MXPA97009657A - Preparation of an acil protein dust - Google Patents

Abstract

The present invention relates to a method for recovering acylated protein as a powder, especially in cases where the acylated protein is one that resists isolation by isoelectric precipitation from the aqueous solutions of the protein, the method comprises in combination: adjust the aqueous solution to a value close to the isoelectric pH of the protein and provide a suitable concentration of alcohol to cause the precipitation of the protein in the form of filterable particles at pH adjust

Description

PREPARATION OF AN ACILATED PROTEIN DUST BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is broadly directed to a method for recovering, as an acylated protein, particularly those that resist recovery by precipitation or crystallization and subsequent filtration of aqueous solutions, and especially certain 'acylated proinsulins, acylated insulins and acylated insulin analogues. More particularly, the present invention relates to the preparation of a free flowing powder, of the acylated proinsulins, insulins and insulin analogs, from their aqueous solutions.

Description of Related Art Insulin therapy has been an object for a long time to minimize the model of endogenous insulin secretion in normal individuals. The daily physiological demand for the substances is variable and can be separated into two phases: (a) the phase R? F.026395 absorbent that requires an impulse of insulin to discard excess glucose from the blood related to food or food, and (b) the post-absorbent phase which requires a sustained amount of insulin to regulate the output of the hepatic glucose in order to keep the blood glucose at an optimum level. Consequently, an effective therapy generally involves the combined use of two exogenous insulins: a temporary insulin for fast-acting foods, provided by bolus injections and a long-acting or sustained basal insulin, administered by injection once or twice at a time. day. Recently, a class of acylated insulins has shown that it is promising for use as a long-acting basal insulin therapy. These acylated insulins are prepared by the acylation, selectively with an activated fatty acid derivative, of the free amino group (s) of a monomeric insulin, including a proinsulin, normal insulin and certain insulin analogues. Useful fatty acid derivatives include compounds of the reactive fatty acid type having at least one chain length of six (6) carbon atoms and particularly those derived from fatty acids having 8 to 21 carbon atoms in their chain . Normal mono-acylated human insulin, acylated with a palmitic acid derivative, is a particularly promising candidate. The insulins that are considered within this category are described in Japanese Patent Application No. 1-254,699. As is well understood by those skilled in the art, forms for recovering a protein in a solid form, preferably a free flowing powder, are especially advantageous, if not essential, in many applications. The preparation of a protein as a powder, for example, maximizes the storage and distribution options. This is particularly true in the context of compositions intended for insulin therapy wherein high volumes of the product must be produced to meet the demand. Accidentally, it has long been discovered that normal insulin, including cattle insulin, pig insulin and human insulin, could be recovered as a powder by precipitation, or more precisely by crystallization, of insulin from relatively dilute aqueous solutions of insulin such as a zinc complex or as sodium crystals. In general, the so-called zinc insulin is crystallized from a buffered aqueous solution containing zinc ions and can be easily isolated by filtration.

Unfortunately, attempts to recover the insulins acylated with long chain fatty acid derivatives by filtration have not been as successful. Indeed, known attempts to crystallize acylated fatty acid insulins have been uniformly unsuccessful. It has been postulated that the hydrophobic character of the long chain fatty acid portion of such insulin monomers contributes to this difficulty by interfering inter alia with the protein-protein interactions required for satisfactory crystal formation. In consecuensenot. , other forms must be found to prepare these insulins insulilated in a pulverized, solid form. One approach technically feasible to produce an acylated protein, such as an acylated insulin, in a powdered form, is to lyophilize an aqueous solution of the protein. Although powdered preparations of acylated fatty acid insulins can be produced in this manner, lyophilization techniques are not conveniently adapted for the production of large quantities of commercially needed insulin. Issues of operator safety and product handling are particularly problematic for large volume lyophilization operations.

