims 1-15, wherein said chromatographic stationary phase is a silica or substituted silica material.
19. The process as claimed in claim 18, wherein said chromatographic stationary phase is CI6 or CI8 substituted silica.
20. The process as claimed in claim 18, wherein said chromatographic stationary phase is C4, C8 or phenyl-substituted silica.
21. The process as claimed in claim 18, wherein said chromatographic stationary phase is a polymeric material.
22. The process as claimed in any of the preceding claims, wherein 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, most preferably

from 1 minute to 24 hours, from 1 minute to 5 hours, from 1 minute to 2 hours, from 10 minutes to 60 minutes.
23. The process as claimed in any one of claims 1-11, wherein said chromatographic stationary phase is contacted with said regeneration solution until the pressure drop over the length of the chromatographic column at normal flow rate decreases by at least 10%, preferably at least 25%, most preferably at least 50%.
24. The process as claimed in any of the preceding claims, wherein contacting of said chromatographic stationary phase with said regeneration solution is performed at a temperature in the range from about 5°C to 50°C, preferably from 10°C to 40°C, more preferably from 15°C to 30°C and most preferably from 18°C to 25°C.
25. The process as claimed in any one of claims 1-24, wherein the life time 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.
26. The process as claimed in any one of claims 1-25, wherein said process is applied to said chromatographic stationary phase for every 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.
27. Use of the process as claimed in claims 1- 26 for regenerating a chromatographic stationary phase used for the production of a therapeutic polypeptide.

"AN IMPROVED PROCESS FOR REGENERATING A CHROMATOGRAPHIC STATIONARY PHASE"

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