Another potential strategy involves isoelectric precipitation. The proteins in an aqueous solution are already known to become less soluble when the pH of the environment is adjusted to a value close to the isoelectric point of the protein. This general phenomenon has already been widely exploited in the processes of purification and recovery of proteins. Once insolubilized, such proteins can often be recovered from the resulting aqueous suspension simply by thickening by the action of gravity or by filtration aided by a vacuum or aided by pressure. Unfortunately, attempts to recover insulins acylated with fatty acids by simply adjusting the pH of an aqueous solution of the acylated protein near its isoelectric point, have produced emulsion-like compositions that readily resist solid-liquid separation, such as thickening by the action of gravity or filtration aided by vacuum or aided by pressure. As a result, a new approach to recovering such insulin species acylated in a powdered form must be found. The present invention provides a simple method for recovering acylated proteins by precipitation and filtration, as a free-flowing powder, and especially certain insulins acylated with fatty acids that resist such isolation and recovery by precipitation and filtration. The present invention thus provides a method for recovering acylated fatty acid insulins, such as a free flowing powder, from aqueous solutions. In particular, the present invention provides a process for recovering a powdered acylated insulin with fatty acids, which can be conveniently adapted for production techniques on a relatively large scale. The present invention also pertains to the pulverized protein prepared by this method.

DESCRIPTION OF THE INVENTION All abbreviations of the amino acids used in this description are those accepted by the United States Patent and Trademark Office as described in 37 C.F.R. & 1.822 (B) (2). The terms "insulin" and "normal insulin" when used herein mean human insulin, pig insulin, or cattle insulin. Insulin possesses three free amino groups: B1-Phenylalanine, A1-Glycine, and B29-Lysine. The free amino groups at positions A1 and B1 are the a-amino groups. The free amino group at position B29 is an α-amino group. The term "proinsulin" when used herein is a properly cross-linked protein of the formula: B - C - A wherein: A is the A chain of insulin or a functional derivative thereof; B is the B chain of insulin or a functional derivative thereof which has an α-amino group; and C is the connecting peptide of proinsulin. Preferably, proinsulin is the A chain of human insulin, the B chain of human insulin, and C is the naturally occurring peptide. When proinsulin is the natural sequence, proinsulin has three free amino groups: phenylalanine (1) (a-amino group), lysine (29) (e-amino group) and lysine (64) (group -amino). The term "insulin analogue" when used herein, is an appropriately cross-linked protein that exhibits an insulin activity of the formula: A - B where: A is the insulin A chain or a functional derivative of the insulin A chain; and B is the B chain of insulin or a functional derivative of the B chain of insulin having an e-amino group and at least one of A and B contains an amino acid modification of the natural sequence. Preferred insulin analogs include insulin, wherein: the amino acid residue at position B28 is Asp, Lys, Leu, Val, or Ala; the amino acid residue at position B29 is Lys or Pro; the amino acid residue at position B10 is His or Asp; the amino acid residue at position B1 is Phe, Asp, or deleted alone or in combination with a deletion of the residue in position B2; the amino acid residue at position B30 is Thr, Ala, or deleted; and the amino acid residue at position B9 is Being or Asp; provided that any of positions B28 or B29 is Lys. In standard biochemical terms known to those of ordinary skill in the art, the preferred insulin analogues are insulin LysB28ProB29-human (B28 is Lys; B29 is Pro); insulin AspB28-human (B28 is Asp); insulin AspB1-human, insulin ArgB31, B32-human, insulin AspB10-human, insulin ArgA0-human, AspB1, insulin GluB13-human, insulin AlaB26-human, and insulin GlyA21-human. The term "acylation" means the introduction of one or more acyl groups covalently linked to the free amino groups of the protein. The term "fatty acid" means a fatty acid with C6-C2? saturated or not saturated. The term "activated fatty acid ester" means a fatty acid which has been activated using general techniques as described in Methods of Enzymology, 25: 494-499 (1972) and Lapidot et al., In J. of Lipid Res. , 8 ^: 142-145 (1967), the descriptions of which are incorporated herein for reference. Preferred fatty acids are saturated and include myristic acid (Cu), pentadecyl acid (C15), palmitic acid (Cie), heptadecyl acid (C17) and stearic acid (C? 8). More preferably, the fatty acid is palmitic acid. Esters of activated fatty acids include those derived from agents such as hydroxybenzotriazide (HOBT), H-hydroxysuccinimide and derivatives thereof. The preferred activated ester is N-succinimidyl palmitate.

The term "crosslinking" means the formation of the disulfide bonds between the cysteine residues. An appropriately cross-linked insulin proinsulin, insulin or analogue contains three disulfide bridges. The first disulfide bridge is formed between the cysteine residues in positions 6 and 11 of the A chain. The second disulfide bridge binds or binds the cysteine residues in position 7 of the A chain to the cysteine in position 7 of the B chain. The third disulfide bridge binds the cysteine at position 20 of the A chain to the cysteine at position 19 of the B chain. The term "precipitation" refers to a process in which the easily filterable particles they are produced in a liquid and are to be distinguished from simply a change in the solubility of a solute in the solution, which leads to the formation of stable suspensions and / or mixtures of two phases, similar to an emulsion. Filterable particles by themselves, are referred to as a "precipitate". The "easily filterable" phase and other similar phrases refer to a condition in which the particles of a solid-liquid mixture or suspension can be isolated by a closed-end filtration process and the related techniques for a manageable filter cake. , ie a material with a residual moisture content that does not interfere with the handling of the cake as a solid instead of a suspension. In the "closed end" filtration, the suspension is supplied and the filtrate liquid passes substantially perpendicular to the plane of the filter and will be contrasted with the "cross flow" or "tangential flow" filtration in which the main liquid flow It is parallel to 1 surface of the filter medium. Importantly, the method of the present invention produces a suspension of an acylated protein that can be isolated by "closed end" filtration and techniques related to an average or average filtration rate exceeding 5 l / m2 / h ( LMH), and often at an average or average filtration rate greater than 20 LMH, the endpoint of filtration which is usually the point when the cake can be handled as a solid. Such filtration rates are critical for the economy of the operation at a commercial production scale. The term "aqueous" includes cosolvent systems as well as the use of water only as a solvent. The present invention relates to a method of recovering a protein acylated as a powder from an aqueous mixture, especially those acylated proteins that resist isolation and recovery by isoelectric precipitation from an aqueous solution of the acylated protein. The method comprises adjusting an aqueous solution of the acylated protein to a pH which causes the protein in solution to become insoluble and adding sufficient alcohol to the gaseous mixture of the protein to cause precipitation of the protein in the form of filterable particles. said pH. In another aspect, the present invention pertains to a powdered acylated protein, particularly an acylated protein that resists isolation by isoelectric precipitation from an aqueous solution. In particular, the present invention pertains to an acylated fatty acid proinsulin, an acylated insulin of fatty acid or an insulin analog acylated with a fatty acid in the form of a powder, prepared by adjusting an aqueous solution of the insulin protein acylated to a pH that causes the protein in the aqueous solution to become insoluble, and add enough alcohol to the aqueous protein to cause precipitation of the protein in the form of filterable particles at said pH. After this, the protein can be recovered as a zinc-free powdered acylated protein, by filtration and drying. Preferred acylated proteins, prepared in the powder form using the method herein, include insulin N-palmitoyl Lys-human and insulin B28-Ne-palmitoyl-LisB28ProB29-human (B28 is acylated Lys and B29 is Pro). The proinsulin, insulin and insulin analogs used to prepare the acylated proteins that are the main focus of the present invention can be prepared from any of a variety of recognized peptide synthesis techniques including classical (solution) methods, of solid phase, semisynthetic methods, and most recent recombinant DNA methods. For example, Chance et al., U.S. patent application. number 07 / 388,201, EPO publication number 383 472, Brange et al., EPO 214 826, and Belagaje et al., U.S. Pat. 5,304,473 describes the preparation of various proinsulins and insulin analogs and are incorporated herein by reference. The A and B chains of the insulin analogs of the present invention can also be prepared by means of the proinsulin-like precursor molecule using recombinant DNA techniques. See Frank et al., Peptides: Synthesis-Structure-Function, Proc. Seventh Am. Pept. Symp., Eds. D. Rich and E. Gross (1981) which is incorporated herein for reference. In general, proinsulin, insulin and insulin analogs are acylated by reacting them with an activated fatty acid derivative, such as an activated fatty acid ester. Acylation of normal insulin with a fatty acid is described in Japanese Patent Application No. 1,254,699. See also Hashimoto et al., Pharmaceutical Research, 6: 171-176 (1989). These descriptions are incorporated herein for reference. Preferably, the acylation is carried out under basic conditions, that is, at a pH greater than 9.0 and preferably in about 10.5, in a polar solvent. Although the reaction can be carried out in a fully organic polar solvent using a base having an aqueous pKa greater than or equal to 10.75, an aqueous and organic solvent mixed for the reaction medium is preferred. Preferred bases are tetramethylguanidine, diisopropylethylamine or tetrabutylammonium hydroxide. A particularly suitable solvent has been acetonitrile and water, which contains about 50% acetonitrile. Other polar solvents include dimethyl sulfoxide, dimethylformamide and the like. The cosolvent systems also include acetone and water, isopropyl alcohol and water, and ethanol and water. The time and temperature conditions suitable for carrying out the reactions are not narrowly critical. A temperature of 0 to 40 ° C and a reaction time of 15 minutes to 24 hours should be generally adequate. A particularly preferred form of preparation of such insulins acylated with a fatty acid is described in the U.S. Copendiente Serial No. 08/341231 filed on November 17, 1994, the description of which is incorporated herein for reference. Once the reaction is complete, the reaction mixture containing the acylated protein is typically diluted with water and an acid is added to neutralize the alkalinity. The acid is supplied as an aqueous solution to the acylated protein and serves to lower the pH of the solution below the isoelectric point of the protein. Normally, at this point the protein is an aqueous solution buffered appropriately for further processing. Such processing includes particularly purification by standard chromatographic methods such as hydrophobic or reverse phase chromatography, cross flow filtration concentration, solvent exchange by ultrafiltration and the like. For acylated proinsulin, acylated insulin and acylated insulin analogs, particularly human N-palmitoyl insulin LysB29 and insulin B28-Ne-? Almitoyl-LysB28ProB29-human, the pH should normally be adjusted below about 3.0, and preferably to between approximately 1.5 and 2.5, using the acid that is necessary. Suitable acids include HCl, acetic acid, glycine and especially citric acid. The use of citric acid at a concentration of 50 mM has been found adequate. If necessary, the pH can also be readjusted with a base, such as sodium hydroxide, to keep it within the desired range. At this point, the aqueous solution of the acylated protein, isolated and preferably purified, particularly a fatty acid-acylated proinsulin, an insulin acylated with fatty acid or an insulin analog acylated with fatty acid, can be processed according to the present invention to recover the soluble protein as a powder. According to the invention, the pH of the solution of the aqueous protein is adjusted, by the addition of a base, preferably a water-soluble base such as sodium hydroxide, in an amount sufficient to cause the soluble protein to reach be insoluble This is done by adjusting the pH of the solution to near the isoelectric point of the acylated protein, alternatively referred to as the isoelectric pH. In the case of a proinsulin acylated with a fatty acid, or an insulin acylated with a fatty acid and an insulin analog acylated with a fatty acid, particularly the human insulin N-palmitoyl LysB29, the pH adjustment within the range of about 4.0 to 6.0, and preferably up to about 4.5 to 5.5 has been proven to be adequate. At this pH, the net charge of the acylated protein should be at a minimum and the solubility of the protein should be at its lowest point. During pH adjustment, the solution must be agitated to obtain complete mixing. The aqueous solutions of the acylated proteins, which are especially treatable for treatment with the present invention, form a composition similar to an emulsion when the pH is adjusted to near its isoelectric pH. This condition interferes with the isolation of the protein using those solid / liquid separation techniques, such as closed-end filtration, desirable for large-scale processing. A key feature of the present invention relates to the use of an alcohol, especially ethanol, added to the aqueous protein mixture to aid in the formation of an easily filterable precipitate of the acylated protein. The sequence for adjusting the pH of the solution and the addition of alcohol is not critical. The alcohol can be added to the protein solution prior to the pH adjustment required for the insolubilization of the protein and the eventual formation of the precipitate and can also be added after adjusting the pH of the protein solution. Applicants have found that the amount of alcohol that must be added to the acylated protein to obtain an easily filterable precipitate falls within a narrow range. In particular, applicants have discovered that for an insulin acylated with a fatty acid, and especially the human N-palmitoyl insulin LysB29, the desired result, ie, the formation of an easily filtered precipitate, is obtained only during the addition of an amount of alcohol, especially ethanol, in a narrow range to provide a final alcohol concentration of at least about 20% and up to about 35%, and preferably in an approximate form from 25% to 30%, in the slurry or slurry of the aqueous protein . Although ethanol is clearly the alcohol of choice, other alcohols such as methanol and isopropanol should be suitable substitutes in certain circumstances. The addition of too much alcohol to the aqueous protein interferes with obtaining an acceptable level of precipitate formation, although the addition of an insufficient amount does not adequately resolve the composition similar to the emulsion produced during the pH adjustment of the acylated protein in a slurry or paste easily filterable. Actually, at levels higher than the desired levels of alcohol, the protein will redissolve contributing to possible reductions in production. The determination of an amount of alcohol, such as ethanol, necessary to facilitate precipitation and filtration of other proteins acylated with a fatty acid, can be done using routine experimentation. In effect, therefore, the invention is based on the recognition that in order to recover, as a free-flowing powder, an acylated protein, particularly one that is otherwise resistant to isolation and recovery by precipitation and isoelectric filtration of the solutions The following combination of conditions must be produced, (a) a pH at or near the isoelectric pH of the protein and (b) a sufficient alcohol concentration to aid in the precipitation and filtration of the protein facilitating the formation of filterable particles easily. As noted above, in the case of human LysB29 N-palmitoyl insulin, an ethanol concentration of about 20% to 35% by volume of the suspension, and preferably 25% to 30% by volume, has been found to be be particularly suitable for aiding precipitation of the protein in the form of easily filterable particles.

The method by which a mixture of aqueous proteins is widely prepared to have the required concentration of alcohol (for example ethanol) and to exhibit the isoelectric pH of the acylated protein is not critical. For example, such a mixture can be obtained by diluting, with water, a pH-adjusted aqueous solution, of the protein having a higher alcohol concentration than required, that is, in the case of the N-palmitoyl insulin LysB29. human, by diluting an ethanol concentration above 35% by volume, ie from 40 to 45% ethanol, to a concentration of alcohol in the desired range. Preferably, the composition is stirred gently (mixed) at the point where both the alcohol concentration and the pH are within the desired limits to facilitate the formation of the precipitate. The concentration of the acylated protein in the solution prior to the treatment according to the present invention is also not critical. In the case of acylated insulins, also including acylated proinsulin and acylated insulin analogues, a concentration of at least about 1 mg / ml, and preferably at least about 5 mg / ml, should generally be used, although the use of A protein concentration as high as 35 to 50 mg / ml and higher should provide a precipitated protein that can be processed at more acceptable filtration rates. As noted above, the suspension of the protein produced by the combined treatment of the pH adjustment and the addition of the alcohol must exhibit a mean or average filtration range of at least about 5 LMH, and preferably exhibits an average filtration rate of less approximately 15 LMH. Surprisingly, the average filtration rates of approximately 20 LMH and higher have been observed during the treatment of human LysB29 N-palmitoyl insulin solutions according to the present invention. Although not acutely critical, for best results, the temperature during filtration is maintained in the range of 20 ° C to 30 ° C. Temperatures of 0 ° C or below it, however, should be avoided during filtration because such temperatures interfere with the operation at the high, desired filtration rates. Although the filtration is preferably done at an essentially ambient temperature, the steps of adjusting the pH and adding the ethanol, above, can be done at any temperature above the freezing point of the suspension.

At this point, the composition of the aqueous protein is easy to be filtered to isolate the precipitated protein. In some cases, it may be advantageous to thicken or sediment the aqueous protein composition or suspension prior to filtration to minimize the hydraulic load on the filtration equipment. Although centrifugal filtration of the aqueous protein composition may be advantageous at larger treatment scales, eg, production batches greater than about 0.5 kg, any means for liquid / solid separation, including vacuum assisted or aided filtration by the pressure, they can be used to recover the precipitated protein in the broad practice of the present invention. On a smaller scale, for example, bottom lot sizes of about 0.3 to 0.5 kg, pressure-assisted filtration using a filtration device having a porous frit filtration surface, can be used to advantage. Filtration through a porous frit with a nominal pore size of approximately 5.0 μm has proven to be completely effective. The equipment used to filter the suspension is not acutely critical and any of the wide variety of known filtration systems compatible with the processing of the pharmaceutical compositions can be used.

Following the filtration, the filter cake is recovered and dried, generally by evaporation assisted by vacuum. In the broad practice of the present invention, any method for drying a thick paste of solids, not otherwise detrimental to the isolated protein, can be used. The ways to recover the filter cake and other ways to dry the filter cake should be apparent to those skilled in the art and are not critical. The acylated insulin powders of the present invention are useful as a volumetric drug substance (BDS) for preparing the pharmaceutical compositions used in insulin therapy, ie for administration to a patient in need thereof (i.e. , a patient suffering from hyperglycemia). Such pharmaceutical compositions contain an effective amount of the powder in combination with one or more pharmaceutically acceptable excipients or carriers. For these purposes, the pharmaceutical compositions can typically be formulated to contain about 100 units per ml or similar concentrations containing an effective amount of the acylated insulin powder. These compositions can typically be formulated to contain about 100 units per ml or similar concentrations containing an effective amount of the acylated insulin powder. These compositions are typically, but not necessarily, parenteral in nature and can be prepared by any of a variety of techniques using conventional excipients or carriers for parenteral products which are well known in the art.

See, for example, Remington's Pharmaceutical Sciences, 17 / a. Edition, Mack Publishing Company, Easton, PA, USA (1985) which is incorporated herein for reference. For example, dosage forms for parenteral administration can be prepared by suspending or dissolving the desired amount of the protein powder in a non-toxic liquid vehicle suitable for injection, such as an aqueous medium and the sterilization of the suspension or solution. . Alternatively, a measured amount of the powder can be placed in an ampule; and the vial and its contents are sterilized and sealed. An ampoule or companion vehicle may be provided for mixing purposes prior to administration. Pharmaceutical compositions adapted for parenteral administration employ diluents, excipients and carriers such as water and water miscible organic solvents such as glycerin, sesame oil, peanut oil, aqueous propylene glycol, N, N-dimethylformamide and the like. Examples of such pharmaceutical compositions include aqueous, isotonic, sterile saline solutions of the powder, which can be quenched with a pharmaceutically acceptable buffer and which are pyrogen-free. Additionally, the parenteral pharmaceutical formulation may contain preservatives such as metacresol, other agents for adjusting the pH of the final product such as sodium hydroxide or hydrochloric acid and stabilizers such as zinc salts. The following example is presented to illustrate and explain the invention. Although the invention is illustrated by the reference to the recovery of human LysB29 N-palmitoyl insulin as a powder, the scope of the invention should not be considered to be limited to this example. Unless stated otherwise, all references to parts and percentages are based on weight and all temperatures are expressed in degrees centigrade.

EXAMPLE An aqueous solution (8.8 1) of an Insulin Human Biosynthetic acylated with a fatty acid (insulin Human LysB29 N-palmitoyl), which has a protein concentration of 50 mg / ml and a pH of 2.6, is treated at a temperature of 2-8 ° C with 530 ml of 10% sodium hydroxide which increases the pH to 5.7. Following the agitation of the solution adjusted in the pH to obtain a complete mixing, absolute ethanol, in an amount of 0.46 liters per liter of the solution adjusted in the pH (up to a volume of the total solution of 13 1), was added, with gentle agitation, to help the precipitation of the acylated insulin. To the suspension of the acylated protein the water was removed on a filter assisted by the pressure that has a stainless steel frit, with a nominal pore size of 5.0 μ. The average filtration rate observed was approximately 25 LMH. The filtrate was discarded. The filter cake is washed with an aqueous solution containing 30% ethanol by volume, and the washed filter cake is recovered from the filter and dried under a vacuum to give a zinc-free powder. This powder has been found to have excellent storage stability making it suitable as a pharmaceutical volumetric drug substance. Substantially all of the protein was recovered in the filter cake with very little loss through the filtrate. The principles, preferred embodiments, and modes of operation of the present invention have been described with particularity in the foregoing specification primarily with reference to proinsulin, insulin, and insulin analogs acylated with a fatty acid, particularly N-insulin. palmitoyl LysB29 human. The invention which is proposed to be protected here, however, will not be constructed to be limited to the particular forms described, since they will be considered as illustrative rather than restrictive. Changes and variations may be made by those skilled in the art without departing from the spirit of the invention as defined in the following claims.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (19)

1. A method for recovering a protein acylated as a powder from an aqueous solution of the acylated protein, the method is characterized in that it comprises adjusting the pH of the aqueous solution of the acylated protein to a pH which causes the acylated protein to become insoluble and provide a suitable alcohol concentration to aid in the formation of the easily filterable particles of the acylated protein.

2. The method according to claim 1, characterized in that the protein has been acylated with a fatty acid derivative.

3. The method according to claim 1, characterized in that the acylated protein comprises a protein that resists isolation by isoelectric precipitation from the aqueous solutions of the protein.

4. The method according to claim 3, characterized in that the acylated protein is selected from the group consisting of a proinsulin acylated with a fatty acid, an insulin acylated with a fatty acid and an insulin analog acylated with a fatty acid.

5. The method according to claim 4, characterized in that the pH of the aqueous solution is adjusted to a pH of about 4.0 to 6.0.

6. The method according to claim 5, characterized in that the acylated protein is insulin N-palmitoyl LysB29 human.

7. The method according to claim 6, characterized in that the alcohol is ethanol and the ethanol is added in an amount to give an alcohol concentration of 20% to 35% by volume.

8. The method according to claim 7, characterized in that the concentration of the alcohol is from 25% to 30% by volume.

9. A method for recovering a protein acylated as a powder from an aqueous solution of the acylated protein, the method is characterized in that it comprises, in combination, adjusting the pH of the aqueous solution of the acylated protein to a pH close to the isoelectric pH of the acylated protein and provide a suitable alcohol concentration to aid in the formation of easily filterable particles of the acylated protein.

10. The method according to claim 9, characterized in that the acylated protein is one that resists the isolation by the isoelectric precipitation of the aqueous solutions.

11. The method according to claim 10, characterized in that the acylated protein is selected from the group consisting of a proinsulin acylated with a fatty acid, an insulin acylated with a fatty acid and an insulin analog acylated with a fatty acid.

12. The method according to claim 10, characterized in that the pH of the solution. aqueous is adjusted to a pH of about 4.0 to 6.0.

13. The method according to claim 12, characterized in that the acylated protein is the human N-palmitoyl insulin LysB29.

14. The method according to claim 13, characterized in that the alcohol is ethanol and the ethanol is added in an amount to give an alcohol concentration of 25% to 30% by volume.

15. A powdered acylated protein, characterized in that it is prepared by a combination of (a) adjusting an aqueous solution of the acylated protein to a pH at or near the isoelectric pH of the protein and (b) providing a suitable concentration of the alcohol to assist the formation of filterable particles easily followed by recovery of the filterable particles as a powder of acylated protein, by filtration and drying.

16. The powdered acylated protein according to claim 15, characterized in that the acylated protein comprises a protein that resists isolation by isoelectric precipitation from an aqueous solution.

17. The powdered acylated protein according to claim 16, characterized in that the acylated protein is selected from the group consisting of a proinsulin acylated with a fatty acid, an insulin acylated with a fatty acid and an insulin analog acylated with a fatty acid.

18. The powdered acylated protein according to claim 17, characterized in that the pH of the aqueous solution is adjusted to a pH of about 4.0 to 6.0.

19. The acylated protein according to claim 18, characterized in that the alcohol is ethanol and the ethanol is added in an amount to give an alcohol concentration of 25% to 30% by volume.